Overview of Protein Folding Mechanisms: Experimental and Theoretical Approaches to Probing Energy Landscapes

Abstract

We present an overview of the current experimental and theoretical approaches to studying protein folding mechanisms, set against current models of the folding energy landscape. We describe how stability and folding kinetics can be determined experimentally and how this data can be interpreted in terms of the characteristic features of various models from the simplest two-state pathway to a multi-state mechanism. We summarize the pros and cons of a range of spectroscopic methods for measuring folding rates and present a theoretical framework, coupled with protein engineering approaches, for elucidating folding mechanisms and structural features of folding transition states. A series of case studies are used to show how experimental kinetic data can be interpreted in the context of non-native interactions, populated intermediates, parallel folding pathways, and sequential transition states. We also show how computational methods now allow transient species of high energy, such as folding transition states, to be modeled on the basis of experimental Φ-value analysis derived from the effects of point mutations on folding kinetics.

Observing a late folding intermediate of Ubiquitin at atomic resolution by NMR

“…Molecular dynamics (MD) simulations can also potentially hint towards the existence of higher energy intermediates . However, molecular details of these intermediates are unavailable because experimentally solved atomic resolution structures of the intermediates are rare …”

“…12 However, molecular details of these intermediates are unavailable because experimentally solved atomic resolution structures of the intermediates are rare. 13,14 Ubiquitin (Ub) is a small (76 residues) eukaryotic protein that has been extensively studied to understand protein folding due to its high stability.…”

Single-molecule studies of riboswitch folding

“…A grand challenge of biology has been to build predictive models of organisms by leveraging our understanding of the structure-function relationship. One milestone in this pursuit would be the ability to predict the three-dimensional structures of proteins and nucleic acids directly from their genetic sequences [1–6]. But static structural information alone is insufficient: biomolecules are also highly dynamic.…”

Single-Molecule Protein Folding Experiments Using High-Precision Optical Tweezers

“…Accessing folding stability and kinetics of proteins is crucial for understanding their working mechanisms, functions, and dysfunctions. Various experimental approaches have been developed to study protein folding (4–6). They differ in the proteins of interest and ways to induce protein unfolding and to detect subsequent folding.…”

“…These methods also slow down protein folding, facilitating its detection. Once proteins are unfolded, their synchronized folding kinetics can be monitored by various spectroscopic techniques (4), such as circular dichroism (8), fluorescence (11), absorbance, Raman scattering (12). These approaches have greatly contributed to our understanding on protein folding.…”