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PDBsum entry 1k5r
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Signaling protein
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PDB id
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1k5r
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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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Using flexible loop mimetics to extend phi-Value analysis to secondary structure interactions.
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Authors
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N.Ferguson,
J.R.Pires,
F.Toepert,
C.M.Johnson,
Y.P.Pan,
R.Volkmer-Engert,
J.Schneider-Mergener,
V.Daggett,
H.Oschkinat,
A.Fersht.
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Ref.
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Proc Natl Acad Sci U S A, 2001,
98,
13008-13013.
[DOI no: ]
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PubMed id
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Abstract
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Chemical synthesis allows the incorporation of nonnatural amino acids into
proteins that may provide previously untried probes of their folding pathway and
thermodynamic stability. We have used a flexible thioether linker as a loop
mimetic in the human yes kinase-associated protein (YAP 65) WW domain, a
three-stranded, 44-residue, beta-sheet protein. This linkage avoids problems of
incorporating sequences that constrain loops to the extent that they
significantly change the nature of the denatured state with concomitant effects
on the folding kinetics. An NMR solution structure shows that the thioether
linker had little effect on the global fold of the domain, although the loop is
apparently more dynamic. The thioether variants are destabilized by up to 1.4
kcal/mol (1 cal = 4.18 J). Preliminary Phi-value analysis showed that the first
loop is highly structured in the folding transition state, and the second loop
is essentially unstructured. These data are consistent with results from
simulated unfolding and detailed protein-engineering studies of structurally
homologous WW domains. Previously, Phi-value analysis was limited to studying
side-chain interactions. The linkers used here extend the protein engineering
method directly to secondary-structure interactions.
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Figure 1.
Fig. 1. Amino acid sequence of the YAP 65 WW domain and
its thioether analogues  S24, S24G25 and
I33. In the
sequences, Z refers to the nonnatural amino acid
(2-amino-ethylsulfanyl)-acetic acid.
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Figure 6.
Fig. 6. Thermal unfolding simulations of the FBP28 WW
domain. The  -strands are
colored as follows: 1 (residues
8-12), red; 2 (res.
18-22), green; and 3 (res.
27-30), blue. This figure was made by using MIDASPLUS (38). The
transition states for unfolding are near 2 ns.
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