The phenomenon of macromolecular crowding alters the properties of molecules in a

solution Solution may refer to: * Solution (chemistry), a mixture where one substance is dissolved in another * Solution (equation), in mathematics ** Numerical solution, in numerical analysis, approximate solutions within specified error bounds * Solutio ...

when high concentrations of

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 such as

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, res ...

s are present. Such conditions occur routinely in living

cells Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery w ...

; for instance, the

cytosol The cytosol, also known as cytoplasmic matrix or groundplasm, is one of the liquids found inside cells ( intracellular fluid (ICF)). It is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondri ...

of ''

Escherichia 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 '' Esc ...

'' contains about 300– of macromolecules. Crowding occurs since these high concentrations of macromolecules reduce the volume of

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 ...

available for other molecules in the solution, which has the result of increasing their effective concentrations. Crowding can promote formation of a

biomolecular condensate In biochemistry, biomolecular condensates are a class of lipid bilayer, membrane-less organelles and organelle subdomains, which carry out specialized functions within the cell (biology), cell. Unlike many organelles, biomolecular condensate com ...

colloid A colloid is a mixture in which one substance consisting of microscopically dispersed insoluble particles is suspended throughout another substance. Some definitions specify that the particles must be dispersed in a liquid, while others extend ...

al phase separation. This crowding effect can make molecules in cells behave in radically different ways than in test-tube assays. Consequently, measurements of the properties of

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 ...

s or processes in

metabolism Metabolism (, from el, μεταβολή ''metabolē'', "change") is the set of life-sustaining chemical reactions in organisms. The three main functions of metabolism are: the conversion of the energy in food to energy available to run ...

that are made in the laboratory (''

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 a ...

'') in dilute solutions may be different by many orders of magnitude from the true values seen in living cells (''

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 p ...

''). The study of biochemical processes under realistically crowded conditions is very important, since these conditions are a ubiquitous property of all cells and crowding may be essential for the efficient operation of metabolism. Indeed, ''

'' studies have shown that crowding greatly influences binding stability of proteins to DNA.

Cause and effects

The interior of cells is a crowded environment. For example, an ''

'' cell is only about 2

micrometre The micrometre (American and British English spelling differences#-re, -er, international spelling as used by the International Bureau of Weights and Measures; SI symbol: μm) or micrometer (American and British English spelling differences# ...

s (μm) long and 0.5 μm in diameter, with a cell volume of 0.6 - 0.7 μm³. However, ''E. coli'' can contain up to 4,288 different types of proteins, and about 1,000 of these types are produced at a high enough level to be easily detected. Added to this mix are various forms of RNA and the cell's DNA chromosome, giving a total concentration of macromolecules of between 300 and 400 mg/ml. In

eukaryote Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacter ...

s the cell's interior is further crowded by the

protein filament In biology, a protein filament is a long chain of protein monomers, such as those found in hair, muscle, or in flagella. Protein filaments form together to make the cytoskeleton of the cell. They are often bundled together to provide support, st ...

s that make up the

cytoskeleton The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in the cytoplasm of all cells, including those of bacteria and archaea. In eukaryotes, it extends from the cell nucleus to the cell membrane and is co ...

, this meshwork divides the cytosol into a network of narrow pores. These high concentrations of macromolecules occupy a large proportion of the volume of the cell, which reduces the volume of solvent that is available for other macromolecules. This

excluded volume The concept of excluded volume was introduced by Werner Kuhn in 1934 and applied to polymer molecules shortly thereafter by Paul Flory. Excluded volume gives rise to depletion forces. In liquid state theory In liquid state theory, the 'excluded ...

effect increases the effective concentration of macromolecules (increasing their

chemical activity In chemical thermodynamics, activity (symbol ) is a measure of the "effective concentration" of a species in a mixture, in the sense that the species' chemical potential depends on the activity of a real solution in the same way that it would depen ...

