Is Protein Folding a Thermodynamically Unfavorable, Active, Energy-Dependent Process?

Abstract

The prevailing current view of protein folding is the thermodynamic hypothesis, under which the native folded conformation of a protein corresponds to the global minimum of Gibbs free energy G. We question this concept and show that the empirical evidence behind the thermodynamic hypothesis of folding is far from strong. Furthermore, physical theory-based approaches to the prediction of protein folds and their folding pathways so far have invariably failed except for some very small proteins, despite decades of intensive theory development and the enormous increase of computer power. The recent spectacular successes in protein structure prediction owe to evolutionary modeling of amino acid sequence substitutions enhanced by deep learning methods, but even these breakthroughs provide no information on the protein folding mechanisms and pathways. We discuss an alternative view of protein folding, under which the native state of most proteins does not occupy the global free energy minimum, but rather, a local minimum on a fluctuating free energy landscape. We further argue that ΔG of folding is likely to be positive for the majority of proteins, which therefore fold into their native conformations only through interactions with the energy-dependent molecular machinery of living cells, in particular, the translation system and chaperones. Accordingly, protein folding should be modeled as it occurs in vivo, that is, as a non-equilibrium, active, energy-dependent process.

Keywords: co-translational protein folding; energy-dependent protein folding; entropy; free energy; free energy landscape; molecular chaperones; physical model of protein folding; protein folding.

Figure 1

Protein folding energy landscapes in vitro and in vivo. Blue areas are occupied by “perfectly unfolded” conformations with no stable interactions between non-contiguous residues. Yellow and purple areas are populated by more compact protein conformations. Red zones are thought to arise mostly as the result of interactions between the protein and cellular components in a crowded environment. Green zones correspond to proteins in native conformation. (a) Canonical funnel-shaped energy landscape that most likely applies only to folding of small, thermodynamically stable proteins as it occurs spontaneously, in vitro, in isolation from all cellular compounds. (b) Folding energy landscape for a protein that folds in vivo is poorly understood, but most likely, is complex, rugged, dynamic, and shaped by interactions of the folding polypeptide with multiple cellular components. (c) Folding energy landscape of the same small protein as in (a) is most likely substantially different and far more complex when folding occurs in a crowded cellular environment. (d) Native conformations of most proteins are likely to occupy local thermodynamic minima with higher Gibbs free energy than their unfolded conformations (positive ΔG of folding). Such native conformation can only arise as a result of active, energy dependent folding process.