![Protein structure](https://upload.wikimedia.org/wikipedia/commons/0/05/Protein_structure.png)
Protein folding is the
physical process
Physical changes are changes affecting the form of a chemical substance, but not its chemical composition. Physical changes are used to separate mixtures into their component compounds, but can not usually be used to separate compounds into chem ...
by which a
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
chain is
translated
Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. The English language draws a terminological distinction (which does not exist in every language) between ''transla ...
to its native
three-dimensional structure, typically a "folded"
conformation by which the protein becomes biologically functional. Via an expeditious and reproducible process, a
polypeptide
Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides.
A p ...
folds into its characteristic three-dimensional structure from a
random coil
In polymer chemistry, a random coil is a conformation of polymers where the monomer subunits are oriented randomly while still being bonded to adjacent units. It is not one specific shape, but a statistical distribution of shapes for all the cha ...
.
Each protein exists first as an unfolded polypeptide or random coil after being translated from a sequence of
mRNA
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein.
mRNA is ...
to a linear chain of
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
s. At this stage the polypeptide lacks any stable (long-lasting) three-dimensional structure (the left hand side of the first figure). As the polypeptide chain is being synthesized by a
ribosome
Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to ...
, the linear chain begins to fold into its three-dimensional structure.
Folding of many proteins begins even during translation of the polypeptide chain. Amino acids interact with each other to produce a well-defined three-dimensional structure, the folded protein (the right hand side of the figure), known as the
native state
In biochemistry, the native state of a protein or nucleic acid is its properly folded and/or assembled form, which is operative and functional. The native state of a biomolecule may possess all four levels of biomolecular structure, with the s ...
. The resulting three-dimensional structure is determined by the amino acid sequence or primary structure (
Anfinsen's dogma
Anfinsen's dogma, also known as the thermodynamic hypothesis, is a postulate in molecular biology. It states that, at least for a small globular protein in its standard physiological environment, the native structure is determined only by the pro ...
).
The correct three-dimensional structure is essential to function, although some parts of functional proteins
may remain unfolded, so that
protein dynamics Proteins are generally thought to adopt unique structures determined by their amino acid sequences. However, proteins are not strictly static objects, but rather populate ensembles of (sometimes similar) conformations. Transitions between these stat ...
is important. Failure to fold into native structure generally produces inactive proteins, but in some instances misfolded proteins have modified or toxic functionality. Several
neurodegenerative
A neurodegenerative disease is caused by the progressive loss of structure or function of neurons, in the process known as neurodegeneration. Such neuronal damage may ultimately involve cell death. Neurodegenerative diseases include amyotrophic ...
and other
disease
A disease is a particular abnormal condition that negatively affects the structure or function of all or part of an organism, and that is not immediately due to any external injury. Diseases are often known to be medical conditions that a ...
s are believed to result from the accumulation of
amyloid
Amyloids are aggregates of proteins characterised by a Fibril, fibrillar morphology of 7–13 Nanometer, nm in diameter, a beta sheet (β-sheet) Secondary structure of proteins, secondary structure (known as cross-β) and ability to be Staining, ...
fibrils
Fibrils (from the Latin ''fibra'') are structural biological materials found in nearly all living organisms. Not to be confused with fibers or filaments, fibrils tend to have diameters ranging from 10-100 nanometers (whereas fibers are micro ...
formed by misfolded proteins, infectious varieties of which are known as
prions
Prions are misfolded proteins that have the ability to transmit their misfolded shape onto normal variants of the same protein. They characterize several fatal and transmissible neurodegenerative diseases in humans and many other animals. It i ...
.
Many
allergies
Allergies, also known as allergic diseases, refer a number of conditions caused by the hypersensitivity of the immune system to typically harmless substances in the environment. These diseases include hay fever, food allergies, atopic derma ...
are caused by incorrect folding of some proteins, because the
immune system
The immune system is a network of biological processes that protects an organism from diseases. It detects and responds to a wide variety of pathogens, from viruses to parasitic worms, as well as cancer cells and objects such as wood splinte ...
does not produce
antibodies
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
for certain protein structures.
Denaturation of proteins is a process of transition from the folded to the
unfolded state
In biochemistry, denaturation is a process in which proteins or nucleic acids lose the quaternary structure, tertiary structure, and secondary structure which is present in their native state, by application of some external stress or compound ...
. It happens in
cooking
Cooking, cookery, or culinary arts is the art, science and craft of using heat to Outline of food preparation, prepare food for consumption. Cooking techniques and ingredients vary widely, from grilling food over an open fire to using electric ...
, in
burn
A burn is an injury to skin, or other tissues, caused by heat, cold, electricity, chemicals, friction, or ultraviolet radiation (like sunburn). Most burns are due to heat from hot liquids (called scalding), solids, or fire. Burns occur mainl ...
s, in
proteinopathies, and in other contexts.
The duration of the folding process varies dramatically depending on the protein of interest. When studied
outside the cell, the slowest folding proteins require many minutes or hours to fold primarily due to
proline isomerization, and must pass through a number of intermediate states, like checkpoints, before the process is complete. On the other hand, very small single-
domain
Domain may refer to:
Mathematics
*Domain of a function, the set of input values for which the (total) function is defined
**Domain of definition of a partial function
**Natural domain of a partial function
**Domain of holomorphy of a function
* Do ...
proteins with lengths of up to a hundred amino acids typically fold in a single step. Time scales of milliseconds are the norm and the very fastest known protein folding reactions are complete within a few microseconds. The folding time scale of a protein depends on its size,
contact order The contact order of a protein is a measure of the locality of the inter-amino acid contacts in the protein's native state tertiary structure. It is calculated as the average sequence distance between residues that form native contacts in the folde ...
and
circuit topology
The circuit topology of a folded linear polymer refers to the arrangement of its intra-molecular contacts. Examples of linear polymers with intra-molecular contacts are nucleic acids and proteins. Proteins fold via formation of contacts of variou ...
.
Understanding and simulating the protein folding process has been an important challenge for
computational biology
Computational biology refers to the use of data analysis, mathematical modeling and computational simulations to understand biological systems and relationships. An intersection of computer science, biology, and big data, the field also has fo ...
since the late 1960s.
Process of protein folding
Primary structure
The
primary structure of a protein, its linear amino-acid sequence, determines its native conformation.
The specific amino acid residues and their position in the polypeptide chain are the determining factors for which portions of the protein fold closely together and form its three-dimensional conformation. The amino acid composition is not as important as the sequence.
The essential fact of folding, however, remains that the amino acid sequence of each protein contains the information that specifies both the native structure and the pathway to attain that state. This is not to say that nearly identical amino acid sequences always fold similarly. Conformations differ based on environmental factors as well; similar proteins fold differently based on where they are found.
Secondary structure
![BetaPleatedSheetProtein](https://upload.wikimedia.org/wikipedia/commons/e/e4/BetaPleatedSheetProtein.png)
Formation of a
secondary structure
Protein secondary structure is the three dimensional conformational isomerism, form of ''local segments'' of proteins. The two most common Protein structure#Secondary structure, secondary structural elements are alpha helix, alpha helices and beta ...
is the first step in the folding process that a protein takes to assume its native structure. Characteristic of secondary structure are the structures known as
alpha helices
The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...
and
beta sheet
The beta sheet, (β-sheet) (also β-pleated sheet) is a common motif of the regular protein secondary structure. Beta sheets consist of beta strands (β-strands) connected laterally by at least two or three backbone hydrogen bonds, forming a g ...
s that fold rapidly because they are stabilized by
intramolecular hydrogen bond
In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing a ...
s, as was first characterized by
Linus Pauling
Linus Carl Pauling (; February 28, 1901August 19, 1994) was an American chemist, biochemist, chemical engineer, peace activist, author, and educator. He published more than 1,200 papers and books, of which about 850 dealt with scientific top ...
