John T. Gerig

Reactions of 2,6-dinitro-4-trifluoromethylbenzenesulfonate with human serum albumin

The reaction of the title compound with human serum albumin has been examined at various concentrations of the sulfonate. Kinetic data suggest that there are two highly reactive lysine amino groups on the protein, five lysine residues which are less reactive and an undetermined number of additional nucleophilic groups that react very slowly with the reagent at pH 7.5. One of the rapidly reacting lysines is tentatively identified as lysine-199 in the protein sequence. Fluorine NMR experiments indicate the presence of tight binding sites on the protein for the sulfonate which are not near reactive functional groups.

Modification of human serum albumin with N-(2,5-dinitro-4-fluorophenyl)-4-amino-2,2,6,6-tetramethylpiperidinooxy radical

Human serum albumin has been treated with the spin-labeling reagent indicated in the title. Ultraviolet spectral studies of the protein so modified suggest that reaction takes place at lysine and tyrosine sidechains; kinetic experiments indicate that there are two especially reactive amino groups of the protein which are preferentially modified. Evidence is presented that these groups include the one acetylated by aspirin (Lys-199) or those arylated by 2.6-dinitro-4-trifluoromethylbenzenesulfonate. Esr experiments show that bound spin labels have about the same correlation time expected for overall tumbling of the protein; ESR observations indicate that molecular freedom near the spin labels is not increased when the protein is transferred to 8 M urea.

Fluorine-NMR studies of chimpanzee hemoglobin.

It has been demonstrated that 4-fluorophenylalanine, a known inhibitor of protein synthesis, becomes incorporated into hemoglobin when present in the diet of a chimpanzee. 19F-NMR spectra of various forms of this protein show well-resolved lines, each line presumably corresponding to a unique phenylalanine/fluorophenylalanine position of the primary sequence. Fluorine chemical shifts and, by implication, tertiary structures vary with the oxidation state and ligand.

Interactions of 2,2,2-trifluoroethanol with melittin

Melittin dissolved in 42% trifluoroethanol‐water at pH 2 has been shown to be α‐helical between residues 6 and 12 and between residues 13 and 25, with the two helical regions separated by a bend at the Leu13 residue. The inter‐helix angle was found to be 154 ± 3° at 0 °C and 135 ± 3° at 25 °C. The dominant conformation of the peptide is thus similar to those observed by previous workers for the peptide in a variety of media. At 25 °C, intermolecular nuclear Overhauser effects arising from nuclear spin dipole‐dipole interactions between melittin hydrogens and fluorines of the solvent are essentially those expected for a system that is homogeneous as regards concentration and translational diffusion of the peptide and fluoroalcohol components. However, at 0 °C, peptide‐trifluoroethanol cross‐relaxation terms are negative, a result consistent with the conclusion that fluoroalcohol molecules associate with the peptide for times (∼1 ns) that are long compared to the time of a typical peptide‐fluoroalcohol diffusive encounter (∼0.2 ns). Such interactions may be responsible for the reduction of the translational diffusion coefficient of trifluoroethanol produced by dissolved peptides. Copyright

Solvent Interactions with [Val5]angiotensin II in Ethanol-Water

Intermolecular NOE experiments have been used to explore the interactions of water and ethanol molecules in 35% ethanol/65% water (v/v) with the octapeptide hormone [val (5)]angiotensin II at temperatures from 0 to 25 degrees C. Magnetic dipole-dipole cross relaxation terms sigma(HH)(NOE) and sigma(HH)(ROE) for interaction of both solvent components suggest that ethanol molecules interact with the peptide backbone atoms strongly enough to associate for times comparable to the rotational correlation time of the peptide; comparison of observed ROE and NOE cross relaxation terms indicate that lifetimes of these interactions are of the order 0.4 ns at 5 degrees C. Formation of such peptide-ethanol complexes can also account for larger-than-expected values of the cross relaxation terms at higher temperatures. Alternative explanations of the observations reported are shown to be unlikely, primarily because they require unreasonable and highly localized concentrations of the ethanol near the peptide. Side chains of the peptide appear to experience no unusual interactions with ethanol. Cross relaxation terms for water-peptide backbone interactions indicate long-lived interactions of water with the backbone atoms although the nonpolar side chains of the peptide (Val3, Val5, Pro7, and possibly Phe8) do not interact in any specific way with water molecules. Cross relaxation terms for protons of the polar (Tyr4 and His6) side chains may reflect strong interactions with water, but analysis of these is confounded by solvent proton exchange and possible spin diffusion effects.

