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

**Irina Sorokina 1,\*, Arcady R. Mushegian 2,3 and Eugene V. Koonin 4,\***


**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 Δ*G* of folding is likely to be positive for the majority of proteins, which therefore fold into their native conformations only through interactions with the energydependent 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:** protein folding; entropy; free energy; free energy landscape; energy-dependent protein folding; co-translational protein folding; molecular chaperones; physical model of protein folding