. Formation of intramolecular hydrogen bonds provides another important contribution to protein stability.
α-helices are formed by hydrogen bonding of the
backbone
The backbone is the vertebral column of a vertebrate.
Arts, entertainment, and media Film
* ''Backbone'' (1923 film), a 1923 lost silent film starring Alfred Lunt
* ''Backbone'' (1975 film), a 1975 Yugoslavian drama directed by Vlatko Gilić
M ...
to form a spiral shape (refer to figure on the right).
The β pleated sheet is a structure that forms with the backbone bending over itself to form the hydrogen bonds (as displayed in the figure to the left). The hydrogen bonds are between the amide hydrogen and carbonyl oxygen of the
peptide bond
In organic chemistry, a peptide bond is an amide type of covalent chemical bond linking two consecutive alpha-amino acids from C1 (carbon number one) of one alpha-amino acid and N2 (nitrogen number two) of another, along a peptide or protein cha ...
. There exists anti-parallel β pleated sheets and parallel β pleated sheets where the stability of the hydrogen bonds is stronger in the anti-parallel β sheet as it hydrogen bonds with the ideal 180 degree angle compared to the slanted hydrogen bonds formed by parallel sheets.
Tertiary structure
The α-Helices and β-Sheets are commonly amphipathic, meaning they have a hydrophilic and a hydrophobic portion. This ability helps in forming tertiary structure of a protein in which folding occurs so that the hydrophilic sides are facing the aqueous environment surrounding the protein and the hydrophobic sides are facing the hydrophobic core of the protein.
Secondary structure hierarchically gives way to tertiary structure formation. Once the protein's tertiary structure is formed and stabilized by the hydrophobic interactions, there may also be
covalent bond
A covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atoms ...
ing in the form of
disulfide bridges
In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...
formed between two
cysteine
Cysteine (symbol Cys or C; ) is a semiessential proteinogenic amino acid with the formula . The thiol side chain in cysteine often participates in enzymatic reactions as a nucleophile.
When present as a deprotonated catalytic residue, sometime ...
residues. These non-covalent and covalent contacts take a specific
topological
In mathematics, topology (from the Greek words , and ) is concerned with the properties of a geometric object that are preserved under continuous deformations, such as stretching, twisting, crumpling, and bending; that is, without closing h ...
arrangement in a native structure of a protein. Tertiary structure of a protein involves a single polypeptide chain; however, additional interactions of folded polypeptide chains give rise to quaternary structure formation.
Quaternary structure
Tertiary structure may give way to the formation of
quaternary structure
Protein quaternary structure is the fourth (and highest) classification level of protein structure. Protein quaternary structure refers to the structure of proteins which are themselves composed of two or more smaller protein chains (also refe ...
in some proteins, which usually involves the "assembly" or "coassembly" of subunits that have already folded; in other words, multiple polypeptide chains could interact to form a fully functional quaternary protein.
Driving forces of protein folding
![225 Peptide Bond-01](https://upload.wikimedia.org/wikipedia/commons/2/26/225_Peptide_Bond-01.jpg)
Folding is a
spontaneous process In thermodynamics, a spontaneous process is a process which occurs without any external input to the system. A more technical definition is the time-evolution of a system in which it releases free energy and it moves to a lower, more thermodynamica ...
that is mainly guided by hydrophobic interactions, formation of intramolecular
hydrogen bond
In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing a ...
s,
van der Waals forces
In molecular physics, the van der Waals force is a distance-dependent interaction between atoms or molecules. Unlike ionic bond, ionic or covalent bonds, these attractions do not result from a Chemical bond, chemical electronic bond; they are c ...
, and it is opposed by
conformational entropy
In chemical thermodynamics, conformational entropy is the entropy associated with the number of conformations of a molecule. The concept is most commonly applied to biological macromolecules such as proteins and RNA, but also be used for poly ...
. The process of folding often begins
co-translationally, so that the
N-terminus
The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the ami ...
of the protein begins to fold while the
C-terminal
The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When the protein is ...
portion of the protein is still being
synthesized by the
ribosome
Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to ...
; however, a protein molecule may fold spontaneously during or after
biosynthesis
Biosynthesis is a multi-step, enzyme-catalyzed process where substrates are converted into more complex products in living organisms. In biosynthesis, simple compounds are modified, converted into other compounds, or joined to form macromolecules. ...
. While these
macromolecule
A macromolecule is a very large molecule important to biophysical processes, such as a protein or nucleic acid. It is composed of thousands of covalently bonded atoms. Many macromolecules are polymers of smaller molecules called monomers. The ...
s may be regarded as "
folding themselves", the process also depends on the
solvent
A solvent (s) (from the Latin '' solvō'', "loosen, untie, solve") is a substance that dissolves a solute, resulting in a solution. A solvent is usually a liquid but can also be a solid, a gas, or a supercritical fluid. Water is a solvent for ...
(
water
Water (chemical formula ) is an inorganic, transparent, tasteless, odorless, and nearly colorless chemical substance, which is the main constituent of Earth's hydrosphere and the fluids of all known living organisms (in which it acts as a ...
or
lipid bilayer
The lipid bilayer (or phospholipid bilayer) is a thin polar membrane made of two layers of lipid molecules. These membranes are flat sheets that form a continuous barrier around all cells. The cell membranes of almost all organisms and many vir ...
), the concentration of
salts
In chemistry, a salt is a chemical compound consisting of an ionic assembly of positively charged cations and negatively charged anions, which results in a compound with no net electric charge. A common example is table salt, with positively cha ...
, the
pH, the
temperature
Temperature is a physical quantity that expresses quantitatively the perceptions of hotness and coldness. Temperature is measured with a thermometer.
Thermometers are calibrated in various temperature scales that historically have relied o ...
, the possible presence of cofactors and of molecular
chaperones.
Proteins will have limitations on their folding abilities by the restricted bending angles or conformations that are possible. These allowable angles of protein folding are described with a two-dimensional plot known as the
Ramachandran plot
In biochemistry, a Ramachandran plot (also known as a Rama plot, a Ramachandran diagram or a ,ψplot), originally developed in 1963 by G. N. Ramachandran, C. Ramakrishnan, and V. Sasisekharan, is a way to visualize energetically allowed region ...
, depicted with psi and phi angles of allowable rotation.
Hydrophobic effect
Protein folding must be thermodynamically favorable within a cell in order for it to be a spontaneous reaction. Since it is known that protein folding is a spontaneous reaction, then it must assume a negative
Gibbs free energy
In thermodynamics, the Gibbs free energy (or Gibbs energy; symbol G) is a thermodynamic potential that can be used to calculate the maximum amount of work that may be performed by a thermodynamically closed system at constant temperature and pr ...
value. Gibbs free energy in protein folding is directly related to
enthalpy
Enthalpy , a property of a thermodynamic system, is the sum of the system's internal energy and the product of its pressure and volume. It is a state function used in many measurements in chemical, biological, and physical systems at a constant ...
and
entropy
Entropy is a scientific concept, as well as a measurable physical property, that is most commonly associated with a state of disorder, randomness, or uncertainty. The term and the concept are used in diverse fields, from classical thermodynam ...
