George D. Rose

The deep entanglement of biomolecular structure and function with aqueous systems supports the view that water actively sculpted both molecules and processes during the origins of life and continues to constrain evolution today. Nature's rules of biochemistry and biophysics have survived for nearly 4 billion years. The in vivo roles of water, the architectures of biopolymer backbones and side chains, the structure and function of ribose, ATP, the translation system, the genetic code, and certain inorganic cations persist unchanged. Hydrogen bonding and the hydrophobic effect predate life on Earth altogether. Here we examine fundamental forces that established, shaped, and continue to constrain biochemistry and biophysics. The results support a model in which life emerged through water‐based selection among diverse molecules and molecular ensembles, with molecular fitness defined by behaviors in and interactions with water. Life is thus composed of molecules that cooperate with, resist, and exploit the unique properties of water. Biological molecules employ chemical strategies that enable selective and controlled persistence in aqueous environments, a phenomenon we classify as recalcitrance. Molecular assembly reduces conformational heterogeneity, constrains dynamics, and sterically excludes reactive agents, including water and hydrolytic enzymes. By this mechanism, lifetimes of folded proteins, structured RNAs, assembled phospholipids, and polysaccharides are mediated by their organizational states.

The protein folding problem dates back to Pauling’s insights almost a century ago, but the first venture into actual protein structure was the Pauling–Corey–Brandson α-helix in 1951, a proposed model that was confirmed almost immediately using X-ray crystallography. Many subsequent efforts to predict protein helices from the amino acid sequence met with only partial success, as discussed here. Surprisingly, in 2021, these efforts were superseded by deep-learning artificial intelligence, especially AlphaFold2, a machine learning program based on neural nets. This approach can predict most protein structures successfully at or near atomic resolution. Deservedly, deep-learning artificial intelligence was named Science magazine’s 2021 “breakthrough of the year.” Today, ~200 million predicted protein structures can be downloaded from the AlphaFold2 Protein Structure Database. Deep learning represents a deep conundrum because these successfully predicted macromolecular structures are based on methods that are completely devoid of a hypothesis or of any physical chemistry. Perhaps we are now poised to transcend five centuries of reductive science.

As proposed here, β‐turns play an essential role in protein self‐assembly. This compact, four‐residue motif affects protein conformation dramatically by reversing the overall chain direction. Turns are the “hinges” in globular proteins. This new proposal broadens a previous hypothesis that globular proteins solve the folding problem in part by filtering conformers with unsatisfied backbone hydrogen bonds, thereby preorganizing the folding population. Recapitulating that hypothesis: unsatisfied conformers would be dramatically destabilizing, shifting the U(nfolded) ⇌ N(ative) equilibrium far to the left. If even a single backbone polar group is satisfied by solvent when unfolded but buried and unsatisfied when folded, that energy penalty alone, approximately +5 kcal/mol, would rival almost the entire free energy of protein stabilization at room temperature. Consequently, globular proteins are built on scaffolds of hydrogen‐bonded α‐helices and/or strands of β‐sheet, motifs that can be extended indefinitely, with intra‐segment hydrogen bond partners for their backbone polar groups and without steric clash. Scaffolds foster a protein‐wide hydrogen‐bonded network, and, of thermodynamic necessity, they self‐assemble cooperatively. Unlike elements of repetitive secondary structure, α‐helices and β‐sheet, a four‐residue β‐turn has only a single hydrogen bond (from i + 3 → i), not a cooperatively formed assembly of hydrogen bonds. As such, turns can form autonomously and are poised to initiate assembly of scaffold elements by bringing them together in an orientation and registration that promotes cooperative “zipping”. The overall effect of this self‐assembly mechanism is to induce substantial preorganization in the thermodynamically accessible folding population and, concomitantly, to reduce the folding entropy.

