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Solution structures of mutant Zn fingers containing aromatic substitutions in
the hydrophobic core are determined by 2D-NMR spectroscopy and
distance-geometry/simulated annealing (DG/SA). The wild-type domain (designated
ZFY-6) is derived from the human male-associated protein ZFY and represents a
sequence motif (Cys-X2-Cys-X-Ar-X7-Leu-X2-His-X4-His) that differs from the
consensus (Cys-X2,4-Cys-X3-Phe-X5-Leu-X2-His-X3-His) in the location ("aromatic
swap") and diversity (Ar = tyrosine, phenylalanine, or histidine) of the central
aromatic residue (underlined). In a given ZFY domain the choice of a particular
aromatic residue is invariant among vertebrates, suggesting that alternative
"swapped" aromatic residues are functionally inequivalent. 2D-NMR studies of
analogues containing tyrosine, phenylalanine, or histidine at the swapped site
yield the following results. (i) The three DG/SA structures each retain the beta
beta alpha motif and exhibit similar staggered-horizontal packing between the
variant aromatic residue and the proximal histidine in the hydrophobic core.
(ii) The structures and stabilities of the tyrosine and phenylalanine analogues
are essentially identical, differing only by local exposure of polar (Tyr p-OH)
or nonpolar (Phe p-H) surfaces. (iii) The dynamic stability of the histidine
analogue is reduced as indicated by more rapid protein-deuterium exchange of
hydrogen bonds related to secondary structure and amide-sulfur coordination
(slowly exchanging amide resonances in D2O) and by more extensive averaging of
main-chain dihedral angles (3J alpha NH coupling constants). An aspartic acid in
the putative DNA recognition surface, whose configuration is well-defined as a
possible helix N-cap in the tyrosine and phenylalanine analogues, exhibits
multiple weak main-chain contacts in the NOESY spectrum of the histidine
analogue; such NOEs are geometrically inconsistent and so provide complementary
evidence for structural fluctuations. (iv) Because the three DG ensembles have
similar apparent precision, the finding of reduced dynamic stability in the
histidine analogue emphasizes the importance of experiments that directly probe
fluctuations at several time scales. Our results provide insight into the design
of biological metal-binding sites and the relationship of protein sequence to
structure and dynamics.
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