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Photocleavage of the polypeptide backbone by 2-nitrophenylalanine.

ABSTRACT: Photocleavage of the polypeptide backbone is potentially a powerful and general method to activate or deactivate functional peptides and proteins with high spatial and temporal resolution. Here we show that 2-nitrophenylalanine is able to photochemically cleave the polypeptide backbone by an unusual cinnoline-forming reaction. This unnatural amino acid was genetically encoded in E. coli, and protein containing 2-nitrophenylalanine was expressed and site-specifically photocleaved.

SUBMITTER: Peters FB

PROVIDER: S-EPMC2714363 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Photocleavage of the polypeptide backbone by 2-nitrophenylalanine.

Peters Francis B FB Brock Ansgar A Wang Jiangyun J Schultz Peter G PG

Chemistry & biology 20090201 2

Photocleavage of the polypeptide backbone is potentially a powerful and general method to activate or deactivate functional peptides and proteins with high spatial and temporal resolution. Here we show that 2-nitrophenylalanine is able to photochemically cleave the polypeptide backbone by an unusual cinnoline-forming reaction. This unnatural amino acid was genetically encoded in E. coli, and protein containing 2-nitrophenylalanine was expressed and site-specifically photocleaved. ...[more]

PMID: 19246005

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Project description:Model-free-based NMR dynamics studies have been undertaken for polypeptide backbone amide N-H bond vectors for both the deoxy and carbonmonoxy forms of chain-specific, isotopically (15N and 2H) labeled tetrameric hemoglobin (Hb) using 15N-relaxation parameters [longitudinal relaxation rate (R1), transverse relaxation rate (R2), and heteronuclear nuclear Overhauser effect (NOE)] measured at two temperatures (29 and 34 degrees C) and two magnetic field strengths (11.7 and 14.1 T). In both deoxy and carbonmonoxy forms of human normal adult hemoglobin (Hb A), the amide N-H bonds of most amino acid residues are rigid on the fast time scale (nanosecond to picosecond), except for the loop regions and certain helix-helix connections. Although rigid in deoxy-Hb A, beta146His has been found to be free from restriction of its backbone motions in the CO form, presumably due to the rupture of its hydrogen bond/salt bridge network. We now have direct dynamics evidence for this structural transition of Hb in solution. While remarkably flexible in the deoxy state, alpha31Arg and beta123Thr, neighbors in the intradimer (alpha1beta1) interface, exhibit stiffening upon CO binding. These findings imply a role for alpha31Arg and beta123Thr in the intradimer communication but contradict the results from X-ray crystallography. We have also found that there is considerable flexibility in the intradimer (alpha1beta1) interface (i.e., B, G, and H helices and the GH corner) and possible involvement of several amino acid residues (e.g., alpha31Arg, beta3Leu, beta41Phe, beta123Thr, and beta146His) in the allosteric pathway. Several amino acid residues at the intradimer interfaces, such as beta109Val, appear to be involved in possible conformational exchange processes. The dynamic picture derived from the present study provides new insights into the traditional description of the stereochemical mechanism for the cooperative oxygenation of Hb A based on X-ray crystallographic results.

Project description:In aqueous solutions with high concentrations of chemical denaturants such as urea and guanidinium chloride (GdmCl) proteins expand to populate heterogeneous conformational ensembles. These denaturing environments are thought to be good solvents for generic protein sequences because properties of conformational distributions align with those of canonical random coils. Previous studies showed that water is a poor solvent for polypeptide backbones, and therefore, backbones form collapsed globular structures in aqueous solvents. Here, we ask if polypeptide backbones can intrinsically undergo the requisite chain expansion in aqueous solutions with high concentrations of urea and GdmCl. We answer this question using a combination of molecular dynamics simulations and fluorescence correlation spectroscopy. We find that the degree of backbone expansion is minimal in aqueous solutions with high concentrations of denaturants. Instead, polypeptide backbones sample conformations that are denaturant-specific mixtures of coils and globules, with a persistent preference for globules. Therefore, typical denaturing environments cannot be classified as good solvents for polypeptide backbones. How then do generic protein sequences expand in denaturing environments? To answer this question, we investigated the effects of side chains using simulations of two archetypal sequences with amino acid compositions that are mixtures of charged, hydrophobic, and polar groups. We find that side chains lower the effective concentration of backbone amides in water leading to an intrinsic expansion of polypeptide backbones in the absence of denaturants. Additional dilution of the effective concentration of backbone amides is achieved through preferential interactions with denaturants. These effects lead to conformational statistics in denaturing environments that are congruent with those of canonical random coils. Our results highlight the role of side chain-mediated interactions as determinants of the conformational properties of unfolded states in water and in influencing chain expansion upon denaturation.

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Photocleavage of the polypeptide backbone by 2-nitrophenylalanine.

Publications

Photocleavage of the polypeptide backbone by 2-nitrophenylalanine.

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