Sakurai K , Goto Y PNAS 2007;104:15346-15351

1. 24 May 2011 Principal Component Analysis of the pH-dependent Conformational Transitions of Bovine β-lactoglobulin Monitored by Heteronuclear NMR Zeinab Mokhtari Sakurai K , Goto Y PNAS 2007;104:15346-15351

2. Introduction

3. Introduction

4. Introduction

5. pH-dependent conformational stability of proteins Introduction 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.

6. Introduction 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.

7. Introduction A monomeric form with a high stability at acidic pH A number of pH-induced structural transitions as well as changes in the associationstate and stability, between pH 2 and 8. Experimental design. a four-state mechanism pKa,M-Q = 3 pKa,Q-N = 5 pKa,N-R = 7 Dimerization with little alteration in structure at around pH=3 conversion from the acidic Q state to the native (N) dimeric state between pH 4.5 and 6 (changes in compactness) Tanford transition : a conformational change of the EF loop (residues 85–90), which might be caused by the cleavage of hydrogen bonds between the F and G strands (pH=7)

8. Procedure • 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

9. superposition of the HSQC spectra obtained at various pH values 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.

10. Hue plotpH titration monitored by HSQC spectrum Individual residues show their own transitions, whose midpoints do not necessarily converge to common pKavalues, indicating that the four-state transition is not a result of highly cooperative transitions throughout the molecule. The pH-dependent conformational change of β-LG might be a result of collective conformational changes of many residues. From pH 2 to pH 5, the signal of Gln-5 moves toward the top right of the spectrum whereas, from pH 5 to pH 8, it moves downwards. Gln-5

11. PCA of pH Titration Data SVD

12. PCA of pH Titration Data 3dominant PCs

13. Fitting of PCs PCA of pH Titration Data 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.

14. PCA of pH Titration Data 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

15. Reconstructed four basis spectra PCA of pH Titration Data

16. PCA of pH Titration Data PCA assumed a linear combination of the basis spectra. For NMR chemical shifts, this means the fast exchange between different conformational states.

17. Thanks