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Molecular dynamics simulations at 298 and 273 K were carried out in order to explore the ability of calculations to predict NMR spin–lattice relaxation rates of the protons of liquid acetonitrile. The six-site model of Nikitin and Lyubartsev (J. Computat. Chem. 28 (2007), pp. 2020–2026) was used with systems containing up to 13,824 molecules. The effects of employing a cut-off for intermolecular interactions were explored. It was found that the model and procedures used predict intramolecular and intermolecular spin–lattice relaxation rates that are within ∼25% of experimental values at both temperatures.

The fluorinated organic solvents 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and hexafluoroacetone trihydrate (HFA) have been successfully used to produce silk fibroin fibers with high strength. To understand the reasons for the difference in strength between regenerated silk fibers prepared from these two solvents, we analyzed and compared properties of the native silk fibroin and (AGSGAG)2, a model for the crystalline part of fibroin, in the two solvents. The 13C and 1H chemical shifts obtained from 1H−13C HSQC spectra of silk fibroin in HFIP, HFA, and water indicates that silk fibroin formed helix-like structure in the fluorinated alcohols. A similar tendency was observed for (AGSGAG)2 although the chemical shift change is smaller. Intramolecular 1H−1H NOE data for (AGSGAG)2 imply the presence of helical structures in the middle part of the peptide in HFIP and but not in HFA, although an equilibrating collection of conformations likely are present in both solvents. For the peptide dissolved in HFIP, cross...

Tritium NMR spectroscopy has been used to examine the complexformed by [4-3H]benzenesulfon-amide and human carbonicanhydrase I. The results show that in solution the inhibitor forms a 1:1complex with the enzyme. A 100-spin computational model of the system,constructed with reference to crystallographic results, was used tointerpret tritium relaxation behavior and 3H{1H}NOEs. The analysis shows that the rate of dissociation of theenzyme–sulfonamide complex is 0.35 s−1 and thatthe aromatic ring of the inhibitor undergoes rapid rotation while complexed.

Tritium nmr spectroscopy of specifically tritiated tosylchymotrypsin has been used to examine the properties of the tosyl group in this protein. The unavoidable presence of several tritiated isotopomers complicates analysis of experiments and extensive computer simulations of relaxation behavior of tritiated species present were used in conjunction with models developed from crystallographic results to interpret the observations made. These analyses suggest that the tosyl group of tosylchymotrypsin at pH 4 is highly mobile in solution and occupies the lacation in the protein that is observed in the crystalline state only about 50% of the time.

Nuclear magnetic resonance spectroscopy has evolved in complexity to the extent that it is often useful to carry out computer simulations of experiments as a way to refine experimental conditions, to maximize usable information, or to interpret results reliably. The starting point for such simulations is an appropriate quantum mechanical formulation. This article presents many of the physical ideas that are important in high-resolution NMR spectroscopy and then develops expressions for the contribution of each of these to the Hamiltonian for the system under consideration. The Hamiltonians for the Zeeman interaction, scalar coupling, and the chemical shielding effect are discussed with the aim of explaining the quantum mechanics of NMR in a manner understandable to a nontheoretician. Consequently, much of the discussion is explicit and includes points skimmed over in other presentations in the literature.

The consequences of substitution of fluorine for the para hydrogen of a phenylalanine residue in ribonuclease-S were investigated by conformational energy calculations using the AMBER force field. Both the fluorine-containing protein and the corresponding nonfluorinated material were subjected to conformational adjustment through energy minimization and the minimum energy structures so defined were compared. Fluorine substitution leads to small alterations in many atomic positions in the protein, with adjustments at at sites more than 0.5 nm from the fluorine appearing to be somewhat larger than those within the immediate vicinity of the fluorine. Several atoms proximate to the fluorine atom were observed to move toward the fluorine while others in the same vicinity move away. The greater bulk of the fluorine atom and the strongly different electronic properties of fluorine compared to hydrogen thus appear to be insufficient to cause a consistent, unidirectional change in nearest-neighbor interactions upon introduction of a fluorine atom into a protein structure. Virtually all changes in atomic positions that are predicted by these calculations would be barely detectable by a crystallographic study with a resolution of 0.2 nm.

Tritium NMR spectroscopy has been used to examine the complex formed by [4-3H]benzenesulfon-amide and human carbonic anhydrase I. The results show that in solution the inhibitor forms a 1:1 complex with the enzyme. A 100-spin computational model of the system, constructed with reference to crystallographic results, was used to interpret tritium relaxation behavior and 3H{1H} NOEs. The analysis shows that the rate of dissociation of the enzyme-sulfonamide complex is 0.35 s-1 and that the aromatic ring of the inhibitor undergoes rapid rotation while complexed.

Fluorine NMR lineshape, relaxation and Overhauser effect data collected at 282 and 470 MHz have been used to obtain information about the nature of complexes formed between N-trifluoroacetyl-4-fluorophenylalanine and the enzyme chymotrypsin. Systems involving both enantiomers have been examined as well as derivatives of these in which the aromatic ring hydrogens have been replaced by deuterium. The enzyme-induced fluorine chemical shift effects and the dynamics of molecular motions of the fluorophenyl ring at the respective binding sites appear to be similar in both complexes and, where comparable, the results are in agreement with data obtained at lower frequencies that have been reported by other workers. The dynamics of the fluoroaromatic ring in these complexes are significantly different from those observed in a closely related acylated enzyme.

