Equilibrium denaturation experiments were performed on human growth hormone (hGH) under acidic conditions (pH 1.5-3.0) and different protein concentrations. At 0.1 mg/ml hGH using intrinsic tryptophan fluorescence and far-UV circular dichroism (CD) detection, midpoint values of 4.6 M GdnHCl were observed that are identical to those obtained at neutral pH. However, the delta G values were reduced at pH 2.5 relative to pH 8.0 (10.5 vs. 15 kcal/mol). Increasing the protein concentration to 1 mg/ml resulted in a biphasic denaturation profile by far-UV CD detection at 222 nm, while near-UV CD measurements at 295 nm yielded a cooperative transition with a midpoint value of 3.6 M GdnHCl. These results indicate that equilibrium intermediates having a propensity to aggregate are highly populated under acid conditions. Static light scattering measurements performed under partial unfolding conditions (4.5 M GdnHCl) at pH 2.5 confirmed the existence of a large molecular weight (congruent to 80 kDa) self-associated intermediate. No evidence of aggregation was found for hGH under acid conditions in the absence of denaturant, indicating that self-association results from the formation of an intermediate. Equilibrium GdnHCl concentration-jump experiments confirmed that association only occurs from an intermediate species and not from any other conformational state, and formation of the self-associated intermediate can lead to irreversible loss of protein due to precipitation. These results demonstrate that acid stabilizes equilibrium folding intermediates of hGH. Study holds ProTherm entries: 9361, 9362, 9363, 9364, 9365, 9366, 9367, 9368, 9369 Extra Details: pH; denaturation; intermediates; protein folding;,self-association; growth hormone; (human)
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:37 p.m.
|Number of data points||18|
|Proteins||Growth hormone receptor ; Somatotropin|
|Assays/Quantities/Protocols||Experimental Assay: Cm pH:8.0 ; Experimental Assay: dG_H2O pH:8.0 ; Experimental Assay: Cm pH:8.0 ; Experimental Assay: dG_H2O pH:8.0 ; Experimental Assay: Cm pH:7.5 ; Experimental Assay: dG_H2O pH:7.5 ; Experimental Assay: Cm pH:3.0 ; Experimental Assay: dG_H2O pH:3.0 ; Experimental Assay: Cm pH:2.0 ; Experimental Assay: dG_H2O pH:2.0 ; Experimental Assay: Cm pH:1.5 ; Experimental Assay: dG_H2O pH:1.5 ; Experimental Assay: Cm pH:2.5 ; Experimental Assay: dG_H2O pH:2.5 ; Experimental Assay: Cm pH:2.5 ; Experimental Assay: dG_H2O pH:2.5|
|Libraries||Mutations for sequence A:FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCG/B:EPKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQLFYTRRNTQEWTQEWKECPDYVSAGENSCYFNSSFTSIWIPYCIKLTSNGGTVDEKCFSVDEIVQPDPPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWKMMDPILTTSVPVYSLKVDKEYEVRVRSKQRNSGNYGEFSEVLYVTLPQM/C:EPKFTKCRSPERETFSCHWTDEVHHGTKNLGPIQLFYTRRNTQEWTQEWKECPDYVSAGENSCYFNSSFTSIWIPYCIKLTSNGGTVDEKCFSVDEIVQPDPPIALNWTLLNVSLTGIHADIQVRWEAPRNADIQKGWMVLEYELQYKEVNETKWKMMDPILTTSVPVYSLKVDKEYEVRVRSKQRNSGNYGEFSEVLYVTLPQM|