), which in turn alters the

rates Rate or rates may refer to: Finance * Rates (tax), a type of taxation system in the United Kingdom used to fund local government * Exchange rate, rate at which one currency will be exchanged for another Mathematics and science * Rate (mathema ...

and

equilibrium constant The equilibrium constant of a chemical reaction is the value of its reaction quotient at chemical equilibrium, a state approached by a dynamic chemical system after sufficient time has elapsed at which its composition has no measurable tendency ...

s of their reactions. In particular this effect alters

dissociation constant In chemistry, biochemistry, and pharmacology, a dissociation constant (K_D) is a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, as when a complex ...

s by favoring the association of macromolecules, such as when multiple proteins come together to form

protein complex A protein complex or multiprotein complex is a group of two or more associated polypeptide chains. Protein complexes are distinct from multienzyme complexes, in which multiple catalytic domains are found in a single polypeptide chain. Protein ...

es, or when

DNA-binding protein DNA-binding proteins are proteins that have DNA-binding domains and thus have a specific or general affinity for single- or double-stranded DNA. Sequence-specific DNA-binding proteins generally interact with the major groove of B-DNA, becaus ...

s bind to their targets in the

genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ...

. Crowding may also affect enzyme reactions involving small molecules if the reaction involves a large change in the shape of the enzyme. The size of the crowding effect depends on both the

molecular mass The molecular mass (''m'') is the mass of a given molecule: it is measured in daltons (Da or u). Different molecules of the same compound may have different molecular masses because they contain different isotopes of an element. The related quant ...

and shape of the molecule involved, although mass seems to be the major factor – with the effect being stronger with larger molecules. Notably, the size of the effect is non-linear, so macromolecules are much more strongly affected than are small molecules such as

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 ...

s or simple sugars. Macromolecular crowding is therefore an effect exerted by large molecules on the properties of other large molecules.

Importance

Macromolecular crowding is an important effect in

biochemistry Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...

and

cell biology Cell biology (also cellular biology or cytology) is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living an ...

. For example, the increase in the strength of interactions between proteins and DNA produced by crowding may be of key importance in processes such as transcription and

DNA replication In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritan ...

. Crowding has also been suggested to be involved in processes as diverse as the aggregation of

hemoglobin Hemoglobin (haemoglobin BrE) (from the Greek word αἷμα, ''haîma'' 'blood' + Latin ''globus'' 'ball, sphere' + ''-in'') (), abbreviated Hb or Hgb, is the iron-containing oxygen-transport metalloprotein present in red blood cells (erythroc ...

sickle-cell disease Sickle cell disease (SCD) is a group of blood disorders typically inherited from a person's parents. The most common type is known as sickle cell anaemia. It results in an abnormality in the oxygen-carrying protein haemoglobin found in red ...

, and the responses of cells to changes in their volume. The importance of crowding in

protein folding Protein folding is the physical process by which a protein chain is translated to its native three-dimensional structure, typically a "folded" conformation by which the protein becomes biologically functional. Via an expeditious and reproduc ...

is of particular interest in

biophysics Biophysics is an interdisciplinary science that applies approaches and methods traditionally used in physics to study biological phenomena. Biophysics covers all scales of biological organization, from molecular to organismic and populations. ...

. Here, the crowding effect can accelerate the folding process, since a compact folded protein will occupy less volume than an unfolded protein chain. However, crowding can reduce the yield of correctly folded protein by increasing protein aggregation. Crowding may also increase the effectiveness of

chaperone proteins 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 ...

such as

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 com ...

in the cell, which could counteract this reduction in folding efficiency. It has also been shown that macromolecular crowding affects protein-folding dynamics as well as overall protein shape where distinct conformational changes are accompanied by secondary structure alterations implying that crowding-induced shape changes may be important for protein function and malfunction in vivo. A particularly striking example of the importance of crowding effects involves the

crystallin In anatomy, a crystallin is a water-soluble structural protein found in the lens and the cornea of the eye accounting for the transparency of the structure. It has also been identified in other places such as the heart, and in aggressive breast c ...

s that fill the interior of the

lens A lens is a transmissive optical device which focuses or disperses a light beam by means of refraction. A simple lens consists of a single piece of transparent material, while a compound lens consists of several simple lenses (''elements ...

