Maltose-binding protein (MBP) is a part of the maltose/

maltodextrin Maltodextrin is a polysaccharide that is used as a food ingredient. It is produced from vegetable starch by partial hydrolysis and is usually found as a white hygroscopic spray-dried powder. Maltodextrin is easily digestible, being absorbed as ...

system 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 ...

'', which is responsible for the uptake and efficient catabolism of maltodextrins. It is a complex regulatory and transport system involving many

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 and protein complexes. MBP has an approximate molecular mass of 42.5 kilodaltons.

Structure and folding

MBP is encoded by the ''malE''

gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...

of ''Escherichia coli''. The ''malE'' gene codes for a precursor polypeptide (396

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 a ...

residues) which yields the mature MBP (370 residues) upon cleavage of the NH₂-terminal extension (26 residues). The precursor and mature forms of MBP do not contain any cysteine residues. MBP is a monomeric protein. Crystal structures have shown that MBP is divided into two distinct globular domains that are connected by three short polypeptide segments. The two domains are separated by a deep groove that contains the maltose/maltodextrin binding site. Comparison of the structures of the liganded and unliganded forms of MBP has shown that the binding of maltose induces a major conformational change that closes the groove by a rigid motion of the two domains around the linking polypeptide hinge. Both precursor and mature forms of MBP are functional for the binding of maltose. The NH₂-terminal extension decreases the

folding Fold, folding or foldable may refer to: Arts, entertainment, and media * ''Fold'' (album), the debut release by Australian rock band Epicure * Fold (poker), in the game of poker, to discard one's hand and forfeit interest in the current pot *Abov ...

rate of the precursor form of MBP relative to its mature form by at least 5 fold, but it has no effect on the unfolding rate. The equilibrium unfolding of MBP can be modelled by a two-state mechanism with a stability ∆G(H₂O) equal to 9.45 kcal mol⁻¹ at 25 °C, pH 7.6.

Localization and export

MBP is exported into the

periplasmic space The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the ''periplasmic space'' in gram-negative bacteria. Using cryo-electron microscopy it has been found tha ...

of ''E. coli''. The NH₂-terminal extension of MBP, also termed signal peptide, has two roles: (i) it slows down folding of the newly synthesized polypeptide, and (ii) it directs this polypeptide to the membrane and SecYEG

translocon The translocon (also known as a translocator or translocation channel) is a complex of proteins associated with the translocation of polypeptides across membranes. In eukaryotes the term translocon most commonly refers to the complex that transport ...

. Once folded, the precursor can no longer enter the translocation pathway. The introduction of a charged amino-acid residue or a proline residue within the hydrophobic core of the signal peptide is sufficient to block export. The defective exports of the mutant MBPs are consistent with the alpha-helical conformation and hydrophobic interactions of the signal peptide in its interaction with the translocon motor protein

SecA The SecA protein is a cell membrane associated subunit of the eubacterial Sec or Type II secretory pathway, a system which is responsible for the secretion of proteins through the cell membrane. Within this system the SecA ATPase forms a translo ...

Control of expression

The ''malE'' gene, coding for MBP, belongs to the Mal regulon of ''E. coli'', which consists of ten genes whose products are geared for the efficient uptake and utilization of maltose and maltodextrins. All the gene involved in the transport of maltose/maltodextrin, including ''malE'', are clustered in the ''malB'' region of ''E. coli'' and organized in two divergent

operons In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splic ...

: ''malE-malF-malG'' and ''malK-lamB''. The

transcription Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, the fir ...

start sites at the ''malEp'' and ''malKp'' promoters are distant of 271 base pairs. The ''malEp'' and ''malKp'' promoters are synergistically activated by protein MalT, the activator of the Mal regulon and by the

cAMP receptor protein cAMP receptor protein (CRP; also known as catabolite activator protein, CAP) is a regulatory protein in bacteria. CRP protein binds cAMP, which causes a conformational change that allows CRP to bind tightly to a specific DNA site in the promot ...

CRP. This activation is a coupled process that involves, going from ''malEp'' towards ''malKp'': two MalT binding sites; three CRP binding site, and two overlapping sets of three MalT binding sites, staggered by three base pairs. Transcription activation requires the binding of

adenosine triphosphate Adenosine triphosphate (ATP) is an organic compound that provides energy to drive many processes in living cells, such as muscle contraction, nerve impulse propagation, condensate dissolution, and chemical synthesis. Found in all known forms o ...

