Maltose-binding protein

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Maltose/maltodextrin-binding periplasmic protein
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Maltose-binding protein (MBP) is a part of the maltose/maltodextrin system of Escherichia coli, which is responsible for the uptake and efficient catabolism of maltodextrins. It is a complex regulatory and transport system involving many proteins and protein complexes. MBP has an approximate molecular mass of 42.5 kilodaltons.

Structure and folding

MBP is encoded by the malE gene of Escherichia coli. The malE gene codes for a precursor polypeptide (396 amino acid 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.[2]

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.[3][4]

Both precursor and mature forms of MBP are functional for the binding of maltose.[5] The NH2-terminal extension decreases the folding 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.[6][7] 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.[8]

Localization and export

MBP is exported into the

periplasmic space of E. coli.[9] 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. Once folded, the precursor can no longer enter the translocation pathway.[10] 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.[11] 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.[12][13][14]

Control of expression

The malE gene, coding for MBP, belongs to the

transcription start sites at the malEp and malKp promoters are distant of 271 base pairs.[16]

The malEp and malKp promoters are synergistically activated by protein MalT, the activator of the Mal regulon and by the

cyclic AMP to the dimer of CRP.[19] The unliganded form of MalT is monomeric whereas its liganded form, in the presence of ATP and maltotriose, is oligomeric.[20]

Use as a protein and peptide vector

MBP is used to increase the solubility of

affinity tag for purification of recombinant proteins. The fusion protein binds to amylose columns 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 and can then be separated from MBP by affinity chromatography
.

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

plasmid vectors were developed to facilitate the expression and purification of such fusion proteins.[26]

When the recombinant MBP includes a

Klenow polymerase and the dimeric Gene V protein of phage M13.[24][29] When the recombinant MBP includes either a defective or no signal peptide the fusion protein remains within the bacterial cytoplasm 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.[24][30] 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 using molecular replacement on a known MBP structure.[31]

See also

References

  1. PMID 11237621
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  2. PMID 6088507
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  3. PMID 2002054
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  4. PMID 1420181
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  5. PMID 385604
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  6. PMID 3278378
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  7. PMID 15035631
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  8. PMID 8407916
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  9. PMID 4215651
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  10. S2CID 28503725
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  11. S2CID 4253747
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  12. PMID 7312870
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  13. PMID 16046390
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  14. PMID 18022369
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  15. PMID 9529892
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  16. ^
    PMID 6185687
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  17. PMID 6417341
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  18. PMID 11013224
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  20. PMID 10559195
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  23. PMID 11115689
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  24. ^
    PMID 3278900
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  25. PMID 9220983
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  26. PMID 2843437
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  27. PMID 8170930
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  28. PMID 28330113
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  29. PMID 2226455
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  30. PMID 10452611
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  31. PMID 26682969
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External links
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