PubMed Health⌕ Search

Biomedical subjects

T M Rothgeb

Publications and source records attributed to T M Rothgeb.

16 recordsLinked to original sources
PubMed1983

Nuclear magnetic resonance studies of amino acids and proteins. Side-chain mobility of methionine in the crystalline amino acid and in crystalline sperm whale (Physeter catodon) myoglobin.

We have obtained deuterium (2H) nuclear magnetic resonance (NMR) spectra and spin-lattice relaxation times (T1) of L-[epsilon-2H3]methionine, L-[epsilon-2H3]methionine in a D,L lattice, and [S-methyl-2H3]methionine in the crystalline solid state, as a function of temperature, in addition to obtaining 2H T1 and line-width results as a function of temperature on [epsilon-2H3]methionine-labeled sperm whale (Physeter catodon) myoglobins by using the method of magnetic ordering [Rothgeb, T. M., & Oldfield, E. (1981) J. Biol. Chem. 256, 1432-1446]. The results indicate that in the L-amino acid, methyl rotation having an activation energy (delta E) of 8.3 +/- 1 kJ dominates T1 at low temperatures (less than or equal to 10 degrees C), while at higher temperatures an additional large-amplitude side-chain motion occurs which causes changes in the 2H NMR line shape and T1. This motion is inhibited in the D,L lattice, indicating that lattice effects may have a strong effect on the mobility of anhydrous amino acids in the solid state. Further substitution at S delta to form the sulfonium salt [S-methyl-2H3]-methionine causes a large increase in delta E, to 15.9 +/- 2 kJ, a value comparable to the 14-16 kJ found in valine and leucine, which contain the structurally similar isopropyl moiety. These results suggest that the very low barriers to methyl rotation in the methionine side chain are due to long C-S bond lengths and the presence of only two substituents on sulfur, while the anomalous high-temperature behavior is due to a lattice-packing effect. 2H T1 results with methionine-labeled myoglobin are complex, reflecting the presence of fast large-amplitude side-chain motions, in addition to rapid methyl rotation. Our data indicate that Met-55 and Met-131 are motionally inequivalent in crystalline cyanoferrimyoglobin, in contrast to solution NMR results. We have also recorded 13C cross-polarization "magic-angle" sample-spinning NMR spectra of [epsilon-13C]methionine-labeled crystalline cyanoferrimyoglobin (at 37.7 MHz, corresponding to a magnetic field strength of 3.52 T) and of the same protein in aqueous solution. Cross-polarization transfer rates and proton rotating-frame relaxation time results again indicate that Met-55 and Met-131 are motionally inequivalent in the solid state, and the TCH data indicate that Met-55 is more solidlike. However, we find that 13C chemical shifts in solution and those in the crystalline solid state are in very close agreement, suggesting that the average solution and crystal conformations are the same, in the area of Met-55 and Met-131.

Animals↗
PubMed1982

Glucagon carboxyl-terminal derivatives: preparation, purification, and characterization.

Chemical and enzymatic methods have been used to prepare the following series of seven glucagon derivatives modified in the carboxyl-terminal region important for hormone-receptor binding: [des-Asn28,Thr29](homoserine lactone27)glucagon, [des-Asn28,Thr29](homoserine27)glucagon, (S-methyl-Met27)glucagon, [des-Thr29](S-methyl-Met27)-glucagon, [des-Thr29]glucagon,[des-Asn28,Thr29](S-methyl-Met27)glucagon, and [des-Asn28,Thr29]glucagon. The derivatives were isolated in high yield, extensively purified, and chemically characterized. All were found to be full agonists of native glucagon. Binding affinity was evaluated by displacement of mono[125I]iodoglucagon prepared by new methods. Binding and biological activities closely correlated, indicating that most modifications affected the relative binding affinity and relative biological potency of glucagon to a comparable extent. Circular dichroism measured in dilute acid solution resembled that of native glucagon except for [des-Asn28,Thr29]glucagon which displayed increased alpha helicity (25%). All derivatives formed helical structures in 2-chloro-ethanol, although the amount of helicity induced was not closely correlated with biological activity. Binding and biological activities were not affected by removal of Thr-29, though both were reduced 20-fold when Asn-28 was also removed, irrespective of whether homoserine or native methionine remained at the carboxyl terminus. Lactone formation was associated with a further 5-fold reduction in binding affinity but not in activity. Methylation of Met-27 had essentially the same effect as removing the two carboxyl-terminal residues, although the combined effect of both modifications was greater than 100-fold reduction in binding and activity. These findings provide additional insight concerning glucagon structure-function relationships.

Adenylyl Cyclases↗
PubMed1982

High-resolution solid-state NMR of quadrupolar nuclei.

We report the observation of high-resolution solid-state NMR spectra of (23)Na (I = [unk]), (27)Al (I = [unk]) and (51)V (I = [unk]) in various inorganic systems. We show that, contrary to popular belief, relatively high-resolution ( approximately 10 ppm linewidth) spectra may be obtained from quadrupolar systems, in which electric quadrupole coupling constants (e(2)qQ/h) are in the range approximately 1-5 MHz, by means of observation of the ((1/2), -(1/2)) spin transition. The ((1/2), -(1/2)) transition for all nonintegral spin quadrupolar nuclei (I = [unk], [unk], [unk], or [unk]) is only normally broadened by dipolar, chemical shift (or Knight shift) anisotropy or second-order quadrupolar effects, all of which are to a greater or lesser extent averaged under fast magic-angle sample rotation. In the case of (23)Na and (27)Al, high-resolution spectra of (23)NaNO(3) (e(2)qQ/h approximately 300 kHz) and alpha-(27)Al(2)O(3) (e(2)qQ/h approximately 2-3 MHz) are presented; in the case of (51)V(2)O(5) (e(2)qQ/h approximately 800 kHz), rotational echo decays are observed due to the presence of a approximately 10(3)-ppm chemical shift anisotropy. The observation of high-resolution solid-state spectra of systems having spins I = [unk], [unk], and [unk] in asymmetric environments opens up the possibility of examining about two out of three nuclei by solid-state NMR that were previously thought of as "inaccessible" due to the presence of large (a few megahertz) quadrupole coupling constants. Preliminary results for an I = [unk] system, (93)Nb, having e(2)qQ/h approximately 19.5 MHz, are also reported.

Journal Article↗
PubMed1981

Nuclear magnetic resonance of heme protein crystals. General aspects.

A new technique capable of determining the static and dynamic structures of heme protein crystals is reported. It is shown that microcrystals of a variety of paramagnetic heme proteins, suspended in approximately 90% saturated (NH4)2SO4, may be perfectly aligned by an intense static external magnetic field, H0, due to the large anisotropy in the magnetic susceptibility of the protein caused by the paramagnetic center. Myoglobin from sperm whale (Physeter catodon) was isotopically enriched at the C epsilon methyl groups of methionine residues 55 and 131 with either 13C or 2H and studied in the crystalline solid state by 2H-quadrupole echo and 13C-Fourier transform nuclear magnetic resonance spectroscopy. It was found that suspensions of both high (S = 5/2) and low (S = 1/2) spin ferric forms of the labeled protein were ordered, the axis of ordering being approximately perpendicular to the low temperature minimum g tensor valve, even though upper Kramers levels are populated at room temperature. The paramagnetic CoII derivative "coboglobin" showed similar ordering behavior, but the diamagnetic carboxymyoglobin was unaffected. The magnetic ordering method permits the recording of "single crystal" NMR spectra from microcrystalline arrays of proteins which cannot be prepared in large enough form (approximately 1 cm3) for single crystal NMR spectroscopy and thereby allows the resolution and assignment of numerous single atom sites in the crystalline solid state. The information from a "single crystal" NMR spectrum combined with that obtained on the crystal powder allows for the direct determination of (i) the spatial orientation of the particular labeled residue within the protein crystal and (ii) the rates and types of side chain motion. Resonances were assigned by spin label broadening experiments and by use of existing x-ray data to predict 2H-NMR spectra. This new technique opens up the possibility of determining directly the dynamic structure of protein crystals and of comparing the structures of proteins in the crystalline solid state with that in solution and is applicable to other heme proteins, e.g. catalase.

