PubMed · 6034235
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M Ptashne. 1967-04-15. Specific binding of the lambda phage repressor to lambda DNA.. https://doi.org/10.1038/214232a0
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To address a number of conflicting reports in the literature, we undertook an infrared spectroscopic study to test for the presence of native-like secondary structures in thermally denatured ribonuclease A. Ribonuclease A does not aggregate at high temperatures, and the infrared spectrum shows a completely featureless amide I band contour. Using 13C-labeled urea, we were also able to obtain the infrared spectrum of the chemically denatured protein, which is practically identical with that of the heat-denatured protein. To the best of our knowledge, this is the first study that uses 13C-labeled urea as a chemical denaturant which circumvents the problem encountered with the strong absorption of urea in the conformation-sensitive amide I region of proteins; it opens up the possibility of investigating protein folding/unfolding processes in the presence of high concentrations of chemical denaturants. From an analysis of the amide I region of the infrared spectra of thermally and chemically denatured RNase A, it was concluded that heat-denatured ribonuclease A does not contain any significant amount of authentic hydrogen-bonded secondary structures. Furthermore, a comparison of the infrared spectra of ribonuclease A with those of ribonuclease T1 demonstrates that in spite of major differences between their native structures there are practically no differences between their heat-denatured states. This would not be expected if there were residual native-like secondary structures in the thermally denatured state of one or both of these proteins.
We report the results of a comparative study of the molecular order and dynamics of phosphatidylcholine (PC) bilayer membranes in the absence and presence of cholesterol, ergosterol and lanosterol, using deuterium (2H) nuclear magnetic resonance (NMR) of deuterated phospholipid molecules, in addition to solid state 13C and 31P-NMR. Using dimyristoylphosphatidylcholines (DMPCs) specifically labeled at positions 2', 3', 4', 6', 8', 10' and 12' of the sn-2 chain together with the perdeuterated 2-[2H27]DMPC derivative, the order profile for 9 of the 13 methylene groups of the sn-2 chain was established at 25 degrees C for DMPC, DMPC/cholesterol, DMPC/ergosterol and DMPC/lanosterol membranes, at a fixed sterol/phospholipid mol ratio of 30%, and in the presence of excess water. The overall ordering effects were found to be ergosterol > cholesterol >> lanosterol. Transverse relaxation (T2e) studies of these systems indicated that while for DMPC, DMPC/cholesterol and DMPC/ergosterol the relative relaxation rates were in qualitative agreement with models which assume cooperative motions of the bilayer molecules as the main relaxation mechanism, those in DMPC/lanosterol were anomalously high, suggesting alterations of lipid packing. Using dipalmitoylphosphatidylcholine (DPPC) deuterated at the trimethylammonium group of the choline moiety, we found that the differential ordering and motional effects induced by the sterols in the acyl chains were also reflected in the headgroup, both in the gel (L beta) and liquid-crystalline phases. 13C and 1H spin dynamics studies of these systems, including cross-polarization, rotating frame longitudinal relaxation and dipolar echo relaxation rates showed that the mobility of the different regions of the phospholipid molecules in the binary lipid systems were inversely correlated with the ordering effects induced by the sterols. A novel combination of C-D bond order parameters (obtained by 2H-NMR) and 13C-1H cross polarization rates confirmed these results. The effects of the same sterols at the same molar proportion on the unsaturated lipid 1-[2H31]palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (2H31-POPC) at 25 and 35 degrees C were different from those observed on DMPC and showed ordering effects which are largest for cholesterol, while ergosterol and lanosterol produced significantly smaller effects. Transverse relaxation studies indicate that while cholesterol does not perturb cooperative motions in POPC, both ergosterol and lanosterol do. Again, high-resolution solid state 13C-NMR studies support the conclusions of the 2H-NMR experiments.(ABSTRACT TRUNCATED AT 400 WORDS)
Proton sharing between acidic groups has been observed in the active sites of several enzymes, including bacteriorhodopsin, aspartic proteases, and ribonuclease HI. We here report NMR observations suggestive of proton sharing between cysteine thiols in the active site of the oxidation-reduction enzyme thioredoxin. The pKas of the two cysteine thiols in the Escherichia coli protein are removed from the expected value of 8.4 by approximately 1 pH unit in either direction, upward and downward. Further, the C beta resonances of both residues show clearly the effects of both of these pKas, indicating that the titrations of the two thiol groups are intimately linked. This behavior strongly suggests that the low pKa ascribed to the deprotonation of the Cys 32 thiol most likely arises through the interaction and close approach of the thiol of Cys 35, with the thiolate anion of Cys 32 stabilized through the sharing of the remaining thiol proton, nominally attached to Cys 35. These observations provide a rationale for the mediation of active site pH control, an important aspect of the mechanism of thioredoxin and other proteins with catalytic thioredoxin domains, such as protein disulfide isomerases.