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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., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The analysis of protein folding reactions by monitoring the kinetic effects of specifically designed single-point mutations, the so-termed phi-value analysis, has been a favorite technique to experimentally probe the mechanisms of protein folding. The idea behind phi-value analysis is that the effects that mutations have on the folding and unfolding rate constants report on the energetic/structural features of the folding transition state ensemble (TSE), which is the highest point in the free energy surface connecting the native and unfolded states, and thus the rate limiting step that ultimately defines the folding mechanism. For single-domain, two-state folding proteins, the general procedure to perform the phi-value analysis of protein folding is relatively simple to implement in the lab. Once the mutations have been produced and purified, the researcher needs to follow a few specific guidelines to perform the experiments and to analyze the data so produced. In this chapter, a step-by-step description of how to measure and interpret the effects induced by site-directed mutations on the folding and unfolding rate constants of a protein of interest is provided. Some possible solutions to the most typical problems that arise when performing phi-value analysis in the lab are also provided., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The macromolecular machines of life use allosteric control to self-assemble, dissociate and change shape in response to signals. Despite enormous interest, the design of nanoscale allosteric assemblies has proven tremendously challenging. Here we present a proof of concept of allosteric assembly in which an engineered fold switch on the protein monomer triggers or blocks assembly. Our design is based on the hyper-stable, naturally monomeric protein CI2, a paradigm of simple two-state folding, and the toroidal arrangement with 6-fold symmetry that it only adopts in crystalline form. We engineer CI2 to enable a switch between the native and an alternate, latent fold that self-assembles onto hexagonal toroidal particles by exposing a favorable inter-monomer interface. The assembly is controlled on demand via the competing effects of temperature and a designed short peptide. These findings unveil a remarkable potential for structural metamorphosis in proteins and demonstrate key principles for engineering protein-based nanomachinery.

Theory and simulations predict that the structural concert of protein folding reactions is relatively low. Experimentally, folding cooperativity has been difficult to study, but in recent years we have witnessed major advances. New analytical procedures in terms of conformational ensembles rather than discrete states, experimental techniques with improved time, structural, or single-molecule resolution, and combined thermodynamic and kinetic analysis of fast folding have contributed to demonstrate a general scenario of limited cooperativity in folding. Gradual structural disorder is already apparent on the unfolded and native states of slow, two-state folding proteins, and it greatly increases in magnitude for fast folding domains. These results demonstrate a direct link between how fast a single-domain protein folds and unfolds, and how cooperative (or structurally diverse) is its equilibrium unfolding process. Reducing cooperativity also destabilizes the native structure because it affects unfolding more than folding. We can thus define a continuous cooperativity scale that goes from the 'pliable' two-state character of slow folders to the gradual unfolding of one-state downhill, and eventually to intrinsically disordered proteins. The connection between gradual unfolding and intrinsic disorder is appealing because it suggests a conformational rheostat mechanism to explain the allosteric effects of folding coupled to binding., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A one-state downhill protein folding process is barrierless at all conditions, resulting in gradual melting of native structure that permits resolving folding mechanisms step-by-step at atomic resolution. Experimental studies of one-state downhill folding have typically focused on the thermal denaturation of proteins that fold near the speed limit (ca. 10(6) s(-1)) at their unfolding temperature, thus being several orders of magnitude too fast for current single-molecule methods, such as single-molecule FRET. An important open question is whether one-state downhill folding kinetics can be slowed down to make them accessible to single-molecule approaches without turning the protein into a conventional activated folder. Here we address this question on the small helical protein BBL, a paradigm of one-state downhill thermal (un)folding. We decreased 200-fold the BBL folding-unfolding rate by combining chemical denaturation and low temperature, and carried out free-diffusion single-molecule FRET experiments with 50-μs resolution and maximal photoprotection using a recently developed Trolox-cysteamine cocktail. These experiments revealed a single conformational ensemble at all denaturing conditions. The chemical unfolding of BBL was then manifested by the gradual change of this unique ensemble, which shifts from high to low FRET efficiency and becomes broader at increasing denaturant. Furthermore, using detailed quantitative analysis, we could rule out the possibility that the BBL single-molecule data are produced by partly overlapping folded and unfolded peaks. Thus, our results demonstrate the one-state downhill folding regime at the single-molecule level and highlight that this folding scenario is not necessarily associated with ultrafast kinetics.

