The Trp37 side chain resides in a well-structured environment.
When located in highly asymmetric (i.e., well-structured)
environments, side chains of aromatic residues exhibit CD signals in
overlapping regions of the near-UV (Phe, 250-270 nm; Tyr, 270-290 nm;
Trp, 280-300
nm).38 As shown
in Figure 2B , b5 displays signals in the near-UV region (250-
350 nm) attributable to the fact that it contains 2 Phe, 4 Tyr, and 2
Trp residues. Comparatively stronger signals, with significant fine
structure, appear in the near-UV CD spectrum of N/b5 (Figure
2B ). The N-terminal region contains only two aromatic residues, Phe9
and Trp37. Given that the strongest feature in the near-UV CD signature
of N/b5 appears at 286 nm, we conclude that the major contributor is
Trp37, which is part of the invariant mammalian Ncb5or
L34MDWIRL40 sequence noted above. In
contrast, the corresponding spectrum of N-term exhibits no such signal
at 286 nm (Figure 2D ).
Aromatic residues in well-ordered regions of proteins often contribute
to far-UV CD spectra as well. In the case of Trp, positive bands in the
225-250 nm region are a particularly common
feature.39,
40 Trp37 is therefore a likely source
of the positive feature in the difference CD spectrum of N-term centered
at 230 nm (Figure 2C ), another conjecture which is supported by
additional CD data described below. Notably, N-term exhibited no signals
in the near-UV region (Figure 2D ) and displays negative
ellipticity in the vicinity of 230 nm (Figure 2C ). The results
described in this section allow us to conclude that interactions between
the N-terminal region and the b5 domain induce secondary structure in
the former, and that this structural transition is accompanied by
adoption of tertiary structure involving Trp37. This conclusion was
further tested with truncation and point mutants of N/b5.