(ATP) and

maltotriose Maltotriose is a trisaccharide (three-part sugar) consisting of three glucose molecules linked with α-1,4 glycosidic bonds. It is most commonly produced by the digestive enzyme alpha-amylase (a common enzyme in human saliva) on amylose in starc ...

to MalT and the binding of

cyclic AMP Cyclic adenosine monophosphate (cAMP, cyclic AMP, or 3',5'-cyclic adenosine monophosphate) is a second messenger important in many biological processes. cAMP is a derivative of adenosine triphosphate (ATP) and used for intracellular signal tra ...

to the dimer of CRP. The unliganded form of MalT is monomeric whereas its liganded form, in the presence of ATP and maltotriose, is oligomeric.

Use as a protein and peptide vector

MBP is used to increase the solubility of

recombinant protein Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating DNA sequence, sequences that would not othe ...

s expressed in ''E. coli''. In these systems, the protein of interest is often expressed as a MBP- fusion protein, preventing aggregation of the protein of interest. The mechanism by which MBP increases solubility is not well understood. In addition, MBP can itself be used as an

affinity tag Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or a ...

for purification of recombinant proteins. The fusion protein binds to

amylose Amylose is a polysaccharide made of α-D-glucose units, bonded to each other through α(1→4) glycosidic bonds. It is one of the two components of starch, making up approximately 20–30%. Because of its tightly packed helical structure, amylose ...

columns A column or pillar in architecture and structural engineering is a structural element that transmits, through compression, the weight of the structure above to other structural elements below. In other words, a column is a compression membe ...

while all other proteins flow through. The MBP-protein fusion can be purified by eluting the column with maltose. Once the fusion protein is obtained in purified form, the protein of interest is often cleaved from MBP with a specific

protease A protease (also called a peptidase, proteinase, or proteolytic enzyme) is an enzyme that catalyzes (increases reaction rate or "speeds up") proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the ...

and can then be separated from MBP by

affinity chromatography Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the ...

. A first study of the relations between structure and functions of MBP was performed by random insertion of a short DNA fragment, coding for a

BamHI ''Bam''HI (pronounced "Bam H one") (from ''Bacillus amyloliquefaciens'') is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 bp) of DNA and specifically cleaving them at a target site. This exhibit focus ...

restriction site Restriction sites, or restriction recognition sites, are located on a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction enzymes. These are generally palindromic sequences (bec ...

, into the ''malE'' gene. Some of the insertions affected the functions of MBP whereas others were permissive. The permissive sites that were internal to MBP, were used to insert antigenic peptides and challenge the immune response in mice. The 3'-OH terminal insertions were used to create fusion proteins and develop the use of MBP as an affinity handle for the purification of foreign proteins and peptides by affinity chromatography on cross-linked amylose and elution with maltose in mild physico-chemical conditions. Several

plasmid vector A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...

s were developed to facilitate the expression and purification of such fusion proteins. When the recombinant MBP includes a signal peptide, the fusion protein can be exported into the

, which facilitate its purification since the periplasmic fluid contains only a limited number of proteins and can be recovered either by an

osmotic shock Osmotic shock or osmotic stress is physiologic dysfunction caused by a sudden change in the solute concentration around a cell, which causes a rapid change in the movement of water across its cell membrane. Under hypertonic conditions - conditions ...

or by permeabilization of the

bacterial outer membrane The bacterial outer membrane is found in gram-negative bacteria. Its composition is distinct from that of the inner cytoplasmic cell membrane - among other things, the outer leaflet of the outer membrane of many gram-negative bacteria includes ...

with

antibiotics An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention o ...

such as

Polymyxin B Polymyxin B, sold under the brand name Poly-Rx among others, is an antibiotic used to treat meningitis, pneumonia, sepsis, and urinary tract infections. While it is useful for many Gram negative infections, it is not useful for Gram positive inf ...

. Such an export of the fusion protein into the periplasmic space enables the 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 in the passenger protein, for example

antibody 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 ...

fragments. Foreign proteins that are exported or secreted in their native organism, can usually be exported into the ''E. coli'' periplasm by fusion with MBP. Examples of cytoplasmic proteins that could be exported by fusion with MBP, include the monomeric Klenow polymerase and the dimeric Gene V protein of phage M13. When the recombinant MBP includes either a defective or no signal peptide the fusion protein remains within the bacterial

cytoplasm In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. ...

from where it can be recovered by breaking open the cells. The fusion of proteins with MBP usually enhances their solubility and facilitates their proper folding so that the fusion proteins are most often bifunctional. In addition, such fusions can facilitate the crystallisation of difficult proteins, e.g. membrane proteins. The crystallized protein can often have their structures solved by

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 ...

using

molecular replacement Molecular replacement (or MR) is a method of solving the phase problem in X-ray crystallography. MR relies upon the existence of a previously solved protein structure which is similar to our unknown structure from which the diffraction data is de ...

on a known MBP structure.

References

External links