.
For a negative delta G to arise and for protein folding to become thermodynamically favorable, then either enthalpy, entropy, or both terms must be favorable.
Minimizing the number of hydrophobic side-chains exposed to water is an important driving force behind the folding process.
The hydrophobic effect is the phenomenon in which the hydrophobic chains of a protein collapse into the core of the protein (away from the hydrophilic environment).
In an aqueous environment, the water molecules tend to aggregate around the hydrophobic regions or side chains of the protein, creating water shells of ordered water molecules. An ordering of water molecules around a hydrophobic region increases order in a system and therefore contributes a negative change in entropy (less entropy in the system). The water molecules are fixed in these water cages which drives the
hydrophobic collapse
Hydrophobic collapse is a proposed process for the production of the Protein structure, 3-D conformation adopted by Peptide, polypeptides and other molecules in polar solvents. The theory states that the nascent polypeptide forms initial Protein se ...
, or the inward folding of the hydrophobic groups. The hydrophobic collapse introduces entropy back to the system via the breaking of the water cages which frees the ordered water molecules.
The multitude of hydrophobic groups interacting within the core of the globular folded protein contributes a significant amount to protein stability after folding, because of the vastly accumulated van der Waals forces (specifically
London Dispersion forces
London dispersion forces (LDF, also known as dispersion forces, London forces, instantaneous dipole–induced dipole forces, fluctuating induced dipole bonds or loosely as van der Waals forces) are a type of intermolecular force acting between at ...
).
The
hydrophobic effect
The hydrophobic effect is the observed tendency of nonpolar substances to aggregate in an aqueous solution and exclude water molecules. The word hydrophobic literally means "water-fearing", and it describes the segregation of water and nonpolar ...
exists as a driving force in thermodynamics only if there is the presence of an aqueous medium with an
amphiphilic
An amphiphile (from the Greek αμφις amphis, both, and φιλíα philia, love, friendship), or amphipath, is a chemical compound possessing both hydrophilic (''water-loving'', polar) and lipophilic (''fat-loving'') properties. Such a compoun ...
molecule containing a large hydrophobic region. The strength of hydrogen bonds depends on their environment; thus, H-bonds enveloped in a hydrophobic core contribute more than H-bonds exposed to the aqueous environment to the stability of the native state.
In proteins with globular folds, hydrophobic amino acids tend to be interspersed along the primary sequence, rather than randomly distributed or clustered together. However, proteins that have recently been born
de novo, which tend to be
intrinsically disordered, show the opposite pattern of hydrophobic amino acid clustering along the primary sequence.
Chaperones
Molecular chaperones
In molecular biology, molecular chaperones are proteins that assist the conformational folding or unfolding of large proteins or macromolecular protein complexes. There are a number of classes of molecular chaperones, all of which function to ass ...
are a class of proteins that aid in the correct folding of other proteins ''
in vivo
Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
''. Chaperones exist in all cellular compartments and interact with the polypeptide chain in order to allow the native three-dimensional conformation of the protein to form; however, chaperones themselves are not included in the final structure of the protein they are assisting in.
Chaperones may assist in folding even when the nascent polypeptide is being synthesized by the ribosome.
Molecular chaperones operate by binding to stabilize an otherwise unstable structure of a protein in its folding pathway, but chaperones do not contain the necessary information to know the correct native structure of the protein they are aiding; rather, chaperones work by preventing incorrect folding conformations.
In this way, chaperones do not actually increase the rate of individual steps involved in the folding pathway toward the native structure; instead, they work by reducing possible unwanted aggregations of the polypeptide chain that might otherwise slow down the search for the proper intermediate and they provide a more efficient pathway for the polypeptide chain to assume the correct conformations.
Chaperones are not to be confused with folding
catalyst
Catalysis () is the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst (). Catalysts are not consumed in the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recyc ...
proteins, which catalyze chemical reactions responsible for slow steps in folding pathways. Examples of folding catalysts are protein
disulfide isomerases and
peptidyl-prolyl isomerase
Prolyl isomerase (also known as peptidylprolyl isomerase or PPIase) is an enzyme () found in both prokaryotes and eukaryotes that interconverts the ''cis'' and ''trans'' isomers of peptide bonds with the amino acid proline. Proline has an unusua ...
s that may be involved in formation of
disulfide bond
In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...
s or interconversion between cis and trans stereoisomers of peptide group.
Chaperones are shown to be critical in the process of protein folding ''in vivo'' because they provide the protein with the aid needed to assume its proper alignments and conformations efficiently enough to become "biologically relevant".
This means that the polypeptide chain could theoretically fold into its native structure without the aid of chaperones, as demonstrated by protein folding experiments conducted ''
in vitro
''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...
'';
however, this process proves to be too inefficient or too slow to exist in biological systems; therefore, chaperones are necessary for protein folding ''in vivo.'' Along with its role in aiding native structure formation, chaperones are shown to be involved in various roles such as protein transport, degradation, and even allow
denatured proteins exposed to certain external denaturant factors an opportunity to refold into their correct native structures.
A fully denatured protein lacks both tertiary and secondary structure, and exists as a so-called
random coil
In polymer chemistry, a random coil is a conformation of polymers where the monomer subunits are oriented randomly while still being bonded to adjacent units. It is not one specific shape, but a statistical distribution of shapes for all the cha ...
. Under certain conditions some proteins can refold; however, in many cases, denaturation is irreversible.
Cells sometimes protect their proteins against the denaturing influence of heat with
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
s known as
heat shock protein
Heat shock proteins (HSP) are a family of proteins produced by cells in response to exposure to stressful conditions. They were first described in relation to heat shock, but are now known to also be expressed during other stresses including expo ...
s (a type of chaperone), which assist other proteins both in folding and in remaining folded.
Heat shock protein
Heat shock proteins (HSP) are a family of proteins produced by cells in response to exposure to stressful conditions. They were first described in relation to heat shock, but are now known to also be expressed during other stresses including expo ...
s have been found in all species examined, from
bacteria
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
to humans, suggesting that they evolved very early and have an important function. Some proteins never fold in cells at all except with the assistance of chaperones which either isolate individual proteins so that their folding is not interrupted by interactions with other proteins or help to unfold misfolded proteins, allowing them to refold into the correct native structure.
This function is crucial to prevent the risk of
precipitation
In meteorology, precipitation is any product of the condensation of atmospheric water vapor that falls under gravitational pull from clouds. The main forms of precipitation include drizzle, rain, sleet, snow, ice pellets, graupel and hail. ...
into
insoluble
In chemistry, solubility is the ability of a substance, the solute, to form a solution with another substance, the solvent. Insolubility is the opposite property, the inability of the solute to form such a solution.