Animals↗
PubMed1980

Electrostatic stabilization in sperm whale and harbor seal myoglobins. Identification of groups primarily responsible for changes in anchoring of the A helix.

The compact, largely helical structure of sperm whale and harbor seal myoglobins undergoes an abrupt one-step transition between pH 4.5 and 3.5 as monitored by changes in either the heme Soret band absorbance or circular dichroism probes of secondary structure, for which a modified Tanford-Kirkwood theory provides identification of certain dominant electrostatic interactions responsible for the loss of stability. A similar treatment permits identification of the electrostatic interactions primarily responsible for a process in which the anchoring of the A helix to other parts of the molecule is weakened. This process is detected with both myoglobins, in a pH range approximately 1 unit higher than the onset of the overall unfolding process, through changes in the circular dichroic spectra near 295 nm which correspond to the L1 O-O band of the only two tryptophan residues in these proteins, residues 7 and 14. In each case protonation of certain sites in neighboring parts of the molecule can be identified as producing destabilizing interactions with components of the A helix, particularly with lysine 6.

Animals↗
PubMed1979

Complete amino acid sequence of the myoglobin from the Pacific sei whale, Balaenoptera borealis.

The complete amino acid sequence of the major component myoglobin from Pacific sei whale, Balaenoptera borealis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. From the sequence analysis of four of these peptides and the apomyoglobin, over 75% of the covalent structure of the protein was obtained. The remainder of the primary structure was determined by the sequence analysis of peptides that resulted from further digestion of the amino-terminal and central cyanogen bromide fragments. The amino-terminal fragment was specifically cleaved at its two tryptophanyl residues with N-chlorosuccinimide and the central cyanogen bromide fragment was cleaved at its glutamyl residues with staphylococcal protease and at its single tyrosyl residue with N-bromosuccinimide. The primary structure of this myoglobin proved identical with that from the gray whale but differs from that of the finback whale at four positions, from that of the minke whale at three positions and from the myoglobin of the humpback whale at one position. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.

Amino Acid Sequence↗
PubMed1979

Motions in proteins.

Explore the source record for details and available documents.

Allosteric Regulation↗
PubMed1979

Aliphatic groups of sperm whale myoglobin: 13C NMR study.

The aliphatic region of the 13C NMR spectrum of sperm whale cyanoferrimyoglobin has been examined at 67.9 MHz. Fifty partially resolved or well-resolved resonances, representing at least half of the aliphatic carbons in the molecule, are observed in the spectral region from 9 to 29 ppm downfield of tetramethylsilane. Analyses of the spin lattice relaxation times (T1) and nuclear Overhauser enhancements for these resonances reveal considerable motion freedom of the aliphatic side chains. In the spectral region from 9 to 15 ppm, eight single carbon resonances are observed and tentatively assigned to Cdelta 1 of eight of the nine isoleucine residues. In at least five cases the reorientational motion of the isoleucine side chains could not be characterized solely by rotation of the Cdelta 1 methyl groups. The simplest model consistent with the data is a restricted diffusion model with two degrees of internal rotation [Wittenbort, R. J. & Szabo, A. (1978) J. Chem. Phys. 69, 1722--1736]. In light of the packing densities within the myoglobin molecule these results are taken to imply concerted motions of the buried aliphatic residues.

Animals↗
PubMed1978

The quantitation of carbamino adduct formation of angiotensin II and bradykinin.

The two equilibrium constants that define the extent of carbamino adduct formation with amines for all values of pH and PCO2 are determined for the alpha-amino groups of the peptide hormones angiotensin II(AII) and bradykinin (BK) by nuclear magnetic resonance techniques. From these constants the variation of carbamino adduct formation has been calculated over the pH range 6.60--8.00 with variable PCO2, and the results are superimposed upon standard pH-bicarbonate diagrams. PCO2, and the results are superimposed upon standard pH-bicarbonate diagrams. The mole fraction, Z, of carbamino adduct form of AII or BK shows a maximum variation in going from metabolic alkalosis, Z congruent to 0.30, to metabolic acidosis, Z congruent to 0.02, with Z near 0.2 for normal acid-base conditions. Adduct formation to hormone may alter the biological effect of the hormone (a) by limiting proteolysis, particularly at the amino-terminal, (b) by altering hormone binding affinity to specific receptors, or (c) by converting the hormone to an antagonist which binds to receptor but does not activate subsequent metabolic events. The requirements for any of these mechanisms to operate are examined in terms of simple equilibrium considerations, and experimental evidence of inhibition of an aminopeptidase model system is presented. These results are consistent with the hypothesis that regulation of some physiological processes through formation of carbamino adduct of peptide hormones is possible.

