Spin wave diffraction and perfect imaging of a grating.

We study the diffraction of Damon-Eshbach-type spin waves incident on a one-dimensional grating realized by microslits in a thin Permalloy film. By means of time-resolved scanning Kerr microscopy, we observe unique diffraction patterns behind the grating which exhibit replications of the spin wave field at the slits. We show that these spin wave images, with details finer than the wavelength of the incident Damon-Eshbach spin wavelength, arise from the strongly anisotropic spin wave dispersion.

Crystal structure of YecO from Haemophilus influenzae (HI0319) reveals a methyltransferase fold and a bound S-adenosylhomocysteine.

The crystal structure of YecO from Haemophilus influenzae (HI0319), a protein annotated in the sequence databases as hypothetical, and that has not been assigned a function, has been determined at 2.2-A resolution. The structure reveals a fold typical of S-adenosyl-L-methionine-dependent (AdoMet) methyltransferase enzymes. Moreover, a processed cofactor, S-adenosyl-L-homocysteine (AdoHcy), is bound to the enzyme, further confirming the biochemical function of HI0319 and its sequence family members. An active site arginine, shielded from bulk solvent, interacts with an anion, possibly a chloride ion, which in turn interacts with the sulfur atom of AdoHcy. The AdoHcy and nearby protein residues delineate a small solvent-excluded substrate binding cavity of 162 A(3) in volume. The environment surrounding the cavity indicates that the substrate molecule contains a hydrophobic moiety and an anionic group. Many of the residues that define the cavity are invariant in the HI0319 sequence family but are not conserved in other methyltransferases. Therefore, the substrate specificity of YecO enzymes is unique and differs from the substrate specificity of all other methyltransferases sequenced to date. Examination of the Enzyme Commission list of methyltransferases prompted a manual inspection of 10 possible substrates using computer graphics and suggested that the ortho-substituted benzoic acids fit best in the active site.

Stability and global fold of the mouse prohormone convertase 1 pro-domain.

We have purified the mouse prohormone convertase 1 (PC1) pro-domain expressed in Escherichia coli cells and demonstrated, using a number of biophysical methods, that this domain is an independent folding unit with a T(m) of 39 degrees C at a protein concentration of 20 microM and pH 7.0. This differs significantly from similar pro-domains in bacteria and human furin, which are unfolded at 25 degrees C and require the catalytic domain in order to be structured [Bryan et al. (1995) Biochemistry 34, 10310-10318; Bhattacharjya et al. (2000) J. Biomol. NMR 16, 275-276]. Using heteronuclear NMR spectroscopy, we have determined the backbone (1)H, (13)C, and (15)N assignments for the pro-domain of PC1. On the basis of (1)H/(13)C chemical shift indices, NOE analysis, and hydrogen exchange measurements, the pro-domain is shown to consist of a four-stranded beta-sheet and two alpha-helices. The results presented here show that both the bacterial pro-domain in complex with subtilisin and the uncomplexed mouse PC1 pro-domain have very similar overall folds despite a lack of sequence homology. The structural data help to explain the location of the secondary processing sites in the pro-domains of the PC family, and a consensus sequence for binding to the catalytic domain is proposed.

Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response in Nicotiana sylvestris.

The hypersensitivity resistance response directed by the N' gene in Nicotiana sylvestris is elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild-type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild-type coat proteins were compared for variations that may explain N' gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and nonelicitor coat proteins. Analytical ultracentrifugation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4 degrees C. Viral reconstitution kinetics at 20 degrees C were used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results reveal a decreased lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences indicating that the mechanism of viral assembly is similar for the two coat protein species. Therefore, an increased rate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20 degrees C using velocity sedimentation analysis. These results are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elicitors that allows N. sylvestris to recognize the invading virus.

New revolutions in the evolution of analytical ultracentrifugation.

The use of the biophysical technique of analytical ultracentrifugation has recently undergone a resurgence. The commercial availability of the Beckman optima XL-A and XL-I analytical ultracentrifuges along with the continued growth in computing ability and analysis software has led to the expanded use of analytical ultracentrifugation and its capabilities. The genetic revolution and the search for further understanding of macromolecular interactions have again brought analytical ultracentrifugation to the forefront of macromolecular characterization.