The deletion series consists of three mutants in which two to six residues were removed in a pairwise fashion, starting with the two serine residues and progressing symmetrically outward. This location was considered favorable in terms of end effects and potential interactions between the inserts and the protein. The site chosen for the modifications encompasses the two adjacent serine residues at positions 72 and 73, which are located in a β-bulge at the tip of L4 that points toward the solvent. Table 1 lists the L4 deletion and insertion mutants that were constructed. The thermodynamically stabilized mutants, as well as the destabilized variants carrying elongated loops, are all deficient in enzymatic activity, suggesting that the native structure of the loop has evolved primarily for function. We provide evidence that supports this hypothesis and show that the enhanced dynamics are accompanied by only relatively small rearrangements in secondary and tertiary structure. Given the above, we postulated that the surplus stabilization arises from an increased conformational entropy of the folded state ensemble. The increased stability is predominantly due to a decrease in the unfolding rate and is attained despite the fact that shortening of the loop is accompanied by considerable losses in enthalpy. 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– 15).Ĭharacterizing 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. 1) and possesses multiple internal and external contacts (refs. 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. 9), through a relatively compact, native-like transition state. 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. The folding stability and dynamics of hmAcP have been studied extensively and are well characterized (ref. 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. The current work originally aimed at studying the effects exerted by changes in the length of a loop region on protein stability and folding.
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