High-resolution nuclear magnetic resonance studies of proteins

The combination of advanced high-resolution nuclear magnetic resonance (NMR) techniques with high-pressure capability represents a powerful experimental tool in studies of protein folding. This review is organized as follows: after a general introduction of high-pressure, high-resolution NMR spectroscopy of proteins, the experimental part deals with instrumentation. The main section of the review is devoted to NMR studies of reversible pressure unfolding of proteins with special emphasis on pressure-assisted cold denaturation and the detection of folding intermediates. Recent studies investigating local perturbations in proteins and the experiments following the effects of point mutations on pressure stability of proteins are also discussed. Ribonuclease A, lysozyme, ubiquitin, apomyoglobin, alpha-lactalbumin and troponin C were the model proteins investigated.     

Pulsed nuclear magnetic resonance studies in sickled erythrocytes

Pulsed proton magnetic resonance studies were made to determine T₁ (spin-lattice) and T₂ (spin-spin) relaxation times for normal and sickled blood cells in the oxygenated and deoxygenated states as a function of temperature between 0°C and 37°C. It was found that the sickling phenomenon affects T₂ values but not T₁ values in these cells. These results are ascribed to a proton-exchange mechanism. The functional relationship found between T₂ and temperature in the sickled cells is similar to that between the formation of hydrophobic bonds and temperature proposed by Nalbandian and Scheraga.     

High resolution nuclear magnetic resonance studies of nigeran

Polymer motion in solution can be studied by ¹³CNMR relaxation methods, which provide information about the correlation time for C-H vectors. ¹³C-Relaxation and Nuclear Overhauser Enhancement (NOE) data may frequently be combined to determine the dipole-dipole relaxation contribution. An alternative method is proposed based on a comparison of the proton spin-lattice relaxation rates of the centre proton resonances of an unlabelled molecule with the relaxation rates of the ¹³C satellites (from ¹³C labelled molecules).Selectively labelled nigeran which is an alternating $\text{1 → 3 and 1 → 4 α-}\text{d}\text{-glucan}$ has been investigated. The discussion in terms of the occurrence of different motions for each of the two units of the polymer requires an unambiguous assignment of the two anomeric carbons. For this reason a detailed assignment of the ¹H and ¹³C Nuclear Magnetic Resonance (NMR) spectra of nigeran in dimethylsulphoxide-d₆ is described, based on T₁ and NOE measurements in addition to selective homonuclear and heteronuclear spin decoupling experiments. These values are correlated with a conformation estimated by HSEA hard-spheres calculation. The measurements of the relaxation parameters for labelled and unlabelled compounds which provide an alternative determination of the ¹³C-¹H dipole-dipole relaxation contribution in a macromolecule agree well with ¹³C-{¹H} NOE experiments.     

Broad-line nuclear magnetic resonance studies of chloroperoxidase.

Chloroperoxidase, a heme glycoprotein isolated from the mold Caldariomyces fumago, was studied by NMR relaxation techniques. Interaction of the chloride ion substrate with the enzyme may be analyzed as consisting of at least three contributions: a weak interaction with the iron atom, nonspecific anion-protein interactions, and a specific interaction generated at low pH. The data indicate that a specific interaction, which develops in parallel with enzyme activity at low pH, does not occur at the iron atom first coordination sphere site. The results are summarized in terms of an enzymatic mechanism not involving chloride ion coordination to the iron atom.     

Carbon-13 nuclear magnetic resonance studies of cobalamins

The carbon-13 nuclear magnetic resonance spectra of aquocobalamin, adenosylcobalamin, methylcobalamin, and (carboxymethyl)cobalamin have been interpreted. The assignments were made by a comparison of the spectra with that of cyanocobalamin, by a study of the pH dependence of the chemical shifts, by an analysis of the effect of the axial ligands on the carbon atoms of the corrin ring, and by a study of the specific line broadening effect of the paramagnetic ions Mn2+ and Gd3+. The chemical shift changes that accompany the "base-on"----"base-off" conversion of the organocobalamins demonstrate that the conformation of the "western" half of the corrin ring and the conformations of the a, b, c, d, f, and g side chains are relatively constant. In contrast, the conformations of the "eastern" half of the corrin ring and the e propionamide side chain are highly variable.     

