Contribution to global protein stabilization of the N-capping box in human growth hormone.


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%

Submission Details

ID: rQm4oNDd

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:15 p.m.

Version: 1

Publication Details
Zhukovsky EA;Mulkerrin MG;Presta LG,Biochemistry (1994) Contribution to global protein stabilization of the N-capping box in human growth hormone. PMID:8060992
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
200.0 B,C Somatotropin P10912 GHR_HUMAN
190.2 B,C Somatotropin Q9XSZ1 GHR_PAPAN
185.4 B,C Somatotropin P79194 GHR_MACMU
181.4 B,C Somatotropin Q95ML5 GHR_SAIBB
100.0 A Somatotropin P01241 SOMA_HUMAN
100.0 A Somatotropin P58756 SOMA_PANTR
96.8 A Somatotropin P33093 SOMA_MACMU
93.7 A Somatotropin P58757 SOM2_PANTR
90.5 A Somatotropin Q9GMB3 SOMA_CALJA
93.2 A Somatotropin P01242 SOM2_HUMAN
90.5 A Somatotropin P58343 SOMA_SAIBB