Solvent interactions with the Trp‐cage peptide in 35% ethanol–water

Intermolecular NOE experiments have been used to explore interactions of water and ethanol molecules in 35% ethanol/65% water (v/v) with the peptide Trp-cage at temperatures from 5 to 25 degrees C. Magnetic dipole-dipole cross-relaxation terms sigma(HH) (NOE) and sigma(HH) (ROE) for interaction of solvent components with spins of the peptide suggest that ethanol molecules associate with backbone atoms for times of the order of nanoseconds at 5 degrees C. Formation of peptide-ethanol complexes can also account for the larger-than-expected values of cross-relaxation terms at higher temperatures. Hydrocarbon side chains of the peptide do not appear to experience such interactions with ethanol. Cross relaxation resulting from water-peptide interactions are consistent with long-lived water interactions with the backbone atoms. Water cross relaxation with nonpolar side chains of the peptide (Leu2, Ile4, Leu7, and proline residues) are only those expected for bulk solvent. However, long-lived association of both water and ethanol with the polar side chains of Tyr3 and Trp6 is indicated by the data.

An automated data collection system for T1 and T2 determinations

Abstract Computer-based schemes for the automatic collection of data from a pulsed NMR spectrometer for the determination of T1 and T2 are described. Emphasis is placed on procedures that are useful in signal-averaging applications with weak signals. A hard-wired multichannel analyzer interfaced to a general purpose minicomputer is used to assemble the appropriate data which is then used in a least squares calculation to obtain the desired relaxation time.

Fluorine nuclear magnetic resonance spectra of rabbit carbonmonoxyhemoglobin

Carbonmonoxyhemoglobin prepared from protein isolated from rabbits maintained on a diet supplemented with 4-fluorophenylalanine (Phe (4F)) has been studied by fluorine NMR spectroscopy. Substitution of Phe(4F) appears to take place randomly at the sixteen nonequivalent phenylalanine positions of the globins; examination of hybrid hemoglobins in which only one type of globin chain contained the fluorinated amino acid, as well as changes in the spectrum upon exposure to oxygen, aided in the assignment of fluorine resonances from the alpha- or beta-globin chains. The effects of modification of Cys-beta-93 with a spin label and variation of pH and sample temperature on the spectrum were also examined. These data in association with theoretical estimates of aromatic ring current and van der Waals effects on chemical shifts were used to support tentative assignments of several signals observed to specific amino acid residues. Evidence suggesting the presence of two conformational forms of the protein, possibly due to disorder of the heme groups, is described.

Interaction of alcohols with [val5]angiotensin in alcohol-water mixtures.

Intermolecular solvent-solute NOE experiments have been used to probe interactions of various alcohols with the peptide hormone [val(5)]angiotensin II at 0 degrees C. It is found that these NOEs are detectable but dependent on the kind of alcohol present and the conformation of the peptide. Solvent-solute NOEs in 100% methanol and 89% methanol-water are basically those predicted by a hard sphere model for intermolecular spin dipole interactions. NOEs at the peptide backbone (N-H, C alpha-H) protons in 25% methanol-water and 36% ethylene glycol-water mixtures indicate that alcohol interactions near these groups are also adequately described by this model. However, in 35% ethanol-water, interactions of alcohol methyl protons with the peptide result in unexpectedly negative NOEs, probably signaling that peptide-alcohol interactions in this solvent take place on a significantly slower time scale than that defined by mutual diffusion of these species. Some side chain-alcohol interactions result in NOEs up to 8 times larger than expected. Possible reasons for these enhanced effects are discussed.

Theoretical treatment of relaxation in the presence of an RF field

Abstract A general description of relaxation of an arbitrary spin system in the presence of an RF field is given. The results show clearly how the presence of the RF field makes new combinations of frequencies important to the relaxation process. Exact solutions of the resulting equations are not possible, but introduction of conventional approximations provides forms that are complicated but amenable to machine solution.