Acid-Base Equilibrium↗
PubMed1977

Methylation of glucagon, characterization of the sulfonium derivative, and regeneration of the native covalent structure.

The methylation of the single methionine residue of glucagon is accomplished at a pH of 3.5 in 8 M urea with methyl iodide. The reaction product is a soluble sulfonium derivative, S-methylglucagon, which can be isolated in a highly purified form. This derivative is characterized by amino acid analysis and its effect on the adenylyl cyclase system of rat liver plasma membranes. S-Methylglucagon does stimulate the adenylyl cyclase system; however, its activity is approximately 500 times less than that observed with the native hormone. The solubility of this derivative is great enough to allow for further modifications of the molecule which can be followed at a later stage by demethylation. Demethylation of S-methylglucagon regenerates the original covalent structure and is accomplished by treatment with Cleland's reagents at a pH of 10.5. The regenerated hormone is indistinguishable from native glucagon by its amino acid composition and its ability to stimulate the adenylyl cyclase system. The entire methylation-demethylation reaction sequence has been carried out with yields that approach 75%. The technique is suitable for the isotopic enrichment of native glucagon and may well be applicable to selected other methionine-containing peptides.

Adenylyl Cyclases↗
PubMed1976

Nuclear magnetic resonance studies of sperm whale myoglobin specifically enriched with 13C in the methionine methyl groups.

The Cepsilon methyl group of the 2 methionine residues in sperm whale myoglobin was enriched with respect to 13C. This was accomplished by treatment of the apomyoglobin at pH 4 at room temperature with a 100-fold proportion of 13CH3I to form an intermediate containing enriched S-methylmethionine. Unselective demethylation to regain the apomyoglobin structure was accomplished by treatment at pH 10.5 with 0.5 M dithioerythritol at 37 degrees for 18 h. Reagents were removed at each stage by dialysis against dilute sodium azide solution. Hemin was reincorporated to form the holoprotein in a way that avoided the presence of an excess of the small molecule. After chromatographic purification the enriched myoglobin was obtained in a yield of between 29 and 60%. The composition, absorbance spectrum, circular dichroism spectrum, isoionic point, electrophoretic behavior, and oxygen-binding behavior following reduction were all indistinguishable from those of the virgin protein. NMR measurements were made at 15.1, 25.2, and 67.9 MHz at 27-30 degrees. The two enriched loci are represented by separate resonances that appear slightly downfield of the spectral position of the corresponding resonance in free methionine. The positions of these resonances are sensitive to pH and to the ligand bound at the heme group which is approximately 17 A distant from each methionine Cepsilon. On the basis of two separate types of experiment the downfield resonance was assigned to methionine 55 and the upfield resonance to methionine 131. Part of the observed variations in chemical shift could be treated as arising from pseudocontact interactions but part was ascribed to structural changes communicated to the environment of each methionine residue as a result of changes in heme ligand, pH, or temperature. The linewidths of the methionine Cepsilon resonances are narrowed by increasing temperature according to an Arrhenius energy of activation of nearly 3 kcal. The spin-lattice relaxation times, T1, of the two methionine Cepsilon resonances at the three spectrometer frequencies were interpreted to indicate the existence of rotational motions in each side chain in addition to that about the Sdelta-Cepsilon bond. The results as a whole show that the two methionine side chains undergo continuous variations in environment, and that these variations are controlled by events at a distance within the protein structure. It is suggested that the structural lability serves the function of facilitating conformational variations and adjustments within the heme pocket.

Amino Acids↗