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PDBsum entry 2p81
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Transcription
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PDB id
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2p81
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References listed in PDB file
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Key reference
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Title
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The helix-Turn-Helix motif as an ultrafast independently folding domain: the pathway of folding of engrailed homeodomain.
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Authors
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T.L.Religa,
C.M.Johnson,
D.M.Vu,
S.H.Brewer,
R.B.Dyer,
A.R.Fersht.
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Ref.
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Proc Natl Acad Sci U S A, 2007,
104,
9272-9277.
[DOI no: ]
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PubMed id
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Abstract
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Helices 2 and 3 of Engrailed homeodomain (EnHD) form a helix-turn-helix (HTH)
motif. This common motif is believed not to fold independently, which is the
characteristic feature of a motif rather than a domain. But we found that the
EnHD HTH motif is monomeric and folded in solution, having essentially the same
structure as in full-length protein. It had a sigmoidal thermal denaturation
transition. Both native backbone and local tertiary interactions were formed
concurrently at 4 x 10(5) s(-1) at 25 degrees C, monitored by IR and
fluorescence T-jump kinetics, respectively, the same rate constant as for the
fast phase in the folding of EnHD. The HTH motif, thus, is an ultrafast-folding,
natural protein domain. Its independent stability and appropriate folding
kinetics account for the stepwise folding of EnHD, satisfy fully the criteria
for an on-pathway intermediate, and explain the changes in mechanism of folding
across the homeodomain family. Experiments on mutated and engineered fragments
of the parent protein with different probes allowed the assignment of the
observed kinetic phases to specific events to show that EnHD is not an example
of one-state downhill folding.
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Figure 1.
Fig. 1. Comparison of chemical shifts between EnHD L16A and
its 16–59 fragment. (a) The ^1H-^15N HSQC spectra of EnHD
residues 16–59 (red) overlayed with that of L16A (blue). Peaks
for L16A residues up to 15 are shown as black contours. (b) The
natural abundance ^1H-^13C HSQC spectra of EnHD residues 16–59
(red) overlayed with that of L16A (blue). The methyl groups in
the fragment were not stereospecifically assigned.
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Figure 4.
Fig. 4. Relaxation kinetics of EnHD residues 16–59
monitored by fluorescence (a, b, and f) and IR (c and d) and
equilibrium properties (e). (a) Kinetic time course for EnHD
residues 16–59 at 25°C, 2 M [NaCl]. The raw data (red)
were fitted after subtracting from the NATA trace (blue), giving
the rate constant of 206,000 s^–1 (4.8 µs). (b) Kinetic
time course for EnHD L16A at 25°C, 2 M [NaCl]. As for the
fragment, the raw data were subtracted from the NATA trace and
fitted to both single (gray) and double (brown) exponentials.
The single-exponential fit gave a rate of 44,000 s^–1, and the
double-exponential fit produced rates of 34,000 s^–1 and
205,000 s^–1. (c) Folding of EnHD L16A (red) and 16–59
fragment (blue), monitored by IR at 25°C, 100 mM [NaCl]. (d)
As in c, but measured at 500 mM [NaCl]. Two kinetic phases were
observed in the folding of L16A and 16–59 fragment at low salt
concentrations when monitored by IR: the fast phase and the
(previously unidentified) ultrafast phase. In addition to these
phases, a slow phase was observed in the folding kinetics of
L16A at 500 mM. No slow phase was observed in the folding
kinetics of the fragment at high salt, as shown in Table 1. (e)
Thermal denaturation of the fragment at increasing salt
concentrations, followed by CD at 222 nm. (f) Rates extracted
from independent traces at multiple temperatures for the
fragment (red) and the L16A mutant (blue/gray) at 2 M [NaCl].
The spread in the data shows the inaccuracy of the measurement
and/or data fitting. The L16A data were fitted to a single and a
double exponential. At higher temperatures, it was not possible
to fit the L16A data to a double exponential.
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