Partly unfolded protein conformations close to the native state may play important roles in protein function and in protein misfolding. Structural analyses of such conformations which are essential for their fully physicochemical understanding are complicated by their characteristic low populations at equilibrium. We stabilize here with a single mutation the equilibrium intermediate of apoflavodoxin thermal unfolding and determine its solution structure by NMR. It consists of a large native region identical with that observed in the X-ray structure of the wild-type protein plus an unfolded region. Small-angle X-ray scattering analysis indicates that the calculated ensemble of structures is consistent with the actual degree of expansion of the intermediate. The unfolded region encompasses discontinuous sequence segments that cluster in the 3D structure of the native protein forming the FMN cofactor binding loops and the binding site of a variety of partner proteins. Analysis of the apoflavodoxin inner interfaces reveals that those becoming destabilized in the intermediate are more polar than other inner interfaces of the protein. Natively folded proteins contain hydrophobic cores formed by the packing of hydrophobic surfaces, while natively unfolded proteins are rich in polar residues. The structure of the apoflavodoxin thermal intermediate suggests that the regions of natively folded proteins that are easily responsive to thermal activation may contain cores of intermediate hydrophobicity., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

A battery of thermodynamic, kinetic, and structural approaches has indicated that the small α-helical protein BBL folds-unfolds via the one-state downhill scenario. Yet, single-molecule fluorescence spectroscopy offers a more conflicting view. Single-molecule experiments at pH 6 show a unique half-unfolded conformational ensemble at mid denaturation, whereas other experiments performed at higher pH show a bimodal distribution, as expected for two-state folding. Here we use thermodynamic and laser T-jump kinetic experiments combined with theoretical modeling to investigate the pH dependence of BBL stability, folding kinetics and mechanism within the pH 6–11 range. We find that BBL unfolding is tightly coupled to the protonation of one of its residues with an apparent pKa of ∼7. Therefore, in chemical denaturation experiments around neutral pH BBL unfolds gradually, and also converts in binary fashion to the protonated species. Moreover, under the single-molecule experimental conditions (denaturant midpoint and 279 K), we observe that proton transfer is much slower than the ∼15 microseconds folding-unfolding kinetics of BBL. The relaxation kinetics is distinctly biphasic, and the overall relaxation time (i.e. 0.2–0.5 ms) becomes controlled by the proton transfer step. We then show that a simple theoretical model of protein folding coupled to proton transfer explains quantitatively all these results as well as the two sets of single-molecule experiments, including their more puzzling features. Interestingly, this analysis suggests that BBL unfolds following a one-state downhill folding mechanism at all conditions. Accordingly, the source of the bimodal distributions observed during denaturation at pH 7–8 is the splitting of the unique conformational ensemble of BBL onto two slowly inter-converting protonation species. Both, the unprotonated and protonated species unfold gradually (one-state downhill), but they exhibit different degree of unfolding at any given condition because the native structure is less stable for the protonated form. [ABSTRACT FROM AUTHOR]

A one-state downhill protein folding process is barrierless at all conditions, resulting in gradual melting of native structure that permits resolving folding mechanisms step-by-step at atomic resolution. Experimental studies of one-state downhill folding have typically focused on the thermal denaturation of proteins that fold near the speed limit (Ca. 106 s-1) at their unfolding temperature, thus being several orders of magnitude too fast for current single-molecule methods, such as single-molecule FRET. An important open question is whether one-state downhill folding kinetics can be slowed down to make them accessible to single-molecule approaches without turning the protein into a conventional activated folder. Here we address this question on the small helical protein BBL, a paradigm of one-state downhill thermal (un)folding. We decreased 200-fold the BBL folding-unfolding rate by combining chemical denaturation and low temperature, and carried out freediffusion single-molecule FRET experiments with 50-its resolution and maximal photoprotection using a recently developed Troloxcysteamine cocktail. These experiments revealed a single conformational ensemble at all denaturing conditions. The chemical unfolding of BBL was then manifested by the gradual change of this unique ensemble, which shifts from high to low FRET efficiency and becomes broader at increasing denaturant. Furthermore, using detailed quantitative analysis, we could rule out the possibility that the BBL single-molecule data are produced by partly overlapping folded and unfolded peaks. Thus, our results demonstrate the onestate downhill folding regime at the single-molecule level and highlight that this folding scenario is not necessarily associated with ultrafast kinetics. [ABSTRACT FROM AUTHOR]