. These proteins have to remain stable and in solution for the lens to be transparent;

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. ...

or aggregation of crystallins causes

cataract A cataract is a cloudy area in the lens of the eye that leads to a decrease in vision. Cataracts often develop slowly and can affect one or both eyes. Symptoms may include faded colors, blurry or double vision, halos around light, trouble ...

s. Crystallins are present in the lens at extremely high concentrations, over 500 mg/ml, and at these levels crowding effects are very strong. The large crowding effect adds to the thermal stability of the crystallins, increasing their resistance to denaturation. This effect may partly explain the extraordinary resistance shown by the lens to damage caused by high temperatures. Crowding may also play a role in diseases that involve protein aggregation, such as

sickle cell anemia Sickle cell disease (SCD) is a group of blood disorders typically inherited from a person's parents. The most common type is known as sickle cell anaemia. It results in an abnormality in the oxygen-carrying protein haemoglobin found in red bl ...

where mutant

forms aggregates and

alzheimer's disease Alzheimer's disease (AD) is a 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 remembering recent events. As ...

, where

tau protein The tau proteins (abbreviated from tubulin associated unit) are a group of six highly soluble protein isoforms produced by alternative splicing from the gene ''MAPT'' (microtubule-associated protein tau). They have roles primarily in maintaining ...

forms

neurofibrillary tangle Neurofibrillary tangles (NFTs) are aggregates of hyperphosphorylated tau protein that are most commonly known as a primary biomarker of Alzheimer's disease. Their presence is also found in numerous other diseases known as tauopathies. Little is ...

s under crowded conditions within neurons.

Study

Due to macromolecular crowding,

enzyme assay Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition. Enzyme units The quantity or concentration of an enzyme can be expressed in molar amounts, as with a ...

s and biophysical measurements performed in dilute solution may fail to reflect the actual process and its kinetics taking place in the cytosol. One approach to produce more accurate measurements would be to use highly concentrated extracts of cells, to try to maintain the cell contents in a more natural state. However, such extracts contain many kinds of biologically active molecules, which can interfere with the phenomena being studied. Consequently, crowding effects are mimicked ''in vitro'' by adding high concentrations of relatively inert molecules such as

polyethylene glycol Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular we ...

, ficoll,

dextran Dextran is a complex branched glucan ( polysaccharide derived from the condensation of glucose), originally derived from wine. IUPAC defines dextrans as "Branched poly-α-d-glucosides of microbial origin having glycosidic bonds predominantly C- ...

, or serum albumin to experimental media. However, using such artificial crowding agents can be complicated, as these crowding molecules can sometimes interact in other ways with the process being examined, such as by binding weakly to one of the components.

Macromolecular crowding and protein folding

A major importance of macromolecular crowding to biological systems stems from its effect on

. The underlying physical mechanism by which macromolecular crowding helps to stabilize proteins in their folded state is often explained in terms of excluded volume - the volume inaccessible to the proteins due to their interaction with macromolecular crowders.{{cite journal, last=Minton, first=A., title=Excluded Volume as a Determinant of Macromolecular Structure and Reactivity, journal=Biopolymers, year=1981, volume=20, issue=10, pages=2093–2120, doi=10.1002/bip.1981.360201006, s2cid=97753189{{cite book, last=Parsegian , first=VA., title=Protein-water interactions., journal=Int. Rev. Cytol., year=2002, volume=215, pages=1–31, doi=10.1016/S0074-7696(02)15003-0, pmid=11952225, series=International Review of Cytology, isbn=9780123646194 This notion goes back to Asakura and Oosawa, who have described depletion forces induced by steric, hard-core, interactions.Asakura">{{cite journal, last=Asakura, first=Sho, author2=Oosawa, F, title=On Interaction between Two Bodies Immersed in a Solution of Macromolecules, journal=The Journal of Chemical Physics, date=1 January 1954, volume=22, issue=7, pages=1255, doi=10.1063/1.1740347, bibcode = 1954JChPh..22.1255A Asakura">{{cite journal, last=Asakura, first=Sho, author2=Oosawa, F., title=Interaction between Particles Suspended in Solutions of Macromolecules, journal=Journal of Polymer Science, year=1958, volume=33, issue=126, pages=183–192, doi=10.1002/pol.1958.1203312618, bibcode = 1958JPoSc..33..183A A hallmark of the mechanism inferred from the above is that the effect is completely a-thermal, and thus completely entropic. These ideas were also proposed to explain why small cosolutes, namely protective

osmolytes Osmolytes are low-molecular weight organic compounds that influence the properties of biological fluids. Their primary role is to maintain the integrity of cells by affecting the viscosity, melting point, and ionic strength of the aqueous solution. ...

