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A study is conducted to compare the stabilization mechanisms of CutA1 proteins from Pyrococcus horikoshii, Thermos thermophilus, and Oryza sativa. The stabilization mechanisms are discussed from a thermodynamic view point, based on the relationship between the crystal structures of the three proteins and their unusually high denaturation temperatures.

A (super 13)C polarization technique, based on the nuclear overhauser effect in rotating solid, is applied uniformly (super 13)C and (super 15)N-labeled protiens. It is proved that in labeled protein samples containing nonlabeled additives, nuclear overhauser polarization (NOP)can eliminate the latter signals, and for this feature, NOP is superior to cross polarization (CP) in many uniformly (super 13)C labeled proteins.

The rate of monomer interchange in a ligand-bound homodimeric protein from NOESY cross peaks has been determined from the HIV protease/KNI-529 complex. It was found that the cross-peaks result from a global process, which affects the exchange of each pair of residues in similar fashion. The exchange rate strongly depended on temperature, increasing from ca. 0.2 to 6 s (super -1) as the temperature increased from 25 to 45 degrees Celsius. In addition, structural information or sequential assignments are required to distinguish dipolar from exchange cross-peaks.

Fragment-based screening using 19 F NMR ( 19 F-FS) is an efficient method for exploring seed and lead compounds for drug discovery. Here, we demonstrate the utility and merits of using 19 F-FS for methionine γ-lyase-binding fragments, together with a 19 F NMR-based competition and mutation assay, as well as enzymatic and in silico methods. 19 F NMR-based assays provided useful information on binding between 19 F-FS hit fragments and target proteins. Although the 19 F-FS and enzymatic assay were weakly correlated, they show that the 19 F-FS hit fragments contained compounds with inhibitory activity. Furthermore, we found that in silico calculations partially account for the differences in activity levels between the 19 F-FS hits as per NMR analysis. A comprehensive approach combining the 19 F-FS and other methods not only identified fragment hits but also distinguished structural differences in chemical groups with diverse activity levels.

Solid-state cross-polarization magic-angle spinning carbon-13 nuclear magnetic resonance ( 13 C CP/MAS NMR) spectroscopy is used to analyze starch derived from plants including wheat, maize, and potato, but few reports have described its application to rice starch. Here, we combined 13 C CP/MAS NMR with deconvolution and subtraction methods to analyze rice lines including mutants that are deficient in at least one enzyme involved in amylose and/or amylopectin biosynthesis. We found that differences in the content of ordered structures between rice lines could be evaluated using C1 signal deconvolution and subtraction. The content of the V-type ordered structure increased with increasing amylose content. Furthermore, starch derived from a starch synthase (SS) IIIa/starch branching enzyme (BE) IIb-deficient mutant formed B- and V-type ordered structures and significantly more nonordered structures than the other rice lines. These data indicate that 13 C CP/MAS NMR analysis is useful for discriminating the genetic backgrounds of starch derived from different rice cultivars., Competing Interests: The authors declare no competing financial interest.

A recently introduced (13)C polarization technique based on the nuclear Overhauser effect in rotating solid (nuclear Overhauser polarization-magic-angle spinning, NOP-MAS) (Takegoshi, K.; Terao, T. J. Chem. Phys. 2002, 117, 1700-1707) is applied to uniformly (13)C, (15)N-labeled proteins. NOP enhancement factors per scan of 1.5 approximately 2.0 are obtained, while that by cross polarization (CP) is less than 1.0. We show that uniform enhancement of all (13)C signals by CP is difficult to attain, while it is easily achieved by NOP, thus enabling quantitative comparison of signal intensities. NOP is easy to carry out under fast MAS and works well even for somewhat mobile molecules, for which CP does not work. Moreover, in labeled protein samples containing nonlabeled additives, NOP can eliminate the latter signals. For these features, NOP is superior to CP in many uniformly (13)C labeled proteins.