 | Figure 3
Nature Structural Biology
9, 425 - 430 (2002)
Published online: 29 April 2002; | doi:10.1038/nsb798
Designing a 20-residue proteinJonathan W. Neidigh, R. Matthew Fesinmeyer
& Niels H. Andersen | | | | Figure 3. NMR spectra and the structure derived for TC5b.
a, Stereo view of the NMR ensemble (38 of 50
structures for TC5b in pH 7 aqueous buffer (Table 1)).
All atoms are displayed for Tyr 22 (orange), Trp 25 (magenta), Leu 26 (cyan),
Pro 31 (dark red), Pro 36 (black), Pro 37 (green) and Pro 38 (blue). For the
remaining residues, only the backbone is displayed. The heavy-atom pairwise
r.m.s. deviation over the key residues in the Trp cage (Tyr 22, Trp 25, Gly 30,
Pro 31, Pro 37 and Pro 38) is 0.46  0.15 Å. The annotated NOESY
segments b, with added TFE and c, without TFE illustrate the
diagnostic long-range NOEs. This is the same color scheme used in (a),
with Leu 21, Gln 24, Gly 30 and Arg 35 also shown in black. In (b), the
unlabeled line at 7.36 p.p.m. is Ile 23-HN. The key long-range NOEs of TC5b
(for example, 22- to 38- , 22- to 38-2, 22-
to 37- 2, 25-1 to 35-/36-/37-, 25-1 to
35-/38-2, 25- 2 to 31-3 and 25- 2 to
31-/37-2-3) were observed in both media (the
22-/38- NOE does not appear in (c)). Trp
25-H1/H3 and H2/H2 are nearly and completely shift
coincident, respectively, in aqueous buffer. Long-range NOEs to these indole
ring resonances are attributed to individual hydrogen sites based on their
occurrence in other Trp-cage constructs under conditions where the resonances
are not shift coincident. d, TC5b is in a temperature dependent
equilibrium with an 'unfolded state' that does not display random coil shifts
because of residual local hydrophobic cluster formation. The increasingly
negative CSDs of 31-3 and 30-3 are rationalized because the
'residual' high temperature hydrophobic cluster between Trp 25 and Pro 31
places these two protons further into the shielding region than their location
in the unmelted Trp cage.
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