Volume 84, February 2024, 102738Author links open overlay panel, Abstract

Relating the native fold of a protein to its amino acid sequence remains a fundamental problem in biology. While computer algorithms have demonstrated recently their prowess in predicting what structure a particular amino acid sequence will fold to, an understanding of how and why a specific protein fold is achieved remains elusive. A major challenge is to define the role of conformational heterogeneity during protein folding. Recent experimental studies, utilizing time-resolved FRET, hydrogen-exchange coupled to mass spectrometry, and single-molecule force spectroscopy, often in conjunction with simulation, have begun to reveal how conformational heterogeneity evolves during folding, and whether an intermediate ensemble of defined free energy consists of different sub-populations of molecules that may differ significantly in conformation, energy and entropy.

Section snippetsHeterogeneity seen through the eyes of experiment and theory

Protein folding occurs by the diffusive motion of the polypeptide chain starting from a highly dynamic U state [8,18,19]. The folding process is therefore expected to be heterogeneous. The idea that heterogeneity exists on protein folding pathways was legitimized when transiently populated partially folded intermediates on the pathways were characterized by hydrogen-exchange-NMR methods [20,21]. Subsequently, intermediates have been detected in the folding and unfolding pathways of many

Single dominant pathway versus multiple pathways

Early experiments investigated the folding reaction only along one or two reaction coordinates, by utilizing only one or two ensemble-averaging experimental probes such as fluorescence or circular dichroism. Hence, they invariably described folding as occurring along a single defined pathway populated by folding intermediates that could be, in many cases, too sparsely populated to be detected [20,38]. Indeed, the notion of a single dominant folding pathway allowed the adoption of the elegant

Assembly of structure can occur in multiple ways under the same folding conditions

The assembly of structural parts during folding can potentially occur in many distinct ways, even under the same folding conditions. It was important to demonstrate this structural distinction because it would validate the existence of concurrently operating folding pathways. Recent multi-site time-resolved FRET (trFRET) studies of MNEI have achieved this [35]. Earlier studies have indicated that MNEI folds and unfolds via multiple intermediates on multiple pathways [65,66]. A more recent study

Gradual conformational change

Both trFRET and HX-MS studies of the folding/unfolding of MNEI, as well as HX-MS studies of the folding/unfolding of a SH3 domain not only indicated that structural change can occur differently on different pathways but that it can occur gradually under some conditions [10,62]. In the case of MNEI, the gradual unfolding and folding reactions on competing pathways could be described adequately by a Rouse-like chain model [69] or by a coarse-grained Markov model [66]. Importantly, in the case of

The role of heterogeneity in protein conformational change

A full understanding of how proteins fold, of the heterogeneity inherent in the folding process, and of the physical and chemical forces that govern folding, is critical for a proper understanding of a variety of protein conformational changes. Does the ribosome modulate co-translational folding [31] by binding to one of several sub-populations present in an intermediate ensemble? What is the role of conformational heterogeneity in determining how the same sequence may fold into two very

Conclusion

Many detailed studies of the kinetic mechanisms of protein folding have shown that a significant number of single-domain and multi-domain proteins utilize multiple pathways to fold (reviewed in references 10, 23). Validation of the existence of competing pathways, by showing how they differ in the manner structure progressively forms on them, is, however, still at its early stages. Much of the kinetic and structural heterogeneity would be present at the early stages of folding, before the

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank past and present members of the JBU laboratory, as well as G. Krishnamoorthy, M.K. Mathew and D. Dhar, for discussions. SB is a recipient of JCC/HHMI postdoctoral research fellowship. JBU is a recipient of a JC Bose National Fellowship from the Government of India. Work in the JBU laboratory has been funded by the Tata Institute of Fundamental Research, the Indian Institute of Science Education and Research Pune, and the Science and Engineering Research Board, Government of India.

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