Oleg Jardetzky

Probability-based protein secondary structure identification using combined NMR chemical-shift data

For a long time, NMR chemical shifts have been used to identify protein secondary structures. Currently, this is accomplished through comparing the observed 1Hα, 13Cα, 13Cβ, or 13C′ chemical shifts with the random coil values. Here, we present a new protocol, which is based on the joint probability of each of the three secondary structural types (β‐strand, α‐helix, and random coil) derived from chemical‐shift data, to identify the secondary structure. In combination with empirical smooth filters/functions, this protocol shows significant improvements in the accuracy and the confidence of identification. Updated chemical‐shift statistics are reported, on the basis of which the reliability of using chemical shift to identify protein secondary structure is evaluated for each nucleus. The reliability varies greatly among the 20 amino acids, but, on average, is in the order of: 13Cα>13C′>1Hα>13Cβ>15N>1HN to distinguish an α‐helix from a random coil; and 1Hα>13Cβ >1HN ∼13Cα∼13C′∼15N for a β‐strand from a random coil. Amide 15N and 1HN chemical shifts, which are generally excluded from the application, in fact, were found to be helpful in distinguishing a β‐strand from a random coil. In addition, the chemical‐shift statistical data are compared with those reported previously, and the results are discussed. A JAVA User Interface program has been developed to make the entire procedure fully automated and is available via http://ccsr3150‐p3.stanford.edu.

Nuclear magnetic resonance spectroscopy of amino acids, peptides, and proteins.

Publisher Summary This chapter discusses the application of nuclear magnetic resonance (NMR) to protein chemistry primarily in terms of the information that the technique can contribute to the protein structure and function. The chapter examines information about proteins that can be derived from NMR spectra, the premises and procedures necessary to interpret the observations. It also discusses whether NMR is merely another method for confirming conclusions reached by other methods or does it, in fact, permit findings not obtainable otherwise. With the success of selective deuteration of proteins, the two major problems of protein NMR spectroscopy—resolution and assignment of resonance lines—have been shown to be soluble at least in principle. It is to be expected that future difficulties in overcoming these problems will be proportional to the complexity of the protein. Success, however, is to be sought in experimental ingenuity rather than through further advances in instrumentation.

Combined use of 1H-NMR and GC-MS for metabolite monitoring and in vivo 1H-NMR assignments

Thirty-three metabolites were observed in perchloric acid extracts of four different tissues by in vitro 1H-NMR, GC-MS and alcohol dehydrogenase assay, and the information was used to interpret an in vivo two-dimensional nuclear Overhauser effect 1H-NMR spectrum. The metabolite profiles of the different tissues indicate a number of potential tissue-specific markers: N-acetylaspartate and gamma-aminobutyric acid for rat brain, glutamine/glutamic acid ratio for dog heart, arginine and sucrose for carrot, and t-aconitate, sucrose, asparagine/aspartic acid concentration ratios for corn roots. gamma-Aminobutyric acid and malate can be regarded as metabolic indicators for stressed corn roots. Concentrations of threonine and valine in corn roots were constant under hypoxic and salt stress, and can serve as internal standards for both in vivo and in vitro NMR studies. The in vitro information was further used to identify 12 compounds from the in vivo 1H-NMR spectra (including the two-dimensional nuclear Overhauser effect spectrum) of a carrot cylinder by correlating the chemical shift and nuclear Overhauser effect information. Thus, our choice of methods with a capability for structural determination allows the characterization of complex tissue extracts with minimum sample preparation, and supports, as well as complements, in vivo 1H-NMR investigations of metabolism.

α-Proton chemical shifts and secondary structure in proteins

Abstract Statistical analyses have been performed on 1H chemical-shift distributions in 32 polypeptides and proteins (ranging from 27 to 129 amino acid residues in length) for which reliable assignments (primarily from analyses of their spectra by 2D NMR methods) and secondary structure information were both available. On the basis of two independent statistical tests, significant differences were found between aH chemical shifts of aliphatic amino acid residues, Ala, Ile, Leu, Lys, and Val, in helical and β-sheet structures. Residues in regular helices have their αH atoms shifted upfield by −0.4 ppm, on average, and those in regular β-sheet structures are shifted downfield by +0.4 ppm with respect to the random coil value.

Determination of metabolite and nucleotide concentrations in proliferating lymphocytes by 1H-NMR of acid extracts

Nuclear magnetic resonance (NMR) studies of extracts have proven to be a powerful window onto the intracellular machinery of cells and tissues. The major advantages of in vitro 1H-NMR, namely chemical preservation, simultaneous detection, identification, and quantitation of compounds, and sensitivity to a large variety of classes of compounds, are employed in this study to characterize the metabolic course of mitogen-stimulated proliferation of human peripheral lymphocytes. A reliable method to quantitate amino acids, metabolic intermediates, soluble membrane lipid precursors, and purine, pyridine and pyrimidine nucleotides is presented, using samples as small as 30 mg wet weight. A total of 53 substances were detected in lymphocytes and other blood cells. During the course of lymphocyte culture, changes in intracellular concentrations of lactate, taurine, inositol and nucleotides, including NAD, IMP and high-energy phosphates, were especially marked. 1H-NMR compares favorably to 31P-NMR and to HPLC, and is especially attractive in light of expectations for future in vivo application.

Extent of intracellular pH changes during H(+) extrusion by maize root-tip cells.

Abstract31P-Nuclear-magnetic-resonance spectra of maize (Zea mays L.) root tips, that had been induced to extrude large amounts of H+ in response to fusicoccin (FC) in the presence of potassium salts, indicate that the cytoplasmic pH does not become higher than that of controls. In fact, the cytoplasmic pH may become slightly (approx. 0.1 pH unit) lower in cells extruding H+. Estimations of the buffer capacity of the cells show that without active intracellular pH regulation, H+ extrusion caused by FC would cause the intracellular pH to rise by at least 0.6 pH unit h-1. Our results indicate that intracellular pH is tightly regulated even during extreme rates of acid extrusion, and that a rise in cytoplasmic pH is not the signal linking H+ extrusion with enhanced organic-acid synthesis or other intracellular responses to H+ pumping.

Dynamics of nuclear Overhauser enhancement in proton decoupled carbon‐13 nuclear magnetic resonance

The contribution of nuclear Overhauser enhancements to signal intensities in proton decoupled 13C nuclear magnetic resonance spectra can be determined by comparing the spectrum obtained with continuous proton irradiation to the spectrum obtained with a gated decoupler technique. The gated decoupler technique utilizes a sequence of acquiring data with the decoupler on followed by a delay time with the decoupler turned off; under appropriate conditions, it gives a fully decoupled spectrum with no nuclear Overhauser enhancements. The mathematical relationship describing the delay requirements for accurate intensity comparisons depends on the ratio of the acquisition and delay times to the longitudinal relaxation time of the carbon, as well as on the magnitude of the nuclear Overhauser enhancement.

Differential mobility of the N-terminal headpiece in the lac-repressor protein.

It is shown by resolution enhancement and relaxation studies of the 360 MHz 1H nuclear magnetic resonance spectra of the lac-repressor of Escherichia coli and the two fragments derived from it by limited tryptic digestion (the N-terminal headpiece and remaining T-core) that the majority of the relatively mobile residues in the intact lac-repressor are located in the headpiece. Although nuclear magnetic resonance data clearly indicate that the headpiece is a highly structured entity, even when isolated, it is a more mobile part of the repressor than the T-core.

Characterization of lipid composition in stimulated human lymphocytes by 1H-NMR.

Recent in vivo NMR studies have raised interest in the structural changes of cellular lipids during proliferative activity. We investigated the changes in plasma membrane lipid and total cell lipid during mitogenically-stimulated proliferation of human peripheral blood lymphocytes by extraction of lipids and assay by 500 MHz 1H-NMR. Resonances were assigned using one- and two-dimensional spectroscopic techniques, and signals unique to certain species of lipid were identified. Choline and ethanolamine-containing lipids, glycerophospholipid backbones, sphingolipids, cholesterol, plasmalogens and triacylglycerols were readily detected. Resolution of a number of lipid species was not possible, despite the use of high-resolution techniques. NMR values for proliferation-induced changes in the most easily determined parameters, namely the total cholesterol to total phospholipid molar ratio, and phosphatidylcholine, phosphatidylethanolamine and sphingolipid composition, were found to agree with traditional methods. Differences in phospholipid and fatty acid profiles were found between plasma membranes and total cell lipid for resting values and for response to mitogen.

Continuous-flow NMR culture system for mammalian cells

A continuous-flow NMR culture system for mammalian cells has been developed on which 31P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by 31P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a delta pH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques.