Forcing Thermodynamically Unfolded Proteins to Fold

Abstract

A growing number of biologically important proteins have been identified as fully unfolded or partially disordered. Thus, an intriguing question is whether such proteins can be forced to fold by adding solutes found in the cells of some organisms. Nature has not ignored the powerful effect that the solution can have on protein stability and has developed the strategy of using specific solutes (called organic osmolytes) to maintain the structure and function cellular proteins in organisms exposed to denaturing environmental stresses (Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., and Somero, G. N. (1982) Science 217, 1214 -1222). Here, we illustrate the extraordinary capability of one such osmolyte, trimethylamine N-oxide (TMAO), to force two thermodynamically unfolded proteins to fold to nativelike species having significant functional activity. In one of these examples, TMAO is shown to increase the population of native state relative to the denatured ensemble by nearly five orders of magnitude. The ability of TMAO to force thermodynamically unstable proteins to fold presents an opportunity for structure determination and functional studies of an important emerging class of proteins that have little or no structure without the presence of TMAO.A growing number of biologically important proteins have been identified as fully or partially disordered under physiological conditions (e.g. different classes of DNA-binding proteins (1), transactivation domains of transcription factors (2-6), non-A␤ component of Alzheimer's disease amyloid plaque precursor implicated in Alzheimer's disease (7), and others (8,9). The issue of shifting a protein or domain from an unfolded to a folded ensemble is a topic of interest not only for these proteins, but also for a host of marginally stable proteins. A question of interest is whether such proteins can be induced to adopt unique and functionally important ordered structures by addition of solutes found in the cells of some organisms.According to Anfinsen, "The native conformation of protein is determined by the totality of interatomic interactions and by the amino acid sequence, in a given environment " (10). Although the statement by Anfinsen acknowledges the importance of both amino acid sequence and the physiological milieu in defining the native (Gibbs energy minimum) conformation of proteins, the overwhelming emphasis in the protein folding field has been on the interatomic interaction aspect of the process (11). Nature, however, has not ignored the powerful effect that the solution can have on protein stability and has developed the strategy of using specific solutes (called organic osmolytes) to maintain the structure of proteins in cells exposed to denaturing environmental stresses (12). Thus, through the power of natural selection, solutes were evolved that have exceptional ability to promote the native states of proteins in the presence of denaturing stresses. The implication is that in the absence of denaturing stresses, osmolytes continue to exert a force t...

Backbone additivity in the transfer model of protein solvation

“…Nevertheless, the calculated and experimental glycine backbone transfer free energies demonstrate reasonable agreement and account for the features of the protein folding ability of TMAO. 15,47,48 We obtained DG tr from the simulated solvation free energy differences between 2M osmolytes and pure water. These calculated transfer free energies of the peptide backbone demonstrated additivity of each incremental unit of the peptide backbone corresponding to a nearly constant change in the free energy for these short oligomers.…”

Why Is Trehalose an Exceptional Protein Stabilizer?

“…Osmolytes and other salting-out agents responsible for protein stabilization are known to induce the formation of secondary structures in proteins under denaturing conditions (40 -42). Naturally occurring osmolytes like sarcosine, proline, sugars, and trimethylamine-N-oxide have been shown to result in the contraction of the denatured state proportional to their stabilizing power (22,43). A relatively compact denatured state would have lesser hydrophobic surface in contact with the solvent leading to a decrease in the apparent heat capacity of protein denaturation.…”

Binary Phases of Aliphatic N-Oxides and Water: Force Field Development and Molecular Dynamics Simulation

“…11 Explanations on the molecular level for these phenomena are only beginning to surface. [12][13][14][15][16][17][18][19] To investigate into the molecular mechanism of these effects by computational methods such as molecular dynamics (MD) simulation techniques, a force field for mixed solvents composed of water and N-oxide species is required that is also compatible with available biopolymer potential energy functions. Noto et al 20 were the first who constructed a force field for a rigid TMAO model in the presence of an aqueous environment based on quantum-chemical calculations of TMAO and a single water molecule within the Hartree-Fock (HF) approximation.…”