pKa values were measured for the 6 carboxylates in the N-terminal domain of L9 (NTL9) by following NMR chemical shifts as a function of pH. The contribution of each carboxylate to the pH dependent stability of NTL9 was estimated by comparing the pKa values for the native and denatured state of the protein. A set of peptides with sequences derived from NTL9 were used to model the denatured state. In the protein fragments, the pKa values measured for the aspartates varied between 3.8 and 4.1 and the pKa values measured for the glutamates varied between 4.1 and 4.6. These results indicate that the local sequence can significantly influence pKa values in the denatured state and highlight the difficulties in using standard pKa values derived from small compounds. Calculations based on the measured pKa values suggest that the free energy of unfolding of NTL9 should decrease by 4.4 kcal mol-1 when the pH is lowered from 6 to 2. In contrast, urea and thermal denaturation experiments indicate that the stability of the protein decreases by only 2.6 kcal mol-1 when the carboxylates are protonated. This discrepancy indicates that the protein fragments are not a complete representation of the denatured state and that nonlocal sequence effects perturb the pKa's in the denatured state. Increasing the salt concentration from 100 to 750 mM NaCl removes the discrepancy between the stabilities derived from denaturation experiments and the stability changes calculated from the pKa values. At high concentrations of salt there is also less variation of the pKa values measured in the protein fragments. Our results argue that in the denatured state of NTL9 there are electrostatic interactions between groups both local and nonlocal in primary sequence. Study holds ProTherm entries: 5766, 5767, 5768, 5769, 5770, 5771, 5772, 5773, 5774, 5775, 5776, 5777 Extra Details: local and nonlocal interactions; carboxylates; local sequence;,free energies of unfolding; electrostatic interactions
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:30 p.m.
|Number of data points||12|
|Proteins||50S ribosomal protein L9 ; 50S ribosomal protein L9|
|Assays/Quantities/Protocols||Experimental Assay: dG ionic:NaCl: 750 mM, pH:7.26 ; Experimental Assay: dG ionic:NaCl: 750 mM, pH:6.56 ; Experimental Assay: dG ionic:NaCl: 750 mM, pH:5.25 ; Experimental Assay: dG ionic:NaCl: 750 mM, pH:4.14 ; Experimental Assay: dG ionic:NaCl: 750 mM, pH:3.15 ; Experimental Assay: dG pH:2.4, ionic:NaCl: 750 mM ; Experimental Assay: dG ionic:NaCl: 100 mM, pH:6.98 ; Experimental Assay: dG ionic:NaCl: 100 mM, pH:5.99 ; Experimental Assay: dG ionic:NaCl: 100 mM, pH:4.51 ; Experimental Assay: dG ionic:NaCl: 100 mM, pH:3.55 ; Experimental Assay: dG pH:2.79, ionic:NaCl: 100 mM ; Experimental Assay: dG pH:2.04, ionic:NaCl: 100 mM|
|Libraries||Mutations for sequence MKVIFLKDVKGKGKKGEIKNVADGYANNFLFKQGLAIEATPANLKALEAQKQKEQRQAAEELANAKKLKEQLEKLTVTIPAKAGEGGRLFGSITSKQIAESLQAQHGLKLDKRKIELADAIRALGYTNVPVKLHPEVTATLKVHVTEQK|