Carbon 13 Nuclear Magnetic Resonance of Pentapeptides of Glycine Containing Central Residues of Methionine, Proline, Arginine, and Lysine

Methionine has been incorporated with high efficiency by Streptococcus faecium var. Durans strain A into dihydrofolate reductase isoenzyme 2. In the l3C NMR spectrum of the purified enzyme the resonances corresponding to the seven methionine residues are partially resolved into three composite peaks. Denaturation with urea collapses these into a single peak centered at 15.32 ppm, whereas the resonance of free methionine is at 15.04 ppm. Spectra of the free enzyme, its complex with methotrexate, and its complex with methotrexate and reduced nicotinamide adenine dinucleotide phosphate (NADPH) have been simulated, permitting more accurate estimates of line widths and nuclear Overhauser enhancement (NOE) values. These, together with the T\ values, cannot be explained solely by the effects of macromolecular tumbling and very rapid rotation of the methionine methyl group about its axis. A model assuming, in addition, the occurrence of free rotation about the methionine CHi-S bond is also unsatisfactory, and it is concluded that internal rotation about the CH2-S bond is highly restricted so that the methyl group oscillates through a relatively narrowangular range. Complex formation with NADPH produced rather small changes in the spectrum of the native enzyme, probably 4 he structure of dihydrofolate reductase is currently under intensive study in a number of laboratories. Interest in this enzyme derives in part from its clinical relevance. The reductase is the target of methotrexate, a drug used in the treatment of various types of cancer, and bacterial reductase is strongly and specifically inhibited by trimethoprim which is now used extensively in combination with sulfa drugs for treating a variety of infectious diseases.Although studies of the amino acid sequence, the effects of chemical modification, and x-ray diffraction of the crystalline protein have made important contributions to understanding the structure of the reductase, NMR studies are able to make a unique contribution. The method does not perturb the structure of the enzyme and is potentially capable of giving information about the internal motions of amino acid residues and about the interactions of residues at the active site with a great variety of substrates and inhibitors in binary or ternary f From the

Section: Discussioncontrasting

confidence: 89%

Nuclear magnetic resonance studies on bacterial dihydrofolate reductase containing [methyl-¹³C]methionine

Blakley¹,

Cocco²,

London³

et al. 1978

“…As stated originally, the nearly constant TVs observed for the a carbons (see Table I) imply that the various parts of the main chain are exhibiting very similar motions on the NMR time scale. This contrasts with observations on a series of pentapeptides where a definite lengthening of the TVs and corresponding increased motional freedom was observed proceeding outward from the central residue toward each terminal residue (Keim et al, 1974). An average of the Ca TVs reveals via eq 3 that tr = 7.09 X 10"10 s for isotropic overall reorientation in the absence of internal motion.…”

Section: Discussioncontrasting

confidence: 81%

Intramolecular microdynamical and conformational parameters of peptides from proton and ¹³C NMR spin-lattice relaxation. Tetragastrin

Bleich¹,

Cutnell²,

Glasel³

1976

Measurements of 1H and 13C spin-lattice relaxation and 13C nuclear Overhauser enhancement factors are reported for dimethyl sulfoxide solutions of tetragastrin, a pharmacologically active tetrapeptide. The use of the dipolar formalism for predicting 1H and quaternary 13C relaxation rates is discussed. Furthermore, the prospect is opened for the use of quaternary 13C and 1H relaxation times to obtain information on the peptide torsion angles phi, psi, and chi in a way supplementing NMR coupling constant methods presently in use.

“…As shown in Figure 2, the l3C NMR spectrum exhibits a single resonance in the guanido carbon region of the spectrum centered at 157.4 ppm, relative to tetramethylsilane, having a line width of about 2 Hz. Occurrence of the guanido carbon resonance of a homoarginine residue at this position compares favorably with the guanido carbon resonance positions of arginine and arginine residues in peptides and proteins which range from 157.4 to 158.1 ppm (Horsley et al, 1970;Glusko et al, 1972;Chien and Wise, 1973;Keim et al, 1974;Oldfield et al, 1975b). No evidence is seen for the natural abundance [,3C]guanido carbon resonance of the single arginine residue in the peptide.…”

Section: Resultsmentioning

confidence: 78%

Characterization of guanidinated cytochrome c by carbon-13 nuclear magnetic resonance spectroscopy

Stellwagen¹,

Smith²,

Cass³

et al. 1977

in which the longest decay component could be unambiguously determined, these measurements represent an important check on the validity of the conclusions drawn from the anti-DNS studies. Furthermore, the measurements were carried out on an antibody population with a different specificity. Thus, Fab segmental flexibility is probably generally present in IgG molecules. 19 homoarginine residues are distributed over a range of about 1 ppm and include two discrete single guanido carbon resonances. Distinct changes in the 13C NMR spectrum are observed upon complexation of guanidinated ferricytochrome c with ferrihexacyanide, upon conversion of the guanidinated ferricytochrome c to its alkaline isomer, and upon reduction to guanidinated ferrocytochrome c. Analysis of the changes in the visible absorbance spectrum accompanying guanidination of ferricytochrome c with O-methylisourea indicates that 18 lysine residues are guanidinated about 3 times more rapidly than the single remaining lysine residue, most likely lysine-79. The guanido carbon of this residue was preferentially enriched with l3C-enriched O-methylisourea. Analysis of the Ferricytochrome c is known to form discrete complexes which facilitate electron exchange with electron acceptors such