RESUMEN
Proteins are constantly undergoing folding and unfolding transitions, with rates that determine their homeostasis in vivo and modulate their biological function. The ability to optimize these rates without affecting overall native stability is hence highly desirable for protein engineering and design. The great challenge is, however, that mutations generally affect folding and unfolding rates with inversely complementary fractions of the net free energy change they inflict on the native state. Here we address this challenge by targeting the folding transition state (FTS) of chymotrypsin inhibitor 2 (CI2), a very slow and stable two-state folding protein with an FTS known to be refractory to change by mutation. We first discovered that the CI2's FTS is energetically taxed by the desolvation of several, highly conserved, charges that form a buried salt bridge network in the native structure. Based on these findings, we designed a CI2 variant that bears just four mutations and aims to selectively stabilize the FTS. This variant has >250-fold faster rates in both directions and hence identical native stability, demonstrating the success of our FTS-centric design strategy. With an optimized FTS, CI2 also becomes 250-fold more sensitive to proteolytic degradation by its natural substrate chymotrypsin, and completely loses its activity as inhibitor. These results indicate that CI2 has been selected through evolution to have a very unstable FTS in order to attain the kinetic stability needed to effectively function as protease inhibitor. Moreover, the CI2 case showcases that protein (un)folding rates can critically pivot around a few key residues-interactions, which can strongly modify the general effects of known structural factors such as domain size and fold topology. From a practical standpoint, our results suggest that future efforts should perhaps focus on identifying such critical residues-interactions in proteins as best strategy to significantly improve our ability to predict and engineer protein (un)folding rates.
Asunto(s)
Mutación , Pliegue de Proteína , Estabilidad Proteica , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Modelos Moleculares , Cinética , Conformación Proteica , PéptidosRESUMEN
Various effects of amino acid substitutions on properties of globular proteins have been described in a large number of research papers. Nevertheless, no definite "rule" has been formulated as of yet that could be used by experimentalists to introduce desirable changes in the properties of proteins. Herein we attempt to establish such a "rule". To this end, a hypothesis is proposed on the effects of substitutions of hydrophobic residues with large number of contacts on free energies of different states of a globular protein. The hypothesis states: Substitutions of hydrophobic residues engaged in a large number of residue-residue contacts would not change the folding rate of a protein but could affect its unfolding rate. This hypothesis was verified by both theoretical and experimental analyses, generating a general rule that can facilitate the work of experimentalists on constructing mutant forms of proteins.
Asunto(s)
Pliegue de Proteína , Proteínas/química , Sustitución de Aminoácidos , Animales , Anhidrasa Carbónica II/química , Anhidrasa Carbónica II/genética , Bovinos , Interacciones Hidrofóbicas e Hidrofílicas , Mutagénesis Sitio-Dirigida , Desnaturalización Proteica , Proteínas/genética , TermodinámicaRESUMEN
Fast-folding WW domains are among the best-characterized systems for comparing experiments and simulations of protein folding. Recent microsecond-resolution experiments and long duration (totaling milliseconds) single-trajectory modeling have shown that even mechanistic changes in folding kinetics due to mutation can now be analyzed. Thus, a comprehensive set of experimental data would be helpful to benchmark the predictions made by simulations. Here, we use T-jump relaxation in conjunction with protein engineering and report mutational Φ-values (Φ(M)) as indicators for folding transition-state structure of 65 side chain, 7 backbone hydrogen bond, and 6 deletion and /or insertion mutants within loop 1 of the 34-residue hPin1 WW domain. Forty-five cross-validated consensus mutants could be identified that provide structural constraints for transition-state structure within all substructures of the WW domain fold (hydrophobic core, loop 1, loop 2, ß-sheet). We probe the robustness of the two hydrophobic clusters in the folding transition state, discuss how local backbone disorder in the native-state can lead to non-classical Φ(M)-values (Φ(M) > 1) in the rate-determining loop 1 substructure, and conclusively identify mutations and positions along the sequence that perturb the folding mechanism from loop 1-limited toward loop 2-limited folding.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Isomerasa de Peptidilprolil/química , Fosfoproteínas/química , Pliegue de Proteína , Secuencia de Aminoácidos , Eliminación de Gen , Humanos , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Temperatura , Termodinámica , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
The folding nucleus (FN) is a cryptic element within protein primary structure that enables an efficient folding pathway and is the postulated heritable element in the evolution of protein architecture; however, almost nothing is known regarding how the FN structurally changes as complex protein architecture evolves from simpler peptide motifs. We report characterization of the FN of a designed purely symmetric ß-trefoil protein by Ï-value analysis. We compare the structure and folding properties of key foldable intermediates along the evolutionary trajectory of the ß-trefoil. The results show structural acquisition of the FN during gene fusion events, incorporating novel turn structure created by gene fusion. Furthermore, the FN is adjusted by circular permutation in response to destabilizing functional mutation. FN plasticity by way of circular permutation is made possible by the intrinsic C3 cyclic symmetry of the ß-trefoil architecture, identifying a possible selective advantage that helps explain the prevalence of cyclic structural symmetry in the proteome.