, which are preferentially excluded from proteins, also shift the protein folding equilibrium towards the folded state. However, it has been shown by various methods, both experimental{{cite journal, last=Politi, first=R, author2=Harries, D., title=Enthalpically Driven Peptide Stabilization by Protective Osmolytes, journal=Chem. Commun., year=2010, volume=46, issue=35, pages=6449–6451, doi=10.1039/c0cc01763a, pmid=20657920{{cite journal, last=Benton, first=L.A., author2=Smith, A.E., author3=Young, G.B., author4=Pielak, G.J., title= Unexpected Effects of Macromolecular Crowding on Protein Stability., journal=Biochemistry, year=2012, volume=51, issue=49, pages=9773–9775, doi=10.1021/bi300909q, pmid=23167542{{cite journal, last=Sukenik, first=S, author2=Sapir, L. , author3=Harries, D. , title=Balance of enthalpy and entropy in depletion forces., journal=Curr. Opin. Colloid Interface Sci., year=2013, volume=18, issue=6, pages=495–501, doi=10.1016/j.cocis.2013.10.002, arxiv=1310.2100, s2cid=18847346 and theoretical,{{cite journal, last=Sapir, first=L, author2=Harries, D., title=Origin of Enthalpic Depletion Forces., journal=J. Phys. Chem. Lett., year=2014, volume=5, issue=7, pages=1061–1065, doi=10.1021/jz5002715, pmid=26274449{{cite journal, last=Sapir, first=L, author2=Harries, D., title=Is the depletion force entropic? Molecular crowding beyond steric interactions., journal=Curr. Opin. Colloid Interface Sci., year=2015, volume=20, pages=3–10, doi=10.1016/j.cocis.2014.12.003{{cite journal, last=Sapir, first=L, author2=Harries, D., title=Macromolecular Stabilization by Excluded Cosolutes: Mean Field Theory of Crowded Solutions., journal=J. Chem. Theory Comput., year=2015, volume=11, issue=7, pages=3478–3490, doi=10.1021/acs.jctc.5b00258, pmid=26575781 that depletion forces are not always entropic in nature.

Macromolecular crowding in regenerative medicine

Satyam et al. from National University of Ireland, Galway (NUI Galway) proposed macromolecular crowding as means to create ECM-rich tissue equivalents. The principle of macromolecular crowding is derived from the notion that ''in vivo'' cells reside in a highly crowded/dense extracellular space and therefore the conversion of the ''de novo'' synthesised procollagen to collagen I is rapid. However, in the even substantially more dilute than body fluids (e.g., urine: 36–50 g/L; blood: 80 g/L) culture conditions (e.g., HAM F10 nutrient medium: 16.55 g/L; DMEM/ F12 medium: 16.78 g/L; DMEM high glucose and L-glutamine medium: 17.22 g/L), the rate limiting conversion of procollagen to collagen I is very slow. It was confirmed that the addition of inert polydispersed macromolecules (presented as spherical objects of variable diameter) in the culture media will facilitate amplified production of ECM-rich living substitutes. Macromolecular crowding, by imitating native tissue localised density, can be utilised to effectively modulate ''in vitro'' microenvironments and ultimately produce ECM-rich cell substitutes, within hours rather than days or months in culture, without compromising fundamental cellular functions.{{cite patent, title=Engineered living tissue substitute, pubdate=Dec 12, 2012, inventor-last=Zeugolis, inventor2-last=Satyam, inventor-first=Dimitrios, inventor2-first=Abhigyan, country=EP, number=2532736

References

{{Reflist, 2

External links

*{{cite journal , author=Rivas G, Ferrone, F, Hertzfeld J. , title=Life in a crowded world: Workshop on the Biological Implications of Macromolecular Crowding , journal=EMBO Reports , volume=5 , issue=1 , pages=23–7 , date=December 2003 , doi=10.1038/sj.embor.7400056 , pmid=14710181 , pmc=1298967 *{{cite journal , author=Satyam A, display-authors=etal, title=Macromolecular Crowding Meets Tissue Engineering by Self-Assembly: A Paradigm Shift in Regenerative Medicine , journal=Advanced Materials , volume=26 , issue=19 , pages=3024–3034 , date=May 2014 , doi=10.1002/adma.201304428 , pmid=24505025, hdl=10379/15414 , hdl-access=free {{DEFAULTSORT:Macromolecular Crowding Physical chemistry Tissue engineering Protein methods Biophysics