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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.
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