Deuteron magnetic resonance studies on the microdynamical behavior of partially deuterated oxytocin with neurophysin

With the use of specifically deuteron-labeled oxytocin and related molecules, deamino oxytocin and prolyl-leucyl-glycinamide, some of the intramolecular micro-mica of the free molecules and their interactions with their biological carriers, the neurophysins, have been studied. The work involves contemporary synthetic and isolation chemical techniques and computer processed nuclear magnetic resonance spectroscopy. The results show that there is considerable intramolecular motion in the “tail” of oxytocin with respect to the remainder of the molecule and that there is a rapid dynamical equilibrium between neurophysin-bound and free hormone in solution. The implications of this work on the biological action of the hormone-protein complex are discussed as well as the interpretation of high resolution nuclear magnetic resonance data in the light of these results.     

Surface plasmon resonance and nuclear magnetic resonance studies of ABAD-Abeta interaction

Abeta binding alcohol dehydrogenase (ABAD) is an NAD-dependent mitochondrial dehydrogenase. The binding between ABAD and Abeta is likely a direct link between Abeta and mitochondrial toxicity in Alzheimer's disease. In this study, surface plasmon resonance (SPR) was employed to determine the temperature dependence of the affinity of the ABAD-Abeta interaction. A van't Hoff analysis revealed that the ABAD-Abeta association is driven by a favorable entropic change (DeltaS = 300 +/- 30 J mol-1 K-1) which overcomes an unfavorable enthalpy change (DeltaH = 49 +/- 7 kJ/mol). Therefore, hydrophobic interactions and changes in protein dynamics are the dominant driving forces of the ABAD-Abeta interaction. This is the first dissection of the entropic and enthalpic contribution to the energetics of a protein-protein interaction involving Abeta. SPR confirmed the conformational changes in the ABAD-Abeta complex after Abeta binding, consistent with differences seen in the crystal structures of free ABAD and the ABAD-Abeta complex. Saturation transfer difference (STD) NMR experiments directly and unambiguously demonstrated the inhibitory effect of Abeta on the ABAD-NAD interaction. Conversely, NAD inhibits the Abeta-ABAD interaction. Binding of Abeta and binding of NAD to ABAD are likely mutually exclusive. Thus, Abeta binding induces conformational and subsequently functional changes in ABAD, which may have a role in the mechanism of Abeta toxicity in Alzheimer's disease.     

Fluorine-19 nuclear magnetic resonance studies of lipid phase transitions in model and biological membranes.

Fluorinated fatty acids of the general formula CH3(CH2)13-mCF2(CH2)m-2COOH are informative spectroscopic probes of the gel to liquid-crystalline phase transitions in phospholipid dispersions and in biological membranes. We present theoretical considerations to suggest that the 19F nuclear magnetic resonance line shapes are very different for frozen and fluid lipid regions. Our studies confirm this expectation for mixed phospholipid multilamellar dispersions containing a trace of difluoromyristate. The method correctly measures the onset and completion temperatures of the transition in the well-studied dimyristoylphosphaditylcholine distearoylphosphatidylcholine system and also describes the motional behavior of the solid and fluid phases within the transition. Lipids extracted from Escherichia coli membranes show similar motional phenomena through the transition-temperature range according to 19F nuclear magnetic resonance studies of difluoromyristate biosynthetically incorporated into the K1060B5 strain, an unsaturated fatty acid auxotroph. Intact cells or membrane vesicles show substantially different behavior from extracted lipids, indicating that membrane proteins significantly perturb the phase transition. Evidence presented in this paper also shows that the 19F resonance from Escherichia coli phospholipids is sensitive to various intramembrane interactions. There is a general decrease in restriction of motion due to neutral lipids and an opposite effect due to the architecture of the native membrane. Neither effect is temperature sensitive. However, there are interactions in the intact membrane, affecting the 19F resonance, that are temperature dependent both due to the phase-transition process and due to processes occurring at high temperatures.     

Quantitative in vivo nuclear magnetic resonance studies of hybridoma metabolism

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of a murine hybridoma cell line at two feed glutamine concentrations, 4.0 and 1.7 mM. Carbon-13 labeling patterns were used in conjunction with nutrient uptake rates to calculate the metabolic fluxes through the glycolytic pathway, the pentose shunt, the malate shunt, lipid biosynthesis, and the tricarboxylic acid (TCA) cycle. Decreasing the feed glutamine concentration significantly decreased glutamine uptake but had little effect on glucose metabolism. A significant incrase in antibody productivity occurred upon decreasing the feed glutamine level. The increased antibody productivity in concert with decreased glutamine uptake and no apparent change in glucolytic metabolism suggests that antibody production was not energy limited. Metabolic flux calculations indicate that (1) approximately 92% of the glucose consumed proceeds directly through glycolysis with 8% channeled through the pentose shunt; (2) lipid biosynthesis appears to be greater than malate shunt activity; and (3) considerable exchange occurs between TCA cycle intermediates and amino acid metabolic pools, leading to substantial loss of (13)C label from the TCA cycle. These results illustrate that (13)NMR spectroscopy is a powerfulf tool in the calculation of metabolic fluxes, particularly for exchange pathways where no net flux occurs. (c) 1994 John Wiley & Sons, Inc.     