The extent of the solubil ...
amorphous aggregates. The external factors involved in protein denaturation or disruption of the native state include temperature, external fields (electric, magnetic),
molecular crowding,
and even the limitation of space (i.e. confinement), which can have a big influence on the folding of proteins. High concentrations of
solutes
In chemistry, a solution is a special type of homogeneous mixture composed of two or more substances. In such a mixture, a solute is a substance dissolved in another substance, known as a solvent. If the attractive forces between the solvent ...
, extremes of
pH, mechanical forces, and the presence of chemical denaturants can contribute to protein denaturation, as well. These individual factors are categorized together as stresses. Chaperones are shown to exist in increasing concentrations during times of cellular stress and help the proper folding of emerging proteins as well as denatured or misfolded ones.
Under some conditions proteins will not fold into their biochemically functional forms. Temperatures above or below the range that cells tend to live in will cause
thermally unstable proteins to unfold or denature (this is why boiling makes an
egg white
Egg white is the clear liquid (also called the albumen or the glair/glaire) contained within an egg. In chickens it is formed from the layers of secretions of the anterior section of the hen's oviduct during the passage of the egg. It forms arou ...
turn opaque). Protein thermal stability is far from constant, however; for example,
hyperthermophilic bacteria have been found that grow at temperatures as high as 122 °C, which of course requires that their full complement of vital proteins and protein assemblies be stable at that temperature or above.
The bacterium ''
E. coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'' is the host for
bacteriophage T4
Escherichia virus T4 is a species of bacteriophages that infect ''Escherichia coli'' bacteria. It is a double-stranded DNA virus in the subfamily '' Tevenvirinae'' from the family Myoviridae. T4 is capable of undergoing only a lytic lifecycle ...
, and the phage encoded gp31 protein () appears to be structurally and functionally homologous to ''E. coli''
chaperone protein
In molecular biology, molecular chaperones are proteins that assist the conformational folding or unfolding of large proteins or macromolecular protein complexes. There are a number of classes of molecular chaperones, all of which function to assi ...
GroES
Heat shock 10 kDa protein 1 (Hsp10), also known as chaperonin 10 (cpn10) or early-pregnancy factor (EPF), is a protein that in humans is encoded by the ''HSPE1'' gene. The homolog in ''E. coli'' is GroES that is a chaperonin which usually works ...
and able to substitute for it in the assembly of bacteriophage T4
virus
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea.
Since Dmitri Ivanovsky's 1 ...
particles during infection.
[Marusich EI, Kurochkina LP, Mesyanzhinov VV. Chaperones in bacteriophage T4 assembly. Biochemistry (Mosc). 1998;63(4):399-406] Like GroES, gp31 forms a stable complex with
GroEL
GroEL is a protein which belongs to the chaperonin family of molecular chaperones, and is found in many bacteria. It is required for the proper folding of many proteins. To function properly, GroEL requires the lid-like cochaperonin protein comp ...
chaperonin that is absolutely necessary for the folding and assembly in vivo of the bacteriophage T4 major capsid protein gp23.
[
]
Fold switching
Some proteins have multiple native structures, and change their fold based on some external factors. For example, the KaiB protein switches fold throughout the day, acting as a clock for cyanobacteria. It has been estimated that around 0.5–4% of PDB (Protein Data Bank
The Protein Data Bank (PDB) is a database for the three-dimensional structural data of large biological molecules, such as proteins and nucleic acids. The data, typically obtained by X-ray crystallography, NMR spectroscopy, or, increasingly, cry ...
) proteins switch folds.
Protein misfolding and neurodegenerative disease
A protein is considered to be misfolded if it cannot achieve its normal native state. This can be due to mutations in the amino acid sequence or a disruption of the normal folding process by external factors. The misfolded protein typically contains β-sheets
The beta sheet, (β-sheet) (also β-pleated sheet) is a common motif of the regular protein secondary structure. Beta sheets consist of beta strands (β-strands) connected laterally by at least two or three backbone hydrogen bonds, forming a gen ...
that are organized in a supramolecular arrangement known as a cross-β structure. These β-sheet-rich assemblies are very stable, very insoluble, and generally resistant to proteolysis. The structural stability of these fibrillar assemblies is caused by extensive interactions between the protein monomers, formed by backbone hydrogen bonds between their β-strands. The misfolding of proteins can trigger the further misfolding and accumulation of other proteins into aggregates or oligomers. The increased levels of aggregated proteins in the cell leads to formation of amyloid
Amyloids are aggregates of proteins characterised by a Fibril, fibrillar morphology of 7–13 Nanometer, nm in diameter, a beta sheet (β-sheet) Secondary structure of proteins, secondary structure (known as cross-β) and ability to be Staining, ...
-like structures which can cause degenerative disorders and cell death. The amyloids are fibrillary structures that contain intermolecular hydrogen bonds which are highly insoluble and made from converted protein aggregates. Therefore, the proteasome pathway may not be efficient enough to degrade the misfolded proteins prior to aggregation. Misfolded proteins can interact with one another and form structured aggregates and gain toxicity through intermolecular interactions.
Aggregated proteins are associated with prion
Prions are misfolded proteins that have the ability to transmit their misfolded shape onto normal variants of the same protein. They characterize several fatal and transmissible neurodegenerative diseases in humans and many other animals. It ...
-related illnesses such as Creutzfeldt–Jakob disease
Creutzfeldt–Jakob disease (CJD), also known as subacute spongiform encephalopathy or neurocognitive disorder due to prion disease, is an invariably fatal degenerative brain disorder. Early symptoms include memory problems, behavioral changes, ...
, bovine spongiform encephalopathy
Bovine spongiform encephalopathy (BSE), commonly known as mad cow disease, is an incurable and invariably fatal neurodegenerative disease of cattle. Symptoms include abnormal behavior, trouble walking, and weight loss. Later in the course of t ...
(mad cow disease), amyloid-related illnesses such as Alzheimer's disease
Alzheimer's disease (AD) is a neurodegeneration, neurodegenerative disease that usually starts slowly and progressively worsens. It is the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in short-term me ...
and familial amyloid cardiomyopathy
Familial amyloid cardiomyopathy (FAC), or transthyretin amyloid cardiomyopathy (ATTR-CM) results from the aggregation and deposition of mutant and wild-type transthyretin (TTR) protein in the heart. TTR is usually circulated as a homo-tetramer—a ...
or polyneuropathy
Polyneuropathy ( poly- + neuro- + -pathy) is damage or disease affecting peripheral nerves (peripheral neuropathy) in roughly the same areas on both sides of the body, featuring weakness, numbness, and burning pain. It usually begins in the hand ...
, as well as intracellular aggregation diseases such as Huntington's and Parkinson's disease
Parkinson's disease (PD), or simply Parkinson's, is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. The symptoms usually emerge slowly, and as the disease worsens, non-motor symptoms becom ...
