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Protein folding and function: the N-terminal fragment in adenylate kinase.

ABSTRACT: Three-dimensional protein folds range from simple to highly complex architectures. In complex folds, some building block fragments are more important for correct protein folding than others. Such fragments are typically buried in the protein core and mediate interactions between other fragments. Here we present an automated, surface area-based algorithm that is able to indicate which, among all local elements of the structure, is critical for the formation of the native fold, and apply it to structurally well-characterized proteins. In particular, we focus on adenylate kinase. The fragment containing the phosphate binding, P-loop (the "giant anion hole") flanked by a beta-strand and an alpha-helix near the N-terminus, is identified as a critical building block. This building block shows a high degree of sequence and structural conservation in all adenylate kinases. The results of our molecular dynamics simulations are consistent with this identification. In its absence, the protein flips to a stable, non-native state. In this misfolded conformation, the other local elements of the structure are in their native-like conformations; however, their association is non-native. Furthermore, this element is critically important for the function of the enzyme, coupling folding, and function.

SUBMITTER: Kumar S

PROVIDER: S-EPMC1301432 | biostudies-other | 2001 May

REPOSITORIES: biostudies-other

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Protein folding and function: the N-terminal fragment in adenylate kinase.

Kumar S S Sham Y Y YY Tsai C J CJ Nussinov R R

Biophysical journal 20010501 5

Three-dimensional protein folds range from simple to highly complex architectures. In complex folds, some building block fragments are more important for correct protein folding than others. Such fragments are typically buried in the protein core and mediate interactions between other fragments. Here we present an automated, surface area-based algorithm that is able to indicate which, among all local elements of the structure, is critical for the formation of the native fold, and apply it to str ...[more]

PMID: 11325743

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Project description:Although molecular simulation methods have yielded valuable insights into mechanistic aspects of protein refolding in vitro, they have up to now not been used to model the folding of proteins as they are actually synthesized by the ribosome. To address this issue, we report here simulation studies of three model proteins: chymotrypsin inhibitor 2 (CI2), barnase, and Semliki forest virus protein (SFVP), and directly compare their folding during ribosome-mediated synthesis with their refolding from random, denatured conformations. To calibrate the methodology, simulations are first compared with in vitro data on the folding stabilities of N-terminal fragments of CI2 and barnase; the simulations reproduce the fact that both the stability and thermal folding cooperativity increase as fragments increase in length. Coupled simulations of synthesis and folding for the same two proteins are then described, showing that both fold essentially post-translationally, with mechanisms effectively identical to those for refolding. In both cases, confinement of the nascent polypeptide chain within the ribosome tunnel does not appear to promote significant formation of native structure during synthesis; there are however clear indications that the formation of structure within the nascent chain is sensitive to location within the ribosome tunnel, being subject to both gain and loss as the chain lengthens. Interestingly, simulations in which CI2 is artificially stabilized show a pronounced tendency to become trapped within the tunnel in partially folded conformations: non-cooperative folding, therefore, appears in the simulations to exert a detrimental effect on the rate at which fully folded conformations are formed. Finally, simulations of the two-domain protease module of SFVP, which experimentally folds cotranslationally, indicate that for multi-domain proteins, ribosome-mediated folding may follow different pathways from those taken during refolding. Taken together, these studies provide a first step toward developing more realistic methods for simulating protein folding as it occurs in vivo.

Project description:Success in evolution depends critically upon the ability of organisms to adapt, a property that is also true for the proteins that contribute to the fitness of an organism. Successful protein evolution is enhanced by mutational pathways that generate a wide range of physicochemical mechanisms to adaptation. In an earlier study, we used a weak-link method to favor changes to an essential but maladapted protein, adenylate kinase (AK), within a microbial population. Six AK mutants (a single mutant followed by five double mutants) had success within the population, revealing a diverse range of adaptive strategies that included changes in nonpolar packing, protein folding dynamics, and formation of new hydrogen bonds and electrostatic networks. The first mutation, AK(BSUB) Q199R, was essential in defining the structural context that facilitated subsequent mutations as revealed by a considerable mutational epistasis and, in one case, a very strong dependence upon the order of mutations. Namely, whereas the single mutation AK(BSUB) G213E decreases protein stability by >25 degrees C, the same mutation in the background of AK(BSUB) Q199R increases stability by 3.4 degrees C, demonstrating that the order of mutations can play a critical role in favoring particular molecular pathways to adaptation. In turn, protein folding kinetics shows that four of the five AK(BSUB) double mutants utilize a strategy in which an increase in the folding rate accompanied by a decrease in the unfolding rate results in additional stability. However, one mutant exhibited a dramatic increase in the folding relative to a modest increase in the unfolding rate, suggesting a different adaptive strategy for thermostability. In all cases, an increase in the folding rates for the double mutants appears to be the preferred mechanism in conferring additional stability and may be an important aspect of protein evolution. The range of overlapping as well as contrasting strategies for success illustrates both the power and subtlety of adaptation at even the smallest unit of change, a single amino acid.

Dataset Information

Protein folding and function: the N-terminal fragment in adenylate kinase.

Publications

Protein folding and function: the N-terminal fragment in adenylate kinase.

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