PubMed · 10295354
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J K Frei, E A Tomeski, A R Tomeski. 1986. Computerized tomography.. https://pubmed.ncbi.nlm.nih.gov/10295354/
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In our 3D NOESY-[(1)H,(15)N,(1)H]-ZQ-TROSY experiment, the TROSY principle (transverse relaxation-optimized spectroscopy) is used in three-dimensional (3D) (15)N-resolved nuclear Overhauser enhancement spectroscopy (NOESY), which enables resonance assignments by sequential nuclear Overhauser effects and the collection of structural constraints in large (15)N- or (2)H,(15)N-labeled proteins. Our experiment affords optimization of the transverse relaxation in all three frequency dimensions, provides suppression of the strong diagonal autorelaxation peaks, which otherwise tend to interfere with the analysis of nearby informative crosspeaks, and yields improved resolution for the entire spectrum when compared with conventional 3D (15)N-resolved-[(1)H,(1)H]-NOESY, because of the narrower lineshapes along both proton dimensions. The key element of this experiment is an approach for correlating the (15)N and (1)H chemical shifts with two-dimensional ZQ-[(15)N,(1)H]-TROSY, where zero-quantum (ZQ) coherence is generated and the remote cross-correlation between the (1)H and (15)N chemical shift anisotropy interactions is used to reduce transverse relaxation during (15)N evolution. Practical applications are illustrated with spectra of a protein with a molecular mass of 110,000 Da.
Using an NMR-based screen, a series of novel phosphotyrosine mimetics were discovered that bind to the SH2 domain of Lck. These compounds may serve as useful leads for the design of nonpeptide inhibitors of SH2 domains with improved bioavailability and metabolic stability compared to the natural ligands that contain phosphotyrosine.
Small monomeric proteins are the best models for studying protein folding, but they are often too stable for denaturation using pressure as the sole perturbant. In the present work we subject [CI-2(1-40).(41-64)], a noncovalent complex formed by the association of two complementary fragments of the chymotrypsin inhibitor-2, to high pressure to investigate the folding mechanism of a model protein. Pressures up to 3.5 kilobar do not affect the intact protein, but it can be unfolded reversibly by pressure in the presence of subdenaturing concentrations of guanidine chloride, with free energy and molar volume changes of 2.5 kcal mol-1 and 42.5 ml mol-1, respectively. In contrast, the complex can be reversibly denatured by high pressure without the addition of chemical denaturants. However, the process is clearly independent of the protein concentration, indicating lack of dissociation. We determined a change in the free energy of 1.4 kcal mol-1 and a molar volume change of 35 ml mol-1 for the pressure denaturation of the complex. A persistent quenching of the tryptophan adds further evidence for the presence of residual structure in the high pressure-denatured state. This state also appears to be compact as the small volume change indicates, compared with pressure denaturation of naturally occurring dimers. Based on observations of a number of pressure-denatured states and on characteristics of large CI-2 fragments with a solvent accessible core but maintaining tertiary interactions, the structure of the pressure-denatured state of the CI-2 complex could be explained by an ordered molten globule-like conformation.