An analytical investigation on tachographic gait records to explore the factors discriminating handicapped gait from normal gait

Abstract

This chapter focuses on ΔΔG°(WT-mut) for BPTI hydrophobic core mutants measurement by hydrogen isotope exchange. In a study described in the chapter, wild-type BPTI was purified by gel filtration on Sephadex G-25 in 20 mM potassium phosphate buffer, pH 7.0, with 0.02% NaN3, filtered through Millipore Millex GV, lyophilized and stored at 4°C. The BPTI mutants were produced from the expression system. Protein samples were dialyzed against distilled water at the same pH of the NMR experiment, filtered and lyophilized. 4-5 mM of samples were adjusted to pH 3.50 ± 0.02 on ice or at room temperature; no correction of isotope effect on pH is made. Nuclear magnetic resonance was performed on a GE 500 MHz spectrometer. Sequential assignment strategy and spin system identification were utilized for resonance assignments in the spectra of the mutants. The temperature dependence of the exchange rate constants for most of the protons in F22A, Y23A, and WT were measured at pH 3.5, and apparent activation energies were estimated. The amide protons of residue Tyr 21, Phe 22, and Tyr 23 have the highest activation energies among all observed -NH protons. They are about 80, 69, and 52 kcal/mol for WT, F22A, and Y23A, respectively.