In this work we have investigated the contribution to protein stability of residues forming the boundaries of alpha-helices. At the N-terminus of helix 2 of human growth hormone there are two residues, Ser71 and Glu74, which form two reciprocal hydrogen bonds between the side chains and the backbone nitrogens of either residue (the N-capping box). In order to evaluate the stabilizing effect of each hydrogen bond, site-directed mutagenesis was employed. In addition, the effect of side-chain negative charge on helix stabilization, via charge dipole interaction, was assessed. Ultraviolet spectroscopy and near- and far-UV CD spectroscopy as well as guanidine hydrochloride protein denaturation were used as assays to monitor the conformational and free energy of stabilization changes induced by the point mutations. The results of these experiments can be summarized as follows: (a) receptor binding studies showed that the tertiary conformation of each mutant was similar to that of the native hormone, (b) far-UV CD spectroscopic analyses showed that the overall alpha-helical content was unchanged in the mutants, (c) UV absorption and CD spectroscopic analyses indicated small alterations in helical packing in those mutants in which the hydrogen bond between the side chain of Ser71 and backbone NH of Glu74 was disrupted, (d) the hydrogen bond involving the side chain of Ser71 contributes at least 1.0 kcal/mol to protein stabilization and has a 2-fold larger stabilizing effect than that of the hydrogen bond involving the Glu74 side chain, and (e) the putative charge-dipole interaction of Glu74 with the alpha-helix dipole does not contribute to the stabilization of the tertiary conformation of human growth hormone. Study holds ProTherm entries: 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 2191, 2192, 2193 Extra Details: ddG values are calculated using m and Cm,dG and ddG were measured in the presence of [GdnHCl]50%
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:15 p.m.
|Number of data points||69|
|Proteins||Growth hormone receptor ; Somatotropin|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: ddG ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence A:FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCG/B:EPKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQLFYTRRNTQEWTQEWKECPDYVSAGENSCYFNSSFTSIWIPYCIKLTSNGGTVDEKCFSVDEIVQPDPPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWKMMDPILTTSVPVYSLKVDKEYEVRVRSKQRNSGNYGEFSEVLYVTLPQM/C:EPKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQLFYTRRNTQEWTQEWKECPDYVSAGENSCYFNSSFTSIWIPYCIKLTSNGGTVDEKCFSVDEIVQPDPPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWKMMDPILTTSVPVYSLKVDKEYEVRVRSKQRNSGNYGEFSEVLYVTLPQM|