. These age onset degenerative diseases are associated with the aggregation of misfolded proteins into insoluble, extracellular aggregates and/or intracellular inclusions including cross-β amyloid
Amyloids are aggregates of proteins characterised by a Fibril, fibrillar morphology of 7–13 Nanometer, nm in diameter, a beta sheet (β-sheet) Secondary structure of proteins, secondary structure (known as cross-β) and ability to be Staining, ...
fibril
Fibrils (from the Latin ''fibra'') are structural biological materials found in nearly all living organisms. Not to be confused with fibers or filaments, fibrils tend to have diameters ranging from 10-100 nanometers (whereas fibers are micro ...
s. It is not completely clear whether the aggregates are the cause or merely a reflection of the loss of protein homeostasis, the balance between synthesis, folding, aggregation and protein turnover. Recently the European Medicines Agency
The European Medicines Agency (EMA) is an agency of the European Union (EU) in charge of the evaluation and supervision of medicinal products. Prior to 2004, it was known as the European Agency for the Evaluation of Medicinal Products or Euro ...
approved the use of Tafamidis
Tafamidis, sold under the brand names Vyndaqel and Vyndamax, is a medication used to delay disease progression in adults with certain forms of transthyretin amyloidosis. It can be used to treat both hereditary forms, familial amyloid cardiomyopa ...
or Vyndaqel (a kinetic stabilizer of tetrameric transthyretin) for the treatment of transthyretin amyloid diseases. This suggests that the process of amyloid fibril formation (and not the fibrils themselves) causes the degeneration of post-mitotic tissue in human amyloid diseases. Misfolding and excessive degradation instead of folding and function leads to a number of proteopathy
In medicine, proteinopathy (; 'pref''. protein -pathy 'suff''. disease proteinopathies ''pl''.; proteinopathic ''adj''), or proteopathy, protein conformational disorder, or protein misfolding disease refers to a class of diseases in which certa ...
diseases such as antitrypsin
Alpha-1 antitrypsin or α1-antitrypsin (A1AT, α1AT, A1A, or AAT) is a protein belonging to the serpin superfamily. It is encoded in humans by the ''SERPINA1'' gene. A protease inhibitor (biology), protease inhibitor, it is also known as alpha1 ...
-associated emphysema
Emphysema, or pulmonary emphysema, is a lower respiratory tract disease, characterised by air-filled spaces ( pneumatoses) in the lungs, that can vary in size and may be very large. The spaces are caused by the breakdown of the walls of the alve ...
, cystic fibrosis
Cystic fibrosis (CF) is a rare genetic disorder that affects mostly the lungs, but also the pancreas, liver, kidneys, and intestine. Long-term issues include difficulty breathing and coughing up mucus as a result of frequent lung infections. O ...
and the lysosomal storage diseases
Lysosomal storage diseases (LSDs; ) are a group of over 70 rare inherited metabolic disorders that result from defects in lysosomal function. Lysosomes are sacs of enzymes within cells that digest large molecules and pass the fragments on to other ...
, where loss of function is the origin of the disorder. While protein replacement therapy has historically been used to correct the latter disorders, an emerging approach is to use pharmaceutical chaperones
A pharmacological chaperone or pharmacoperone is a drug that acts as a protein chaperone. That is, it contains small molecules that enter cells and serve as a molecular scaffolding in order to cause otherwise-misfolded mutant proteins to fold and ...
to fold mutated proteins to render them functional.
Experimental techniques for studying protein folding
While inferences about protein folding can be made through mutation studies, typically, experimental techniques for studying protein folding rely on the gradual unfolding or folding of proteins and observing conformational changes using standard non-crystallographic techniques.
X-ray crystallography
X-ray crystallography
X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...
is one of the more efficient and important methods for attempting to decipher the three dimensional configuration of a folded protein. To be able to conduct X-ray crystallography, the protein under investigation must be located inside a crystal lattice. To place a protein inside a crystal lattice, one must have a suitable solvent for crystallization, obtain a pure protein at supersaturated levels in solution, and precipitate the crystals in solution. Once a protein is crystallized, X-ray beams can be concentrated through the crystal lattice which would diffract the beams or shoot them outwards in various directions. These exiting beams are correlated to the specific three-dimensional configuration of the protein enclosed within. The X-rays specifically interact with the electron clouds surrounding the individual atoms within the protein crystal lattice and produce a discernible diffraction pattern. Only by relating the electron density clouds with the amplitude of the X-rays can this pattern be read and lead to assumptions of the phases or phase angles involved that complicate this method. Without the relation established through a mathematical basis known as Fourier transform
A Fourier transform (FT) is a mathematical transform that decomposes functions into frequency components, which are represented by the output of the transform as a function of frequency. Most commonly functions of time or space are transformed, ...
, the "phase problem
In physics, the phase problem is the problem of loss of information concerning the phase that can occur when making a physical measurement. The name comes from the field of X-ray crystallography, where the phase problem has to be solved for the det ...
" would render predicting the diffraction patterns very difficult. Emerging methods like multiple isomorphous replacement Multiple isomorphous replacement (MIR) is historically the most common approach to solving the phase problem in X-ray crystallography studies of proteins. For protein crystals this method is conducted by soaking the crystal of a sample to be analyz ...
use the presence of a heavy metal ion to diffract the X-rays into a more predictable manner, reducing the number of variables involved and resolving the phase problem.
Fluorescence spectroscopy
Fluorescence spectroscopy
Fluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electron ...
is a highly sensitive method for studying the folding state of proteins. Three amino acids, phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp), have intrinsic fluorescence properties, but only Tyr and Trp are used experimentally because their quantum yield The quantum yield (Φ) of a radiation-induced process is the number of times a specific event occurs per photon absorbed by the system.
Applications
Fluorescence spectroscopy
The fluorescence quantum yield is defined as the ratio of the numb ...
s are high enough to give good fluorescence signals. Both Trp and Tyr are excited by a wavelength of 280 nm, whereas only Trp is excited by a wavelength of 295 nm. Because of their aromatic character, Trp and Tyr residues are often found fully or partially buried in the hydrophobic core of proteins, at the interface between two protein domains, or at the interface between subunits of oligomeric proteins. In this apolar environment, they have high quantum yields and therefore high fluorescence intensities. Upon disruption of the protein's tertiary or quaternary structure, these side chains become more exposed to the hydrophilic environment of the solvent, and their quantum yields decrease, leading to low fluorescence intensities. For Trp residues, the wavelength of their maximal fluorescence emission also depend on their environment.
Fluorescence spectroscopy can be used to characterize the equilibrium unfolding
In biochemistry, equilibrium unfolding is the process of unfolding a protein or RNA molecule by gradually changing its environment, such as by changing the temperature or pressure, pH, adding chemical denaturants, or applying force as with an ...
of proteins by measuring the variation in the intensity of fluorescence emission or in the wavelength of maximal emission as functions of a denaturant value. The denaturant can be a chemical molecule (urea, guanidinium hydrochloride), temperature, pH, pressure, etc. The equilibrium between the different but discrete protein states, i.e. native state, intermediate states, unfolded state, depends on the denaturant value; therefore, the global fluorescence signal of their equilibrium mixture also depends on this value. One thus obtains a profile relating the global protein signal to the denaturant value. The profile of equilibrium unfolding may enable one to detect and identify intermediates of unfolding. General equations have been developed by Hugues Bedouelle to obtain the thermodynamic parameters that characterize the unfolding equilibria for homomeric or heteromeric proteins, up to trimers and potentially tetramers, from such profiles. Fluorescence spectroscopy can be combined with fast-mixing devices such as stopped flow Stopped-flow is an experimental technique for studying chemical reactions with a half time of the order of 1 ms, introduced by Britton Chance and extended by Quentin Gibson (Other techniques, such as the temperature-jump method, are available for m ...
, to measure protein folding kinetics, generate a chevron plot
A chevron plot is a way of representing protein folding kinetic data in the presence of varying concentrations of denaturant that disrupts the protein's native tertiary structure. The plot is known as "chevron" plot because of the canonical ''v' ...
and derive a Phi value analysis Phi value analysis,
\phi
analysis, or \phi-value analysis is an experimental protein engineering technique for studying the structure of the folding transition state of small protein domains that fold in a two-state manner. The structure of the f ...
.
Circular dichroism
Circular dichroism is one of the most general and basic tools to study protein folding. Circular dichroism spectroscopy measures the absorption of circularly polarized light. In proteins, structures such as alpha helices
The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...
and beta sheets
The beta sheet, (β-sheet) (also β-pleated sheet) is a common motif of the regular protein secondary structure. Beta sheets consist of beta strands (β-strands) connected laterally by at least two or three backbone hydrogen bonds, forming a gen ...
are chiral, and thus absorb such light. The absorption of this light acts as a marker of the degree of foldedness of the protein ensemble. This technique has been used to measure equilibrium unfolding
In biochemistry, equilibrium unfolding is the process of unfolding a protein or RNA molecule by gradually changing its environment, such as by changing the temperature or pressure, pH, adding chemical denaturants, or applying force as with an ...
of the protein by measuring the change in this absorption as a function of denaturant concentration or temperature
Temperature is a physical quantity that expresses quantitatively the perceptions of hotness and coldness. Temperature is measured with a thermometer.
Thermometers are calibrated in various temperature scales that historically have relied o ...
. A denaturant melt measures the free energy of unfolding as well as the protein's m value, or denaturant dependence. A temperature melt measures the denaturation temperature (Tm) of the protein. As for fluorescence spectroscopy, circular-dichroism spectroscopy can be combined with fast-mixing devices such as stopped flow Stopped-flow is an experimental technique for studying chemical reactions with a half time of the order of 1 ms, introduced by Britton Chance and extended by Quentin Gibson (Other techniques, such as the temperature-jump method, are available for m ...
to measure protein folding kinetics
Kinetics ( grc, κίνησις, , kinesis, ''movement'' or ''to move'') may refer to:
Science and medicine
* Kinetics (physics), the study of motion and its causes
** Rigid body kinetics, the study of the motion of rigid bodies
* Chemical ki ...
and to generate chevron plot
A chevron plot is a way of representing protein folding kinetic data in the presence of varying concentrations of denaturant that disrupts the protein's native tertiary structure. The plot is known as "chevron" plot because of the canonical ''v' ...
s.
Vibrational circular dichroism of proteins
The more recent developments of vibrational circular dichroism Vibrational circular dichroism (VCD) is a spectroscopic technique which detects differences in attenuation of left and right circularly polarized light passing through a sample. It is the extension of circular dichroism spectroscopy into the infra ...
(VCD) techniques for proteins, currently involving Fourier transform
A Fourier transform (FT) is a mathematical transform that decomposes functions into frequency components, which are represented by the output of the transform as a function of frequency. Most commonly functions of time or space are transformed, ...
(FT) instruments, provide powerful means for determining protein conformations in solution even for very large protein molecules. Such VCD studies of proteins can be combined with X-ray diffraction
X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...
data for protein crystals, FT-IR
Fourier-transform infrared spectroscopy (FTIR) is a technique used to obtain an infrared spectrum of absorption or emission of a solid, liquid, or gas. An FTIR spectrometer simultaneously collects high-resolution spectral data over a wide spectra ...
data for protein solutions in heavy water (D2O), or quantum computations.
Protein nuclear magnetic resonance spectroscopy
Protein nuclear magnetic resonance
Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with a ...
(NMR) is able to collect protein structural data by inducing a magnet field through samples of concentrated protein. In NMR, depending on the chemical environment, certain nuclei will absorb specific radio-frequencies. Because protein structural changes operate on a time scale from ns to ms, NMR is especially equipped to study intermediate structures in timescales of ps to s. Some of the main techniques for studying proteins structure and non-folding protein structural changes include COSY, TOCSY, HSQC The heteronuclear single quantum coherence or heteronuclear single quantum correlation experiment, normally abbreviated as HSQC, is used frequently in NMR spectroscopy of organic molecules and is of particular significance in the field of protein NM ...
, time relaxation (T1 & T2), and NOE. NOE is especially useful because magnetization transfers can be observed between spatially proximal hydrogens are observed. Different NMR experiments have varying degrees of timescale sensitivity that are appropriate for different protein structural changes. NOE can pick up bond vibrations or side chain rotations, however, NOE is too sensitive to pick up protein folding because it occurs at larger timescale.
Because protein folding takes place in about 50 to 3000 s−1 CPMG Relaxation dispersion and chemical exchange saturation transfer have become some of the primary techniques for NMR analysis of folding. In addition, both techniques are used to uncover excited intermediate states in the protein folding landscape. To do this, CPMG Relaxation dispersion takes advantage of the spin echo In magnetic resonance, a spin echo or Hahn echo is the refocusing of spin magnetisation by a pulse of resonant electromagnetic radiation. Modern nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) make use of this effect.
The NMR ...
phenomenon. This technique exposes the target nuclei to a 90 pulse followed by one or more 180 pulses. As the nuclei refocus, a broad distribution indicates the target nuclei is involved in an intermediate excited state. By looking at Relaxation dispersion plots the data collect information on the thermodynamics and kinetics between the excited and ground. Saturation Transfer measures changes in signal from the ground state as excited states become perturbed. It uses weak radio frequency irradiation to saturate the excited state of a particular nuclei which transfers its saturation to the ground state. This signal is amplified by decreasing the magnetization (and the signal) of the ground state.
The main limitations in NMR is that its resolution decreases with proteins that are larger than 25 kDa and is not as detailed as X-ray crystallography
X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...
. Additionally, protein NMR analysis is quite difficult and can propose multiple solutions from the same NMR spectrum.
In a study focused on the folding of an amyotrophic lateral sclerosis
Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND) or Lou Gehrig's disease, is a neurodegenerative disease that results in the progressive loss of motor neurons that control voluntary muscles. ALS is the most comm ...
involved protein SOD1
Superoxide dismutase u-Zn'' also known as superoxide dismutase 1 or hSod1 is an enzyme that in humans is encoded by the ''SOD1'' gene, located on chromosome 21. SOD1 is one of three human superoxide dismutases. It is implicated in apoptosis, fam ...
, excited intermediates were studied with relaxation dispersion and Saturation transfer. SOD1 had been previously tied to many disease causing mutants which were assumed to be involved in protein aggregation, however the mechanism was still unknown. By using Relaxation Dispersion and Saturation Transfer experiments many excited intermediate states were uncovered misfolding in the SOD1 mutants.
Dual-polarization interferometry
Dual polarisation interferometry
Dual-polarization interferometry (DPI) is an analytical technique that probes molecular layers adsorbed to the surface of a waveguide using the evanescent wave of a laser beam. It is used to measure the conformational change in proteins, or othe ...
is a surface-based technique for measuring the optical properties of molecular layers. When used to characterize protein folding, it measures the conformation by determining the overall size of a monolayer of the protein and its density in real time at sub-Angstrom resolution, although real-time measurement of the kinetics of protein folding are limited to processes that occur slower than ~10 Hz. Similar to circular dichroism, the stimulus for folding can be a denaturant or temperature
Temperature is a physical quantity that expresses quantitatively the perceptions of hotness and coldness. Temperature is measured with a thermometer.
