It is now recognized that unfolded states of globular proteins are not random coils but instead can contain significant amounts of residual structure. Here, we combine amide H/D exchange studies and thermodynamic measurements to probe pH dependent structure in the unfolded state of the small, mixed alpha-beta protein CTL9. The m value measured by urea denaturation is strongly dependent upon pD, increasing by 40% from pD 7.5 to 4.85. Likewise, the change in heat capacity upon unfolding, deltaCp(o), increases significantly from pD 7.5 to 5.5. These studies argue that the unfolded state contains interactions, presumably hydrophobic in nature, that lead to a more compact state at high pH. The expansion at lower pH correlates with the estimated unfolded state pKa values of the three histidines in CTL9 with additional contributions from acid side chains at the lower pH. Amide H/D exchange studies were conducted at pD 5.0, 6.0, and 7.0. At pD 5.0, the exchange rates could be measured for 44 residues, 29 of which exchanged by global unfolding. No evidence was found for any super protected sites, that is, sites that exchange at rates slower than those expected for global exchange. The estimated precision for the experiments limits detection to residues that are protected 2.3-fold above the intrinsic exchange rate. Thirty-seven residues could be followed at pD 6 and 27 residues at pD 7. Again no evidence for a significant super protected structure was observed. The properties of CTL9(11) are compared to other structured denatured states. Study holds ProTherm entries: 20112, 20113, 20114, 20115 Extra Details: C-Terminal Domain globular proteins, thermodynamic measurements, pH dependent, urea denaturation, hydrophobic, unfolded state.
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:52 p.m.
|Number of data points||9|
|Proteins||50S ribosomal protein L9 ; 50S ribosomal protein L9|
|Assays/Quantities/Protocols||Experimental Assay: dTm ; Experimental Assay: dG pH:8.0 ; Experimental Assay: dG pH:5.45, temp:25.0 C ; Experimental Assay: dCp prot_conc:8-12 microM, pH:8.0 ; Experimental Assay: Tm prot_conc:8-12 microM, pH:8.0 ; Experimental Assay: dHvH prot_conc:8-12 microM, pH:8.0 ; Experimental Assay: dCp prot_conc:8-12 mM, pH:5.45 ; Experimental Assay: Tm prot_conc:8-12 mM, pH:5.45 ; Experimental Assay: dHvH prot_conc:8-12 mM, pH:5.45|
|Libraries||Mutations for sequence MKVIFLKDVKGKGKKGEIKNVADGYANNFLFKQGLAIEATPANLKALEAQKQKEQRQAAEELANAKKLKEQLEKLTVTIPAKAGEGGRLFGSITSKQIAESLQAQHGLKLDKRKIELADAIRALGYTNVPVKLHPEVTATLKVHVTEQK|