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

Mokhtari

24 May 2011

The analysis of conventional spectroscopic data, such as fluorescence or CD data, can not determine which residues are responsible for the change of stability.

pH conformation of proteins structure and function

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.

Experimental design.

A number of pH-induced structural transitions as well as changes in the association state and stability, between pH 2 and 8.

pKa,M-Q = 3pKa,Q-N = 5pKa,N-R = 7

a four-state mechanism

A monomeric form with a high stability at acidic pH

A monomeric form with a high stability at acidic pH

Dimerization with little alteration in structure at around pH=3

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)

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)

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)

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.

Gln-5Gln-5

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.

Individual residues show their own transitions, whose midpoints do not necessarily converge to common pKa values, 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.

Individual residues show their own transitions, whose midpoints do not necessarily converge to common pKa values, 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.

SVDSVD

3dominant PCs3dominant 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 S⇄ 2 S⇄ 3 S⇄ 4

The relations between these species :

acid dissociation

constantfractions of species i, fSi , as a function of pH

PCs described with the fractions for each species :

3-dimensional vector containing the first, second, and third PCs

a 3-dimensional vector describing the corresponding species

For NMR chemical shifts, this means the fast exchange between different conformational states.

PCA assumed a linear combination of the basis spectra.

Thanks