Thermometers are calibrated in various temperature scales that historically have relied o ...
.
Studies of folding with high time resolution
The study of protein folding has been greatly advanced in recent years by the development of fast, time-resolved techniques. Experimenters rapidly trigger the folding of a sample of unfolded protein and observe the resulting dynamics. Fast techniques in use include neutron scattering
Neutron scattering, the irregular dispersal of free neutrons by matter, can refer to either the naturally occurring physical process itself or to the man-made experimental techniques that use the natural process for investigating materials. Th ...
, ultrafast mixing of solutions, photochemical methods, and laser temperature jump spectroscopy. Among the many scientists who have contributed to the development of these techniques are Jeremy Cook, Heinrich Roder, Harry Gray, Martin Gruebele, Brian Dyer, William Eaton, Sheena Radford, Chris Dobson
Sir Christopher Martin Dobson (8 October 1949 – 8 September 2019) was a British chemist, who was the John Humphrey Plummer Professor of Chemical and Structural Biology in the Department of Chemistry at the University of Cambridge, and Maste ...
, Alan Fersht
Sir Alan Roy Fersht (born 21 April 1943) is a British chemist at the MRC Laboratory of Molecular Biology, Cambridge, and an Emeritus Professor in the Department of Chemistry at the University of Cambridge. He was Master of Gonville and Caius C ...
, Bengt Nölting
Bengt Nölting (1 May 1962 – 16 September 2009) was a German physicist and biophysicist who pioneered various methods in biophysics and engineering. Achievements include studying biological macromolecules, the development of self-evolving comp ...
and Lars Konermann.
Proteolysis
Proteolysis
Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically catalysed by cellular enzymes called protease ...
is routinely used to probe the fraction unfolded under a wide range of solution conditions (e.g. fast parallel proteolysis (FASTpp).
Single-molecule force spectroscopy
Single molecule techniques such as optical tweezers and AFM have been used to understand protein folding mechanisms of isolated proteins as well as proteins with chaperones. Optical tweezers
Optical tweezers (originally called single-beam gradient force trap) are scientific instruments that use a highly focused laser beam to hold and move microscopic and sub-microscopic objects like atoms, nanoparticles and droplets, in a manner simila ...
have been used to stretch single protein molecules from their C- and N-termini and unfold them to allow study of the subsequent refolding. The technique allows one to measure folding rates at single-molecule level; for example, optical tweezers have been recently applied to study folding and unfolding of proteins involved in blood coagulation. von Willebrand factor
Von Willebrand factor (VWF) () is a blood glycoprotein involved in hemostasis, specifically, platelet adhesion. It is deficient and/or defective in von Willebrand disease and is involved in many other diseases, including thrombotic thrombocytopen ...
(vWF) is a protein with an essential role in blood clot formation process. It discovered – using single molecule optical tweezers measurement – that calcium-bound vWF acts as a shear force sensor in the blood. Shear force leads to unfolding of the A2 domain of vWF, whose refolding rate is dramatically enhanced in the presence of calcium. Recently, it was also shown that the simple src SH3 domain accesses multiple unfolding pathways under force.
Biotin painting
Biotin painting enables condition-specific cellular snapshots of (un)folded proteins. Biotin 'painting' shows a bias towards predicted Intrinsically disordered proteins
In molecular biology, an intrinsically disordered protein (IDP) is a protein that lacks a fixed or ordered three-dimensional structure, typically in the absence of its macromolecular interaction partners, such as other proteins or RNA. IDPs rang ...
.
Computational studies of protein folding
Computational studies of protein folding includes three main aspects related to the prediction of protein stability, kinetics, and structure. A 2013 review summarizes the available computational methods for protein folding.
Levinthal's paradox
In 1969, Cyrus Levinthal noted that, because of the very large number of degrees of freedom in an unfolded polypeptide chain, the molecule has an astronomical number of possible conformations. An estimate of 3300 or 10143 was made in one of his papers. Levinthal's paradox
Levinthal's paradox is a thought experiment, also constituting a self-reference in the theory of protein folding. In 1969, Cyrus Levinthal noted that, because of the very large number of degrees of freedom in an unfolded polypeptide chain, the m ...
is a thought experiment based on the observation that if a protein were folded by sequential sampling of all possible conformations, it would take an astronomical amount of time to do so, even if the conformations were sampled at a rapid rate (on the nanosecond
A nanosecond (ns) is a unit of time in the International System of Units (SI) equal to one billionth of a second, that is, of a second, or 10 seconds.
The term combines the SI prefix ''nano-'' indicating a 1 billionth submultiple of an SI unit ( ...
or picosecond
A picosecond (abbreviated as ps) is a unit of time in the International System of Units (SI) equal to 10−12 or (one trillionth) of a second. That is one trillionth, or one millionth of one millionth of a second, or 0.000 000 000  ...
scale). Based upon the observation that proteins fold much faster than this, Levinthal then proposed that a random conformational search does not occur, and the protein must, therefore, fold through a series of meta-stable intermediate states.
Energy landscape of protein folding
The configuration space of a protein during folding can be visualized as an energy landscape
An energy landscape is a mapping of possible states of a system. The concept is frequently used in physics, chemistry, and biochemistry, e.g. to describe all possible conformations of a molecular entity, or the spatial positions of interacting m ...
. According to Joseph Bryngelson and Peter Wolynes
Peter Guy Wolynes is an American theoretical chemist and physicist. Since 2011 he has been a Bullard-Welch Foundation Professor of Science and Professor of Chemistry at Rice University. He is widely recognized for his significant contributions to ...
, proteins follow the ''principle of minimal frustration'', meaning that naturally evolved proteins have optimized their folding energy landscapes, and that nature has chosen amino acid sequences so that the folded state of the protein is sufficiently stable. In addition, the acquisition of the folded state had to become a sufficiently fast process. Even though nature has reduced the level of ''frustration'' in proteins, some degree of it remains up to now as can be observed in the presence of local minima in the energy landscape of proteins.
A consequence of these evolutionarily selected sequences is that proteins are generally thought to have globally "funneled energy landscapes" (a term coined by José Onuchic José Nelson Onuchic is a Brazilian and American physicist, the Harry C & Olga K Wiess Professor of Physics at Rice University.[folding funnel
The folding funnel hypothesis is a specific version of the energy landscape theory of protein folding, which assumes that a protein's native state corresponds to its free energy minimum under the solution conditions usually encountered in cells. A ...](_blank)
" landscape allows the protein to fold to the native state through any of a large number of pathways and intermediates, rather than being restricted to a single mechanism. The theory is supported by both computational simulations of model proteins and experimental studies, and it has been used to improve methods for protein structure prediction
Protein structure prediction is the inference of the three-dimensional structure of a protein from its amino acid sequence—that is, the prediction of its secondary and tertiary structure from primary structure. Structure prediction is different ...
and design
A design is a plan or specification for the construction of an object or system or for the implementation of an activity or process or the result of that plan or specification in the form of a prototype, product, or process. The verb ''to design'' ...
