Stabilization of a protein conferred by an increase in folded state entropy

Abstract

Entropic stabilization of native protein structures typically relies on strategies that serve to decrease the entropy of the unfolded state. Here we report, using a combination of experimental and computational approaches, on enhanced thermodynamic stability conferred by an increase in the configurational entropy of the folded state. The enhanced stability is observed upon modifications of a loop region in the enzyme acylphosphatase and is achieved despite significant enthalpy losses. The modifications that lead to increased stability, as well as those that result in destabilization, however, strongly compromise enzymatic activity, rationalizing the preservation of the native loop structure even though it does not provide the protein with maximal stability or kinetic foldability.protein folding | loop closure entropy | molecular dynamics R educing the difference in entropy between the unfolded and folded states can increase the thermodynamic stability of a protein. This is commonly accomplished by strategies that act to restrict the conformational space available for the unfolded state (e.g., decreasing loop length, macromolecular crowding, and backbone cyclization) (1). In principle, changes that increase the entropy of the folded state can also lead to its stabilization provided that they exceed the loss of enthalpic contributions, if stabilizing interactions are perturbed by the modifications.The current work originally aimed at studying the effects exerted by changes in the length of a loop region on protein stability and folding. As a model, we chose a loop in human muscle acylphosphatase (hmAcP), a small (∼100 aa) enzyme that catalyzes the hydrolysis of the carboxyl-phosphate bond in various acylphosphate compounds and presents an open α/β-sandwich structure (ref. 2, and see, e.g., refs. 3-5). The folding stability and dynamics of hmAcP have been studied extensively and are well characterized (ref. 6-10 and references therein). Excluding a minor cis-trans prolyl isomerization phase, it folds in a two-step process, albeit very slowly (due to abundance of long-range interactions; ref. 9), through a relatively compact, native-like transition state. The loop we chose for the modifications (hereafter referred to as L4) connects between the second helix and the fourth β-strand of the protein (Fig. 1) and possesses multiple internal and external contacts (refs. 3 and 4 and our own contact analysis). The latter contacts are formed predominantly with residues located in the first loop of the protein (L1), which runs along L4 and is involved in the binding of the phosphate group of the substrate (4,(11)(12)(13)(14)(15).Characterizing the properties of hmAcP mutants carrying deletions or insertions in L4 we found that the thermodynamic stability of mutants in which the loop was shortened is increased to an extent significantly larger than that predicted by polymer models for loop closure entropy. The increased stability is predominantly due to a decrease in the unfolding rate and is attained despite the fact that sho...

Entropic stabilization of a deubiquitinase provides conformational plasticity and slow unfolding kinetics beneficial for functioning on the proteasome

“…Loop elongation however, is not always destabilizing for proteins. A recent study showed that polyglycine loop insertion could in fact significantly increase the entropy of the native state of an acylphosphatase, thereby increasing the native state stability without perturbing the transition and denatured states49. In contrast to the artificial manipulation of the loop lengths of model systems, our current results present a biologically relevant example, which has likely evolved to maximize the structural plasticity of UCH-L5 by a longer substrate recognition loop and peripheral helical elements.…”

The alteration of the C‐terminal region of human frataxin distorts its structural dynamics and function

“…In particular, by the reduction of the difference in entropy between unfolded and folded ensembles, proteins can increase their thermodynamic stability [34]. Slow motions (microsecond/millisecond timescale) are related to function (conformational changes) and allostery (intramolecular signal transduction).…”

Folding propensity of anoplin: A molecular dynamics study of the native peptide and four mutated isoforms

“…To better elucidate the above results, we have analyzed the internal fluctuation of the peptide as an indication of the contribution to the total entropy. It is important, also in this case, to remark that the quantitative evaluation of folding entropy is an extremely difficult task and the present results should be only taken as a qualitative indication.…”