th  - 1988 - 51st ENC Conference

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THREE-DIMENSIONAL STRUCTURE OF TURKEY OVOMUCOID THIRD DOMAIN BY 2D-
136 J NMR SPECTROSCOPY, AND DISTANCE GEOMETRY CALCULATIONS. Prashanth
Darba , Andrzej grezel, ~asna Fejzo, S. Macura, Andrew D. Robertson
and 3ohn L. Markley, National Magnetic Resonance Facility at Madison and Department of
Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-
Madison, Madison, WI 53706.
Turkey ovomucoid third domain (OMTKY3) is a small 56-residue protein that inhibits
serine proteinases. Previous studies [1,2] based on chemical shift differences have
revealed that subtle changes in the tertiary structure occur near and remote from the
cleavage site upon hydrolyzing the reactive-site peptide bond. Recently 247 inter-
proton distances were determined from a series of 2D-NMR experiments. Some of the
distances were estimated crudely from the number of contour levels of well resolved
peaks in a single NOESY spectrum; others were determined accurately from a set of
NOESY/ROESY experiments obtained with 7 different mixing times. [3]
These NMR distance constr'aints were used in two different distance geometry
programs, DG900 [4] based on a metric matrix approach and DISMAN [5] which uses a
variable target function, to determine the solution structure of OMTKY3. A comparison
of the structures calculated from these methods with the X-ray structure of OMTKY3 [6]
will be presented. Implications of crude versus accurate distances on the structure
determination and convergence properties of the two distance geometry programs will be
discussed.
[1] A.D. Robertson, W.M. Westler, and 3.L. Markley, Biochemistry, in press. [2] G.I.
Rhyu and J.L. Markley, Biochemistry, in press. [3] 3. Fejzo, S. Macnra, and 3.L.
Markley, unpublished data. [4] 3. Thomason, M. Day, and I.D. Kuntz, nA Vectorized
Distance Geometry Program, n in prep. [5] W. Braun and N. Go, 3. Mol. Biol. 186, 611-
626 (1985). [6] R.$. Read, M. Fujinaga, A.R. Sielecki, and M.N.G. James, Biochemistry
22, 4420-4433 (1983).
[Supported by: NTH Grants RR 02301 and GM 35976 and the University of Wisconsin-
Madison.]
I
-- TWO-DIb~ENSIONAL xIC(XSN}, x3C{x3C} AND XH{x3C} CHEMICAL SHIFT
137 I CORRELATION IN PROTEINS: SI~UENCE-SPECIFIC ASSIGN~[ENT OF
RESONANCES IN xsC AND XSN LABELED S.TREPTOMYCES SUBTILISIN INHIBITOR. William M.
Westler, *~ M. Kainosho, # H. Nagao, # N. Tomonaga,# and 3ohn L. Markley ~. ~Department
of Biochemistry, College of A~ricnltural and Life Sciences, University of Wisconsin-
Madison, Madison, WI 53706. ~Department of Chemistry, Faculty of Science, Tokyo
Metropolitan University, Fnkazawa, Setagaya-ku, Tokyo, 158 Japan.
Applications of heteronuclear two-dimensional NMR to proteins and other
macromolecules are proving to be extremely useful for resonance assignments in large
proteins. Here we demonstrate the use of two-dimensional shift correlation methods
between ssC and ISN, ssC and IsC, and aH and ssC for determinin 8 sequence-specific
assignments in Streptomyces snbtilisin inhibitor (SSI). Two-dimensional ssC{SSN}
correlation spectroscopy is used to detect coupling between selectively ssC labeled
methionine ssC o resonances at position i in the protein sequence and the SSN labeled
amide peak of the next sequential residue (i+1) in [ul 60% SSN, 99% a3C o methionine]
SSI. The known assignments of the methionine xsC o peaks allow sequence-specific
assignment of the XSN a peaks of the i+1 amino acid residue We also report here a
novel method for sequence-specific assignments of carbon and proton resonances in xsC
labeled proteins by the use of 3 two-dimensional methods: xsC[s3C} double-quantum
correlation, SB(ssC} one-bond chemical shift correlation, and SH{ssC] multiple-bond
correlation. By these methods, the NMR spin system assignments of the nine lencine
residues in [85% ul s3C]leucine SSl are extended from the previously assigned
carbonyl carbons to the intraresidne alpha carbons, alpha protons, and to the alpha
protons of the next sequential amino acid. [Supported by Grants in Aid from the
Ministry of Education of Japan (60430033, 60880022, 62220026), NIH grants RR02301 and
RR02781, NSF Grant PLM-845048, the U. S. Department of Agriculture, and the
University of Wisconsin.]
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