. The description of protein folding by the leveling free-energy landscape is also consistent with the 2nd law of thermodynamics. Physically, thinking of landscapes in terms of visualizable potential or total energy surfaces simply with maxima, saddle points, minima, and funnels, rather like geographic landscapes, is perhaps a little misleading. The relevant description is really a high-dimensional phase space in which manifolds might take a variety of more complicated topological forms.
The unfolded polypeptide chain begins at the top of the funnel where it may assume the largest number of unfolded variations and is in its highest energy state. Energy landscapes such as these indicate that there are a large number of initial possibilities, but only a single native state is possible; however, it does not reveal the numerous folding pathways that are possible. A different molecule of the same exact protein may be able to follow marginally different folding pathways, seeking different lower energy intermediates, as long as the same native structure is reached. Different pathways may have different frequencies of utilization depending on the thermodynamic favorability of each pathway. This means that if one pathway is found to be more thermodynamically favorable than another, it is likely to be used more frequently in the pursuit of the native structure. As the protein begins to fold and assume its various conformations, it always seeks a more thermodynamically favorable structure than before and thus continues through the energy funnel. Formation of secondary structures is a strong indication of increased stability within the protein, and only one combination of secondary structures assumed by the polypeptide backbone will have the lowest energy and therefore be present in the native state of the protein. Among the first structures to form once the polypeptide begins to fold are alpha helices and beta turns, where alpha helices can form in as little as 100 nanoseconds and beta turns in 1 microsecond.
There exists a saddle point in the energy funnel landscape where the transition state for a particular protein is found. The transition state in the energy funnel diagram is the conformation that must be assumed by every molecule of that protein if the protein wishes to finally assume the native structure. No protein may assume the native structure without first passing through the transition state. The transition state can be referred to as a variant or premature form of the native state rather than just another intermediary step. The folding of the transition state is shown to be rate-determining, and even though it exists in a higher energy state than the native fold, it greatly resembles the native structure. Within the transition state, there exists a nucleus around which the protein is able to fold, formed by a process referred to as "nucleation condensation" where the structure begins to collapse onto the nucleus.
Modeling of protein folding
'' De novo'' or ''ab initio
''Ab initio'' ( ) is a Latin term meaning "from the beginning" and is derived from the Latin ''ab'' ("from") + ''initio'', ablative singular of ''initium'' ("beginning").
Etymology
Circa 1600, from Latin, literally "from the beginning", from ab ...
'' techniques for computational protein structure prediction
Protein structure prediction is the inference of the three-dimensional structure of a protein from its amino acid sequence—that is, the prediction of its secondary and tertiary structure from primary structure. Structure prediction is different ...
can be used for simulating various aspects of protein folding. Molecular Dynamics
Molecular dynamics (MD) is a computer simulation method for analyzing the physical movements of atoms and molecules. The atoms and molecules are allowed to interact for a fixed period of time, giving a view of the dynamic "evolution" of the ...
(MD) was used in simulations of protein folding and dynamics in silico
In biology and other experimental sciences, an ''in silico'' experiment is one performed on computer or via computer simulation. The phrase is pseudo-Latin for 'in silicon' (correct la, in silicio), referring to silicon in computer chips. It ...
. First equilibrium folding simulations were done using implicit solvent model and umbrella sampling
Umbrella sampling is a technique in computational physics and chemistry, used to improve sampling of a system (or different systems) where ergodicity is hindered by the form of the system's energy landscape. It was first suggested by Torrie and ...
. Because of computational cost, ab initio MD folding simulations with explicit water are limited to peptides and very small proteins. MD simulations of larger proteins remain restricted to dynamics of the experimental structure or its high-temperature unfolding. Long-time folding processes (beyond about 1 millisecond), like folding of small-size proteins (about 50 residues) or larger, can be accessed using coarse-grained models.
Several large-scale computational projects, such as Rosetta@home
Rosetta@home is a volunteer computing project researching protein structure prediction on the Berkeley Open Infrastructure for Network Computing (BOINC) platform, run by the Baker laboratory at the University of Washington. Rosetta@home aims ...
, Folding@home
Folding@home (FAH or F@h) is a volunteer computing project aimed to help scientists develop new therapeutics for a variety of diseases by the means of simulating protein dynamics. This includes the process of protein folding and the movements ...
and Foldit
Foldit is an online puzzle video game about protein folding. It is part of an experimental research project developed by the University of Washington, Center for Game Science, in collaboration with the UW Department of Biochemistry. The objective ...
,FoldIt - Folding Protein Game
/ref> target protein folding.
Long continuous-trajectory simulations have been performed on Anton
Anton may refer to: People
*Anton (given name), including a list of people with the given name
*Anton (surname)
Places
*Anton Municipality, Bulgaria
**Anton, Sofia Province, a village
*Antón District, Panama
**Antón, a town and capital of th ...
, a massively parallel supercomputer designed and built around custom ASIC
An application-specific integrated circuit (ASIC ) is an integrated circuit (IC) chip customized for a particular use, rather than intended for general-purpose use, such as a chip designed to run in a digital voice recorder or a high-efficien ...
s and interconnects by D. E. Shaw Research. The longest published result of a simulation performed using Anton is a 2.936 millisecond simulation of NTL9 at 355 K.
See also
* Chevron plot
A chevron plot is a way of representing protein folding kinetic data in the presence of varying concentrations of denaturant that disrupts the protein's native tertiary structure. The plot is known as "chevron" plot because of the canonical ''v' ...
* Denaturation midpoint
Denaturation midpoint of a protein is defined as the temperature (Tm) or concentration of Denaturation (biochemistry), denaturant (Cm) at which both the Protein tertiary structure, folded and Denaturation (biochemistry), unfolded states are equall ...
* Downhill folding
Downhill folding is a process in which a protein folds without encountering any significant macroscopic free energy barrier. It is a key prediction of the folding funnel hypothesis of the energy landscape theory of proteins.
Overview
Downhill ...
* Folding (chemistry)
In chemistry, folding is the process by which a molecule assumes its shape or conformation. The process can also be described as intramolecular self-assembly, a type of molecular self-assembly, where the molecule is directed to form a specifi ...
* Phi value analysis Phi value analysis,
\phi
analysis, or \phi-value analysis is an experimental protein engineering technique for studying the structure of the folding transition state of small protein domains that fold in a two-state manner. The structure of the f ...
* Potential energy of protein
* Protein dynamics Proteins are generally thought to adopt unique structures determined by their amino acid sequences. However, proteins are not strictly static objects, but rather populate ensembles of (sometimes similar) conformations. Transitions between these stat ...
* Protein misfolding cyclic amplification Protein misfolding cyclic amplification (PMCA) is an amplification technique (conceptually like PCR but not involving nucleotides) to multiply misfolded prions originally developed by Soto and colleagues.Saborio, G.P., Permanne, B. and Soto, C. (20 ...
* Protein structure prediction software
This list of protein structure prediction software summarizes notable used software tools in protein structure prediction, including homology modeling, protein threading, ''ab initio'' methods, secondary structure prediction, and transmembrane ...
* Proteopathy
In medicine, proteinopathy (; 'pref''. protein -pathy 'suff''. disease proteinopathies ''pl''.; proteinopathic ''adj''), or proteopathy, protein conformational disorder, or protein misfolding disease refers to a class of diseases in which certa ...
* Time-resolved mass spectrometry
References
External links
Human Proteome Folding Project
{{Authority control
Protein structure