This Perspective is intended to raise questions about the conventional interpretation of protein folding. According to the conventional interpretation, developed over many decades, a protein population can visit a vast number of conformations under unfolding conditions, but a single dominant native population emerges under folding conditions. Accordingly, folding comes with a substantial loss of conformational entropy. How is this price paid? The conventional answer is that favorable interactions between and among the side chains can compensate for entropy loss, and moreover, these interactions are responsible for the structural particulars of the native conformation. Challenging this interpretation, the Perspective introduces a proposal that high energy (i.e., unfavorable) excluding interactions winnow the accessible population substantially under physical-chemical conditions that favor folding. Both steric clash and unsatisfied hydrogen bond donors and acceptors are classified as excluding interactions, so called because conformers with such disfavored interactions will be largely excluded from the thermodynamic population. Both excluding interactions and solvent factors that induce compactness are somewhat non-specific, yet together they promote substantial chain organization. Moreover, proteins are built on a backbone scaffold consisting of a-helices and strands of b sheet, where the number of hydrogen bond donors and acceptors is exactly balanced. These repetitive secondary structural elements are the only two conformers that can be both completely hydrogen-bond satisfied and extended indefinitely without encountering a steric clash. Putting the “bottom line” at the top: it is likely that hydrogen-bond satisfaction represents a largely overlooked parameter in protein folding models.

It has been a long-standing conviction that a protein’s native fold is selected from a vast number of conformers by the optimal constellation of enthalpically favorable interactions. In marked contrast, this Perspective introduces a different mechanism, one that emphasizes conformational entropy as the principal organizer in protein folding while proposing that the conventional view is incomplete. This mechanism stems from the realization that hydrogen bond satisfaction is a thermodynamic necessity. In particular, a backbone hydrogen bond may add little to the stability of the native state, but a completely unsatisfied backbone hydrogen bond would be dramatically destabilizing, shifting the U(nfolded) ⇌ N(ative) equilibrium far to the left. If even a single backbone polar group is satisfied by solvent when unfolded but buried and unsatisfied when folded, that energy penalty alone, approximately +5 kcal/mol, would rival almost the entire free energy of protein stabilization, typically between −5 and −15 kcal/mol under physiological conditions. Consequently, upon folding, buried backbone polar groups must form hydrogen bonds, and they do so by assembling scaffolds of α-helices and/or strands of β-sheet, the only conformers in which, with rare exception, hydrogen bond donors and acceptors are exactly balanced. In addition, only a few thousand viable scaffold topologies are possible for a typical protein domain. This thermodynamic imperative winnows the folding population by culling conformers with unsatisfied hydrogen bonds, thereby reducing the entropy cost of folding. Importantly, conformational restrictions imposed by backbone···backbone hydrogen bonding in the scaffold are sequence-independent, enabling mutation─and thus evolution─without sacrificing the structure.

This Perspective is intended to raise questions about the conventional interpretation of protein folding. According to the conventional interpretation, developed over many decades, a protein population can visit a vast number of conformations under unfolding conditions, but a single dominant native population emerges under folding conditions. Accordingly, folding comes with a substantial loss of conformational entropy. How is this price paid? The conventional answer is that favorable interactions between and among the side chains can compensate for entropy loss, and moreover, these interactions are responsible for the structural particulars of the native conformation. Challenging this interpretation, the Perspective introduces a proposal that high energy (i.e., unfavorable) excluding interactions winnow the accessible population substantially under physical–chemical conditions that favor folding. Both steric clash and unsatisfied hydrogen bond donors and acceptors are classified as excluding interactions, so called because conformers with such disfavored interactions will be largely excluded from the thermodynamic population. Both excluding interactions and solvent factors that induce compactness are somewhat nonspecific, yet together they promote substantial chain organization. Moreover, proteins are built on a backbone scaffold consisting of α‐helices and strands of β‐sheet, where the number of hydrogen bond donors and acceptors is exactly balanced. These repetitive secondary structural elements are the only two conformers that can be both completely hydrogen‐bond satisfied and extended indefinitely without encountering a steric clash. Consequently, the number of fundamental folds is limited to no more than ~10,000 for a protein domain. Once excluding interactions are taken into account, the issue of “frustration” is largely eliminated and the Levinthal paradox is resolved. Putting the “bottom line” at the top: it is likely that hydrogen‐bond satisfaction represents a largely under‐appreciated parameter in protein folding models.