Ex Vivo MuEtinuciear NMR Spectroscopy of Perfused, Respiring Rat Brain Slices: Model Studies of Hypoxia, Ischemia, and Excitotoxscit

We believe there are important roles for in vivo NMR spectroscopy techniques in studies of protection and treatment in stroke. Perhaps the primary utility of in vivo NMR spectroscopy is to establish the relevance of metabolic integrity, intracellular pH, and intracellular energy stores to concurrent changes occurring both at gross physiological levels (e.g., changes in cerebral blood flow, or blood oxygenation), and at microscopic or cellular levels. It has long been known that the brain is exquisitely sensitive to deprivations of oxygen, glucose, and cerebral blood flow. Routine human surgery on a limb takes place every day with tourniquets stopping all blood flow for up to two hours. In contrast, the deprivation of all blood flow to the brain (global ischemia) for approximately 5 minutes can result in severe, permanent brain damage. Research has gone on for more than 30 years to understand why the brain’s revival time is so much shorter, and to discover brain biochemical interventions that might dramatically extend the brain’s intolerance beyond 5 minutes, and therefore be relevant to protection and treatment of stroke. (Kogure and Hossmann, 1985; 1993) Stroke, defined as a permanent neurologic deficit arising from the death of brain cells, kills ∼ 150,000 people in the U.S.A. each year, and is the third leading cause of death (Feinleib et al., 1993). It is the next malady to escape, once one has dodged death from cardiovascular disease and cancer. Many, if not most, U.S.A. stroke victims will receive neurological clinical care not substantially different from what was provided 30 years ago. Most stroke patients will be put in intensive care units where blood pressure will be regulated and kept in a “safe” range, with the body given supportive care and the brain given an opportunity to heal itself. The problem of stroke is actually quite complex because there are several different kinds of stroke (ischemic, hemorrhagic, etc.), and because numerous systemic physiological factors are of relevance. Nevertheless, exciting advances in brain biochemistry suggest that stroke therapy and prophylaxis axe likely to improve dramatically in the near future (Zivin and Choi, 1991).

Determination by 1H NMR of a Slow Conformational Transition and Hydration Change in the Consensus TATAAT Prsbnow Box

The conformational dynamics and hydration of a DNA 14-mer containing the consensus Pribnow box sequence TATAAT have been measured using rotating frame T1 measurements and NOESY and ROESY in water. The H2 proton resonances of adenines show fast intermediate exchange behavior which can be attributed to a conformational transition that affects the distances between H2 protons of neighboring adenine residues, both sequential and cross-strand. The relaxation rate constant of the transition was measured at 4000s-1 at 25°C. Bound water close to the H2 proton of adenines was observed with residence times of >lns. At low temperature (5°C), the Pribnow box is in a closed state in which hydration water in the minor groove is tightly bound. At higher temperatures, the conformation opens up as judged by the increase in separation between sequential H2 protons of adenines and water exchanges freely from the minor groove. The conformational transition and the altered hydration pattern may be related to promoter function. The control of gene expression in procaryotes depends on the specific recognition by RNA polymerase of a six base-pair sequence (consensus: TTGACA) located at -35 from the transcription site, and a second one, named the Pribnow box (consensus: TATAAT) at about 10 base-pairs upstream the initiation site (Rosenberg and Court, 1979). It has been shown (Hawley and McClure, 1983) that strong promoters exhibit a high degree of homology with the consensus sequences, separated by an optimum consensus spacer length of 17 base pairs. The strength of a promoter depends on, among other thing, the rate of the initiation of transcription. This rate depends on the product between the thermodynamic and kinetic constants KB and k2 (McClure, 1980). The initial binding of RNA polymerase to the promoter results in the formation of a transcriptionally inactive ‘closed’ complex, characterized by the association constant KB. Isomerization to the active ‘open’ complex then occurs, and is characterized by the first order rate constant k2. Hence, the frequency of transcription initiation depends both on the strength of the polymerase-promoter interaction, and the ease with which this complex can isomerize to the productive state. Both of these events are likely to depend on the physical properties of the promoter.