Although core residues can sometimes be replaced by shorter ones without introducing significant changes in protein structure, the energetic consequences are typically large and destabilizing. Many efforts have been devoted to understand and predict changes in stability from analysis of the environment of mutated residues, but the relationships proposed for individual proteins have often failed to describe additional data. We report here 17 apoflavodoxin large-to-small mutations that cause overall protein destabilizations of 0.6-3.9 kcal.mol−1. By comparing two-state urea and three-state thermal unfolding data, the overall destabilizations observed are partitioned into effects on the N-to-I and on the I-to-U equilibria. In all cases, the equilibrium intermediate exerts a 'buffering' effect that reduces the impact of the overall destabilization on the N-to-I equilibrium. The performance of several structure-energetics relationships, proposed to explain the energetics of hydrophobic shortening mutations, has been evaluated by using an apoflavodoxin data set consisting of 14 mutations involving branching-conservative aliphatic side-chain shortenings and a larger data set, including similar mutations implemented in seven model proteins. Our analysis shows that the stability changes observed for any of the different types of mutations (LA, IA, IV, and VA) in either data set are best explained by a combination of differential hydrophobicity and of the calculated volume of the modeled cavity (as previously observed for LA and IA mutations in lysozyme T4). In contrast, sequence conservation within the flavodoxin family, which is a good predictor for charge-reversal stabilizing mutations, does not perform so well for aliphatic shortening ones. [ABSTRACT FROM AUTHOR]

Protein intermediates in equilibrium with native states may play important roles in protein dynamics but, in cases, can initiate harmful aggregation events. Investigating equilibrium protein intermediates is thus important for understanding protein behaviour (useful or pernicious) but it is hampered by difficulties in gathering structural information. We show here that the φ-analysis techniques developed to investigate transition states of protein folding can be extended to determine low-resolution three-dimensional structures of protein equilibrium intermediates. The analysis proposed is based solely on equilibrium data and is illustrated by determination of the structure of the apoflavodoxin thermal unfolding intermediate. In this conformation, a large part of the protein remains close to natively folded, but a 40 residue region is clearly unfolded. This structure is fully consistent with the NMR data gathered on an apoflavodoxin mutant designed specifically to stabilise the intermediate. The structure shows that the folded region of the intermediate is much larger than the proton slow-exchange core at 25°C. It also reveals that the unfolded region is made of elements whose packing surface is more polar than average. In addition, it constitutes a useful guide to rationally stabilise the native state relative to the intermediate state, a far from trivial task. [Copyright &y& Elsevier]

The vast majority of our knowledge on protein stability arises from the study of simple two-state models. However, proteins displaying equilibrium intermediates under certain conditions abound and it is unclear whether the energetics of native/intermediate equilibria is well represented in current knowledge. We consider here that the overall conformational stability of three-state proteins is made of a “relevant” term and a “residual” one, corresponding to the free energy differences of the native to intermediate (N-to-I) and intermediate to denatured (I-to-D) equilibria, respectively. The N-to-I free energy difference is considered to be the relevant stability because protein-unfolding intermediates are likely devoid of biological activity. We use surface charge optimisation to first increase the overall (N-to-D) stability of a model three-state protein (apoflavodoxin) and then investigate whether the stabilisation obtained is realised into relevant or into residual stability. Most of the mutations designed from electrostatic calculations or from simple sequence conservation analysis produce large increases in the overall stability of the protein. However, in most cases, this simply leads to similarly large increases of the residual stability. Two mutations, nevertheless, show a different trend and increase the relevant stability of the protein substantially. When all the mutations are mapped onto the structure of the apoflavodoxin thermal-unfolding intermediate (obtained independently by equilibrium φ-analysis and NMR) they cluster perfectly so that the mutations increasing the relevant stability appear in the small unstructured region of the intermediate and the others in the native-like region. This illustrates the need for specific investigation of N-to-I equilibria and the structure of protein intermediates, and indicates that it is possible to rationally stabilise a protein against partial unfolding once the structure of the intermediate conformation is known, even if at low resolution. [Copyright &y& Elsevier]