ELUCIDATION OF THE ORIGIN OF MULTIPLE CONFORMATIONS OF THE HUMAN ALPHA-3-CHAIN TYPE-VI COLLAGEN C-TERMINAL KUNITZ DOMAIN - THE REORIENTATION OF THE TRP(21) RING

The human alpha 3-chain type VI collagen C-terminal Kunitz domain frag ment (alpha 3(VI)) has been studied by two-dimensional H-1-H-1 and H-1 -C-13 NMR spectroscopy at 303 K. It is shown that the secondary struct ure of the protein is strikingly similar to that of BPTI, and that a n umber of unusual H-alpha chemical shifts, which are highly conserved i n Kunitz-domain proteins, are also observed for alpha 3(VI). Furthermo re, a series of exchange cross peaks observed in H-1-H-1 spectra shows that a large number of protons in the central beta-sheet exist in two different chemical environments, corresponding to two unequally popul ated conformations that are slowly exchanging on the NMR time scale. S everal protons, including Ser(47(53)) H-alpha, Arg(32(38)) H-gamma 1 a nd H-gamma 2, and Gln(48(54)) H-beta 2, all located in the vicinity of the Trp(21(27)) ring in the crystal structure of alpha 3(VI) [Arnoux, B. et al. (1995) J. Mol. Biol., 246, 609-617], have very different ch emical shifts in the two conformations, the most affected being Gln(48 (54)) H-beta 2 (Delta delta = 1.53 ppm), which is placed directly abov e the Trp(21(27)) ring in the crystal structure of alpha 3(VI). It is concluded that the origin of the multiple conformations of the central P-sheet is a reorientation of the Trp(21(27)) ring. From the intensit ies of corresponding signals in the two conformations, the population of the minor conformation was found to be 6.4 +/- 0.2% of that of the major conformation, while a rate constant k(M) = 1.01 +/- 0.05 s(-1) f or the major to minor interconversion was obtained from a series of NO ESY spectra with different mixing times. In addition, it is shown that Cys(14(20))-Cys(38(44)) disulfide bond isomerization, previously obse rved in BPTI [Otting, G. et al. (1993) Biochemistry, 32, 3571-3582], i s also likely to occur in alpha 3(VI).