A single histidine residue has been placed at either the N-terminus or the C-terminus of each of the two alpha-helices of barnase. The pKa of that histidine residue in each of the four mutants has been determined by 1H NMR. The pKas of the two residues at the C-terminus are, on average, 0.5 unit higher, and those of the residues at the N-terminus are 0.8 unit lower, than the pKa of histidines in unfolded barnase at low ionic strength. The conformational stability of the mutant proteins at different values of pH has been measured by urea denaturation. C-Terminal histidine mutants are approximately 0.6 kcal mol-1 more stable when the introduced histidine is protonated, both at low and high ionic strength. N-Terminal mutants with a protonated histidine residue are approximately 1.1 kcal mol-1 less stable at low ionic strength and 0.5 kcal mol-1 less stable at high ionic strength (1 M NaCl). The low-field 1H NMR spectra of the mutant proteins at low pH suggest that the C-terminal histidines form hydrogen bonds with the protein while the N-terminal histidines do not form the same. The perturbations of pKa and stability result from a combination of different electrostatic environments and hydrogen-bonding patterns at either ends of helices. The value of 0.6 kcal mol-1 represents a lower limit to the favorable electrostatic interaction between the alpha-helix dipole and a protonated histidine residue at the C-terminal end of the helix.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4229, 4230, 4231, 4232, 4233, 4234, 4235, 4236, 4237, 4238, 4239, 4240, 4241, 4242 Extra Details: alpha-helices; ionic strength; protonated histidine;,hydrogen bonds; electrostatic interaction; alpha-helix dipole
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:24 p.m.
|Number of data points||24|
|Proteins||Ribonuclease ; Ribonuclease|
|Assays/Quantities/Protocols||Experimental Assay: dG pH:8.9, buffers:Tris: 30 mM ; Experimental Assay: dG buffers:MES: 30 mM, pH:5.9 ; Experimental Assay: dG buffers:Sodium acetate: 30 mM, pH:4.4 ; Derived Quantity: ddG pH:8.9, buffers:Tris: 30 mM ; Derived Quantity: ddG buffers:Sodium acetate: 30 mM, pH:4.4|
|Libraries||Mutations for sequence AQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR|