Structure Of Folding Intermediates Research Articles

Pulsed-alkylation mass spectrometry for the study of protein folding and dynamics: development and application to the study of a folding/unfolding intermediate of bacterial luciferase.

A new method employing the classical techniques of chemical modification of proteins and the new technology of mass spectrometry, known as pulsed-alkylation mass spectrometry (PA/MS), has been developed to probe the dynamic structure of folding intermediates and folded complexes of proteins under a variety of conditions. This method is fast and simple, and the results are easily interpreted. PA/MS may provide an alternative to H/D exchange monitored either by NMR or by electrospray ionization mass spectrometry for some experiments; for others, it may provide access to questions not readily answered by available methods. The objective of PA/MS is to determine simultaneously the location and the extent of labeling of functional groups in a protein by measuring the reactivity of cysteines with N-ethylmaleimide, within the context of the conformation of the protein under specific conditions. The method can also be applied to chemical modification of other amino acid residues employing any of a vast array of reagents, depending upon the specifics of the protein under investigation. The enormous range of reactivity of the thiol groups of the cysteinyl residues in proteins and the change in reactivity upon denaturation or conformational rearrangement afford a large signal change that can be correlated with changes in accessibility of the thiol group. The information obtained from the correlation of observed thiol reactivity with the local environment of each cysteinyl residue in the structure of the folded protein can be supplemented by results obtained from fluorescence, circular dichroism, or other methods, to develop an understanding of the structure and dynamics of altered conformational states. With bacterial luciferase as a model system, we have applied PA/MS to investigate the structural differences between the native heterodimeric enzyme and a folding intermediate that is well-populated in 2 M urea. The thiol residues at positions 307, 324, and 325 of the alpha subunit were much more reactive with N-ethylmaleimide in the presence of 2 M urea than in the native enzyme, suggesting that the C-terminal region of the alpha subunit was less tightly packed in the folding intermediate. The apparent unfolding of the C-terminal region of the alpha subunit of the alphabeta structure in 2 M urea appears to mimic the unfolding of the C-terminal domain of the free alpha subunit, also in 2 M urea, described by Noland, B. W., Dangott, L. J., and Baldwin, T. O. (1999) Biochemistry 38, 16136-16145. The approach described here should be applicable to a wide array of problems that have in common the need to determine the locations of conformational changes in proteins. Application of PA/MS to the investigation of the relative thermodynamic stability of the coordination complexes of zinc within each of the six zinc-finger domains of MRE-binding transcription factor-1 (Zn(6) MTF-zf) in its free and DNA-bound forms is presented in the companion paper in this issue [Apuy, J. L., Chen, X., Russell, D. H., Baldwin, T. O., and Giedroc, D. P. (2001) Biochemistry 40, 15164-15175].

Time‐Resolved Hydroxyl Radical Footprinting of RNA with X‐Rays

AbstractRNA tertiary structure and protein interactions can be detected by protection from hydroxyl radical cleavage. Generation of hydroxyl radicals with a synchrotron X‐ray beam provides analysis on a short timescale (50 msec to 100 sec), which enables the structures of folding intermediates or other transient conformational states to be determined. This unit provides detailed instructions on the use of the synchrotron beamline for hydroxyl radical footprinting.

Importance of two buried salt bridges in the stability and folding pathway of barnase.

The importance of two buried salt bridges in barnase in the stability of its folded state, the major transition rate for unfolding, and a folding intermediate has been analyzed by protein engineering, kinetic, and thermodynamic studies. The aspartate residues in the bridges Arg69-Asp93 and Arg83-Asp75 were replaced by the isosteric analogue asparagine, while various replacements were probed for the positively charged arginine partners. The mutations are very destabilizing, lowering stability by up to 5.4 kcal/mol. A value of 3.0-3.5 kcal/mol was derived for the coupling energy between Arg and Asp from a double mutant cycle analysis. Despite the radical nature of these mutations, they do not appear to alter the pathway of folding. The interaction between Arg69 and Asp93, located in a relatively conserved region among ribonucleases, is predominantly formed in the major transition state along the folding pathway, as found previously from an analysis of more benign mutations; the value of phi(F) for all mutations at positions 69 and 93 are 0.8-0.9 in the major transition state for folding where phi(F) = 0 = fully unfolded and phi(F) = 1 = fully folded interaction energies). In contrast, the interaction between Arg83 and Asp75 in the active site of barnase is formed only in the native state of the protein. The analysis of folding pathways and the structure of folding intermediates by making kinetic and thermodynamic measurements on mutants appears even more robust than expected.

Structures of folding intermediates

The past year has yielded important results in the study of protein-folding intermediates. It has been shown that the equilibrium molten globule has a native-like tertiary fold and is separated from the unfolded state by a first-order phase transition. New equilibrium intermediates have been revealed and substantial progress has been made in the understanding of two main barriers in protein folding.

Autonomous subdomains in protein folding

Proteolytic dissection of native trp repressor and horse heart cytochrome c has been used to infer some of the steps in the folding pathways of the intact proteins. For both proteins, small fragments are capable of undergoing spontaneous noncovalent association to form subdomains with native-like secondary and/or tertiary structural features, suggesting that dissection/reassembly may be a general method to gain insight into the structures of folding intermediates. The importance of this approach is its simplicity and potential applicability to studying the folding pathways of a wide range of proteins. The proteases report on the structure and dynamics of the native state, circumventing the need for prior knowledge of the structures of folding intermediates. The observation that small fragments of proteins can associated noncovalently suggests that protein folding can be viewed as an intramolecular "recognition" process. The results imply that substantial information about protein structure and folding is encoded at the level of subdomains, and that chain connectivity has only a minor role in determining the fold.

Probing the structure of folding intermediates

The description of protein folding mechanisms in terms of the structures of well defined, partially folded intermediates is a major objective in structural biology. Despite the fleeting existence of intermediates and the fact that their structures are unlikely to be uniquely defined, significant advances in our understanding of protein folding pathways have been made in the last year based on a variety of physical methods.

Activation-energy landscape for metastable RNA folding.

We consider the relaxation kinetics for folding intermediate structures in random uncorrelated RNA chains. Folding patterns correspond to intrachain secondary structures. We make use of a Monte Carlo simulation that mimics a Markov process to study the refolding dynamics. This is done to obtain the time-dependent behavior of the activation energies. We conclude that the mechanism of relaxation to the equilibrium secondary structure is compatible with the random-energy model as the thermodynamic limit is approached.

A peptide model of a protein folding intermediate.

It is difficult to determine the structures of protein folding intermediates because folding is a highly cooperative process. A disulphide-bonded peptide pair, designed to mimic the first crucial intermediate in the folding of bovine pancreatic trypsin inhibitor, contains secondary and tertiary structure similar to that found in the native protein. Peptide models like this circumvent the problem of cooperativity and permit characterization of structures of folding intermediates.