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PDBsum entry 1m8c
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Hydrolase inhibitor
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PDB id
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1m8c
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Two conformational states of turkey ovomucoid third domain at low ph: three-Dimensional structures, Internal dynamics, And interconversion kinetics and thermodynamics.
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Authors
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J.Song,
M.Laskowski,
M.A.Qasim,
J.L.Markley.
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Ref.
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Biochemistry, 2003,
42,
6380-6391.
[DOI no: ]
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PubMed id
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Abstract
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Turkey ovomucoid third domain (OMTKY3) is shown to exist at low pH as two
distinctly folded, interconverting conformations. Activation parameters were
determined for the transition, and these were of the type reported previously
for cis/trans isomerizations of prolyl peptide bonds. Multidimensional,
multinuclear NMR spectroscopy was used to determine the three-dimensional
structure of each of the two states of P(5)-Pro(14)Asp OMTKY3 at pH 2.5 and 25
degrees C, under conditions where the two states have equal populations with
interchange rates of 0.25 s(-1). The results showed that the two states differ
by cis/trans isomerization of the P(8)-Tyr(11)-P(7)-Pro(12) peptide bond, which
is cis in the conformer dominant at neutral pH and trans in the conformer
appearing at low pH. The major structural differences were found to be in the
region of the reactive site loop. The core of the protein, including the
antiparallel beta-sheet and a alpha-helix, is preserved in both structures. The
state with the cis peptide bond is similar to previously reported structures of
OMTKY3 determined by NMR spectroscopy and X-ray crystallography. The
cis-to-trans transition results in the relocation of the aromatic ring of
P(8)-Tyr(11), disrupts many interactions between the alpha-helix and the
reactive-site loop, and leads to more open spacing between this loop and the
alpha-helix. In addition, the configurations of two of the three disulfide
bonds, P(11)-Cys(8)- P(20)'-Cys(38), and P(3)-Cys(16)- P(17)'-Cys(35), are
altered such that the C(alpha)-C(alpha) distances for each disulfide bridge are
longer by approximately 1 A in the trans state than in the cis. Mutations at
P(1)-Leu(18), P(6)-Lys(13), and P(5)-Pro(14) influence the position of the cis
<= => trans equilibrium. In P(1)-Leu(18)Xxx OMTKY3 mutants, the trans
state is more favored by P(1)-Gly(18) than by Ala(18) or Leu(18); in
P(6)-Lys(13)Xxx OMTKY3 mutants, the trans state is more favored by P(6)-Glu(13)
and P(6)-Asp(13) than Lys(13) or His(13). Stabilization of the trans state in
P(5)-Pro(14)Xxx OMTKY3 mutants follows the series Xxx = Gly > Asp > Glu
> Ala approximately equal His > Pro. In comparing the state with the trans
peptide bond to that with the cis, the pK(a) values of P(12)-Asp(7) and
P(1)'-Glu(19) are higher and those of P(9)-Glu(10) and P(25)'-Glu(43) are lower.
The pK(a) values of other titrating groups in the molecule are similar in both
conformational states. These pK(a) changes underlie the pH dependence of the
conformational equilibrium and can be explained in part by observed structural
differences. (15)N transverse relaxation results indicate that residues
P(6)-Lys(13)-P(3)-Cys(16) in the trans state undergo a dynamic process on the
microsecond-millisecond time scale not present in the cis state.
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