Proton nuclear magnetic resonance studies of mast cell histamine

The state of histamine in mast cells was studied by 1H NMR spectroscopy. Spectra were measured for histamine in situ in intact mast cells, for histamine in suspensions of mast cell granule matrices that had been stripped of their membranes, and for histamine in solutions of heparin. The 1H NMR spectrum of intact mast cells is relatively simple, consisting predominantly of resonances for intracellular histamine superimposed on a weaker background of resonances from heparin and proteins of the cells. All of the intracellular histamine contributes to the NMR signals, indicating it must be relatively mobile and not rigidly associated with the negatively charged granule matrix. Spectra for intracellular histamine and for histamine in granule matrices are similar, indicating the latter to be a reasonable model for the in situ situation. The dynamics of binding of histamine by granule matrices and by heparin are considerably different; exchange of histamine between the bulk water and the granule matrices is slow on the 1H NMR time scale, whereas exchange between the free and bound forms in heparin solution is fast. The chemical shifts of resonances for histamine in mast cells are pH dependent, decreasing as the intragranule pH increases without splitting or broadening. The results are interpreted to indicate that histamine in mast cells is relatively labile, with rapid exchange between bound histamine and pools of free histamine in water compartments confined in the granule matrix.     

High-resolution proton nuclear magnetic resonance studies of human gastrin

High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz has been used to study the behavior of human gastrin in aqueous solution. A large number of resonances have been assigned by analysis of one- and two-dimensional NMR spectra and the effects of pH and by comparison with the spectrum of des-less than Glu1-gastrin. In gastrin, the ratio of cis to trans conformations around the Gly-2 to Pro-3 peptide bond is 3:7. This is reflected in splitting of the resonances of several neighboring residues and of a residue distant in the sequence, Tyr-12. The pKa of Tyr-12 is 10.7. Sulfation of this residue perturbs the resonances of Tyr-12 and Gly-13 but has very little effect on the rest of the spectrum. A study of the temperature dependence shows that several perturbed resonances move toward their expected positions as the temperature is raised but with a linear dependence on temperature, consistent with a redistribution of populations among accessible local conformations rather than a cooperative conformational change. Addition of Na+ or Ca2+ causes only minor changes in the spectrum. The paramagnetic metal ion Co2+ produces a number of spectral changes, reflecting strong binding to at least one site involving the Glu residues and weaker binding to Asp-16.     

Structure of alamethicin in solution: nuclear magnetic resonance relaxation studies

An NMR relaxation study at 500 MHz of the icosapeptide antibiotic alamethicin is reported. This study lends further support to the partly helical, partly extended, amphiphilic, and dimeric structure recently proposed for this peptide in methanolic solutions [Banerjee, U., Tsui, F. P., Balasubramanian, T. N., Marshall, G. R., & Chan, S. I. (1983) J. Mol. Biol. 165, 757]. The N-acetyl methyl groups toward the N terminus of alamethicin in this solvent system were found to exhibit unusual NMR relaxation behavior. The decay of the transverse magnetization due to these protons was nonexponential, but the spin-lattice relaxation recovery of the longitudinal magnetization was exponential. In a solution saturated with urea, however, both decays were exponential. These observations are shown to be consistent with the proposed structure. Studies in water yielded qualitatively similar but more complex results. The transverse relaxation times suggest further aggregation in water and indicate that the larger aggregates in water may be made up of the smaller units observed in methanol.     

C nuclear magnetic resonance studies of propionate catabolism in methanogenic cocultures

Propionate catabolism was monitored in anaerobic cocultures of propionate-degrading and methanogenic bacteria. Metabolism was monitored by use of C-enriched propionate and succinate. The intermediates identified indicated that the methylmalonyl coenzyme A pathway was used in these cultures. The data also indicated that a transcarboxylation reaction between succinate and propionyl coenzyme A occurred, yielding propionate and methylmalonyl coenzyme A.