Transcript ppt
24 May 2011 Zeinab Mokhtari Sakurai K , Goto Y PNAS 2007;104:15346-15351 pH conformation of proteins structure and function The analysis of conventional spectroscopic data, such as fluorescence or CD data, can not determine which residues are responsible for the change of stability. Heteronuclear NMR spectra, such as the heteronuclear sequential quantum correlation (HSQC) spectrum, monitoring the behavior of essentially all residues, has the potential to address the contributions of individual residues. Bovine-lactoglobulin (β-LG) : consists of 162 amino acid residues (18 kDa) and contains two tryptophan residues, Trp-19 and Trp-61 Predominantly β-sheet protein consisting of nine β-strands (A–I), of which the A–H strands form an up-and-down β-barrel, and one major α-helix at the C terminus of the molecule. A number of pH-induced A monomeric form with a structural transitions as well as changes in stability the association state high at acidic pH and stability, between pH 2 and 8. Experimental design. a four-state mechanism pKa,M-Q = 3 Dimerization with little conversion from the acidic Q pKa,Q-N = 5 alteration in structurestate at to the native (N) dimeric transition : a conformational around pH=3 Tanford state between pH 4.5 and 6 change of the EF loop (residues 85–90), pKa,N-R = 7 (changes in compactness) which might be caused by the cleavage of hydrogen bonds between the F and G strands (pH=7) pH titration and hydrogen/deuterium (H/D) exchange experiments monitored by HSQC to relate the pH-dependent stability with the conformational behavior at the residue level PCA to correlate pH-dependent HSQC spectra with pH-dependent conformational transitions HSQC spectra at pH 2.4–8.1 to examine the four-state conformational transitions It is evident that chemical shifts of many signals change with pH. For these residues, we observed no evident change of peak intensity, suggesting the fast exchange between conformational states. On the other hand, some residues showed a decrease in peak intensity above pH 6 without changing the chemical shift, suggesting a contribution of slow conformational change. Individual residues show their own transitions, whose midpoints do From pH 2 to pH 5, the not necessarily converge to common pKa values, indicating that signal of Gln-5 moves the four-state transition result towardisthenot top aright of theof highly cooperative transitions throughoutspectrum the molecule. whereas, from pH 5 to pH 8, it moves downwards. The pH-dependent conformational change of β-LG might be a result of collective conformational changes of many residues. Gln-5 SVD 3dominant PCs Although we also performed the following fitting with the first four PCs, no apparent improvement was detected, consistent with the profile that the amplitude of PC4 was small over the pH range studied. S1⇄S2⇄S3⇄S4 The relations between these species : acid dissociation constant fractions of species i, fSi , as a function of pH a 3-dimensional vector describing the corresponding species PCs described with the fractions for each species : 3-dimensional vector containing the first, second, and third PCs PCA assumed a linear combination of the basis spectra. For NMR chemical shifts, this means the fast exchange between different conformational states. Thanks