We describe an exercise intended to introduce students to the proton NMR experiments used to define the molecular conformation of biological molecules in solution. Proton NMR spectra of the octapeptide [sar1] angiotensin II are assigned and nuclear Overhauser effects are then used to develop constraints that define the three-dimensional shape of the peptide. The exercise demonstrates the utility of COSY, TOCSY, and NOESY types of experiments, while underscoring the limitations of these experiments in the determination of structure. The spectroscopic data used for the exercise are made available in several formats so that an instructor can design variations of the exercise to emphasize particular aspects related to data handling or the generation or comparison of derived structures. At least one quarter of biochemistry and a year of organic chemistry are reasonable prerequisites for the exercise, presuming that these courses provide the needed level of familiarity with the basic concepts of proton NMR spectroscopy. Junior- or senior-level chemistry and biochemistry students are probably most benefited from this exercise.

We have prepared samples of (a) intact calvaria collagen (cross-linked and mineralized), (b) intact tendon collagen (cross-linked but not mineralized), and (c) reconstituted chick calvaria collagen (not cross-linked and not mineralized) containing [methyl-2H3]methionyl, [4,4-2H2]pyrrolidinyl, (4-fluorophenyl)alanyl, and [6-15N]lysyl residues. Using multinuclear magnetic resonance spectroscopy, we have investigated the molecular dynamics of the labeled amino acids. Guided by model compound studies, we reached the following conclusions regarding collagen side chain dynamics from our analysis of line shapes and relaxation rates. At 22 degrees C, imino residues in all samples have flexible rings with root mean square angular fluctuations in the 11-30 degree range. Nearly all labeled amino acid side chains reorient about at least two side chain single bonds. At temperatures below -30 degrees C, most of these side chain motions are absent in all the samples. Surprisingly, in contrast with results obtained for backbone motions, side chain motions are only marginally more hindered in mineralized samples as compared with nonmineralized samples, a result we discuss with reference to collagen-mineral interactions. We also discuss the possible relationship between collagen dynamics and function.

Powers and co-workers have provided evidence that thiobenzyl N-heptafluorobutyrylanthranilate (I) is an extremely potent inhibitor of serine proteases, especially alpha-chymotrypsin (Teshima, T., Griffin, J. C., and Powers, J. C. (1982) J. Biol. Chem. 257, 5085-5091). We have prepared additional derivatives of this structure in which fluorine substitutions have been made on the aromatic rings and have attempted to carry out fluorine NMR studies of the interaction of Powers' compound and these new derivatives with chymotrypsin. The solubility of all inhibitors examined in solvent systems compatible with the retention of native enzyme structure is extremely low. While some nmr evidence for complex formation could be obtained, preparations of the complexes examined were metastable and precipitation of the inhibitor eventually limits the amount of complex that can be present in solution to such low levels that nmr experiments are impractical. An unusual effect of solvent composition on fluorine chemical shifts suggests that the conformation of the inhibitors in aqueous solution and when bound to the enzyme is different from that in organic solvents.

Empirical intermolecular force field parameters for the fluorine atom of the 4-fluorophenyl group have been generated for use in molecular mechanics programs. Partial charges on the ring system were obtained by Mulliken population analysis of wave functions generated by molecular orbital calculations in the STO-3G approximation. Fluorine van der Waals parameters were obtained by non-linear least-squares fitting of crystallographic data following the procedure of Hagler and Lifson. The parameters developed are compared to fluorine parameters reported previously.

Fluorine NMR has been used to examine complexes formed by 2-fluoro-, 3-fluoro-, and 2,5-difluorobenzenesulfonamide and human carbonic anhydrases I and II. The results show that all three inhibitors form complexes with both isozymes that have 2:1 inhibitor/enzyme stoichiometry. The fluorine spectra observed for all inhibitor-isozyme combinations are consistent either with rapid rotation of the aromatic ring of the inhibitor in the complexes or with preferential binding of only one of the two possible conformations of the inhibitors that are isomeric by virtue of rotation about the C1-C4 bond of the fluoro aromatic ring. Because ring rotation is slow in the case of the pentafluorobenzenesulfonamide-CA I complex, selective binding of rotamers is the explanation of these observations presently favored. A computer graphics study shows that formation of 2:1 complexes of CA I is feasible without appreciable distortion of the protein tertiary structure found in the crystalline state.

Addition of aqueous base to solutions of 2-halc-5-nitropyridines in dimethyl sulfoxide produces a stable substance which forms 2-hydroxy-5-nitropyridine if the molar ratio of hydroxide to pyridine is greater than 2:l. IR and 'H and 13C NMR spectra of the intermediate stable substance indicate that the pyridine ring is cleaved to form this material. An SN(ANRORC) mechanism is proposed for the reaction.

Deacylation of unsubstituted and of o-F-, m-F, p-F, α-F, pentafluoro-, p-methyl-, and p-trifluoromethyl-substituted trans-cinnamoyl- α-chymotrypsins has been studied from pH 4 to 8. The deacylation rate constants were found to depend upon the ionization state of a group on the enzyme with an apparent pK in the range 6.3–7.3. The hydrolysis rates of the correspondingly substituted p-nitrophenylcinnamate esters were determined at pH 10.6. Correlation of the data for these model reactions with the corresponding rates of enzyme deacylation suggests that the p-methyl-substituted acylenzyme is about an order of magnitude more reactive than expected while p-trifluoromethyl substitution results in deacylation at a rate 10 times slower than expected. The remaining substituents exert about the anticipated rate effect on deacylation.