Design and Characterization of New Sequence Specific DNA Ligands

During the early 1980s there were two developments which lead to our studies of sequence specific DNA ligands. The first was the development of sequential assignment methods based on 2D NMR spectra which allowed complete assignment of resonances for proteins (Wüthrich, 1986). The assignments in turn allowed determination of many structural restraints through interpretation of NOESY crosspeaks and coupling constants from COSY type spectra. The second advance was the improvement of the chemistry for direct synthesis of DNA oligomers. With multimilligram samples of DNA oligomers available sequential assignment methods for DNA, paralleling those for proteins, were also worked out. Again with assignments came the possibility of determining DNA structures in solution. Howeverfor double stranded, Watson-Crick paired DNAs the structure can be reasonably approximated by the standard B-form model derived from fiber diffraction. The accurate determination of local conformational features has been somewhat difficult using NMR since tertiary contacts (as are so valuable in determining protein structures) do not occur. However with careful quantitative analysis some of the local details of structure can be determined. These NMR methods also offered the possibility of trying to understand the structural basis for binding of ligands to DNA oligomers. In order to make welldefined complexes we wanted to start with a compound that showed some sequence specificity in binding, and selected distamycin (shown below), a polypyrrole antibiotic which was known to have preference for binding to A-T rich DNA sequences. A close relative, netropsin, had been studied by Dinshaw Patel who showed that the binding is in the minor groove by identifying an NOE between a proton of the ligand and an adenosine H2 in the center of the minor groove (Patel, 1982). We began by making a complex with the self-complementary DNA oligomer: 5'-CGCGAATTCGCG-3', which had been studied extensively by X-- ray crystallography, and also by NMR. Distamycin did form a well-defined complex with this DNA, which was is slow exchange with free DNA during titrations (Klevit et al., 1986).

The Interaction of Antigens and Superantigens with the Human Class II Major Histocompatibility Complex Molecule HLA-DR1

The initiation and maintenance of an immune response to pathogens requires the interactions of cells and proteins that together are able to distinguish appropriate non-self targets from the myriadof self-proteins (Janeway and Bottomly, 1994). This discrimination between self and non-self is in part accomplished by three groups of proteins of the immune system that have direct and specific interactions with antigens: antibodies, T cell receptors (TcR) and major histocompatibility complex (MHC) proteins. Antibodies and TcR molecules are clonally expressed by the B and T cells of the immune system, respectively, defining each progenitor cell with a unique specificity for antigen. In these cell types both antibodies and TcR proteins undergo similar recombination events to generate a variable antigen combining site and thus produce a nearly unlimited number of proteins of different specificities. TcR molecules are further selected to recognize antigenic peptides bound to MHC proteins, during a process known as thymic selection, restricting the repertoire of T cells to the recognition of antigens presented by cells that express MHC proteins at their surface. Thymic selection of TcR and the subsequent restricted recognition of peptide-MHC complexes by peripheral T cells provides a fundamental molecular basis for the discrimination of self from non-sell and the regulation of the immune response (Allen, 1994; Nossal, 1994; von Boehmer, 1994). For example, different classes of T cells are used to recognize and kill infected cells (cytotoxic T cells) arid to provide lymphokiries that induce the niajority of soluble antibody responses of B cells (helper T cells). In contrast to the vast combinatorial and clonal diversity of antibodies and TcRs, a small set of MHC molecules is used to recognize a potentially unlimited universe of foreign peptide antigens for antigen presentation to T cells (Germain, 1994). This poses the problem of how each MHC molecule is capable of recognizing enough peptides to insure an immune response to pathogens. In addition, the specificity of the TcR interaction with MHC-peptide complexes is clearly crucial to the problem of self :non-self discrimination, with implications for both protective immunity and auto-immune disease.

Selective Chemical Deuteration of Aromatic Amino Acids: A Retrospective

In 1970 when we began post-doctoral work in the laboratory of Professor Oleg Jardetzky, selective deuteration of proteins to limit the number of protons present in the system for subsequent analysis was a newly developed and effective technique for NMR exploration of protein structure (Crespi et al., 1968; Markley et al., 1968). This approach allowed more facile assignment of specific resonances and generated the potential to follow the spectroscopic behavior of protons for a specific amino acid sidechain over a broad range of conditions. The primary method for labeling at that time involved growth of microorganisms (generally bacteria or algae) in D2O, followed by isolation of the deuteratedamino acids from a cellular protein hydrolysate. The amino acids isolated were, therefore, completely deuterated. Selective deuteration of a target protein was achieved by growing the producing organism on a mixture of completely deuterated and selected protonated amino acids under conditions that minimized metabolic interconversion of the amino acids. In one-dimensional spectra, aromatic amino acid resonances occur well downfield of the aliphatic resonances, and this region can therefore be examined somewhat independently by utilizing a single protonated aromatic amino acid to simplify the spectrum of the protein. However, the multiple spectral lines generated by aromatic amino acids can be complex and overlapping, precluding unequivocal interpretation. To address this complication, chemical methods were developed to both completely and selectively deuterate side chains of the aromatic amino acids, thereby avoiding the costly necessity of growing large volumes of microorganisms in D2O and subsequent tedious isolation procedures. In addition, selective deuteration of the amino acids simplified the resonance patterns and thereby facilitated assignment and interpretation of spectra. The methods employed were based on exchange phenomena reported in the literature and generated large quantities of material for use in growth of microorganisms for subsequent isolation of selectively labeled protein (Matthews et al., 1977a). The target protein for incorporation of the selectively deuterated aromatic amino acids generated by these chemical methods was the lactose repressor protein from Escherichia coli, and greatly simplified spectra of this 150,000 D protein were produced by this approach.

A Solid-State NMR Approach to Structure Determination of Membrane-Associated Peptides and Proteins

Structural biology relies on detailed descriptions of the three-dimensional structures of peptides, proteins, and other biopolymers to explain the form and function of biological systems ranging in complexity from individual molecules to entire organisms. NMR spectroscopy and X-ray crystallography, in combination with several types of calculations, provide the required structural information. In recent years, the structures of several hundred proteins have been determined by one or both of these experimental methods. However, since the protein molecules must either reorient rapidly in samples for multidimensional solution NMR spectroscopy or form high quality single crystals in samples for X-ray crystallography, nearly all of the structures determined up to now have been of the soluble, globular proteins that are found in the cytoplasm and periplasmof cells and fortuitously have these favorable properties. Since only a minority of biological properties are expressed by globular proteins, and proteins, in general, have evolved in order to express specific functions rather than act as samples for experimental studies, there are other classes of proteins whose structures are currently unknown but are of keen interest in structural biology. More than half of all proteins appear to be associated with membranes, and many cellular functions are expressed by proteins in other types of supramolecular complexes with nucleic acids, carbohydrates, or other proteins. The interest in the structures of membrane proteins, structural proteins, and proteins in complexes provides many opportunities for the further development and application of NMR spectroscopy. Our understanding of polypeptides associated with lipids in membranes, in particular, is primitive, especially compared to that for globular proteins. This is largely a consequence of the experimental difficulties encountered in their study by conventional NMR and X-ray approaches. Fortunately, the principal features of two major classes of membrane proteins have been identified from studies of several tractable examples. Bacteriorhodopsin (Henderson et al., 1990), the subunits of the photosynthetic reaction center (Deisenhofer et al., 1985), and filamentous bacteriophage coat proteins (Shon et al., 1991; McDonnell et al., 1993) have all been shown to have long transmembrane hydrophobic helices, shorter amphipathic bridging helices in the plane of the bilayers, both structured and mobile loops connecting the helices, and mobile N- and C-terminal regions.

Early Days of Biochemical NMR

It was my pleasure to participate in Oleg’s 65th birthday celebration and to reminisce about the early days of Biochemical NMR. Oleg was always there. I remember in the summer in the early 1960s sitting on lawn chairs at a Gordon Conference and discussing the need for a meeting on biochemical NMR. This was to convene those with common interests, and out of this grew the 1964 meeting in Boston, which was the first International Conference on Magnetic Resonance in Biological Systems. In organizing the 1964 meeting Oleg was stalwart, in charge of the local arrangements at the old mansion, home of the American Academy of Arts and Sciences. The venue was much appreciated by the more than 100 attendees, and the smooth arrangements and elegant, although somewhat dowdy locale, contributed to the sense, generated by the meeting, that the field had a coherent scientific core and a meaningful future. In the early days of the 1960s the field of magnetic resonance in biological systems, brought together biannually by the society, had a coherence that was nurtured by the society. In those days the NMR and ESR methods were much less developed than they soon became, so that any reasonably competent spectroscopist could understand all the methods employed. Additionally, because the earlier studies concentrated upon the better understood biological molecules or processes, the breadth of the applications did not baffle a slightly informed biochemist. The rapid advances in definite understanding were thrilling to practitioners in the field, and individual efforts were motivated by a sense that the field was going to grow. By that time NMR was firmly established as a quantitative method in chemistry, solid state physics, and other material sciences so that with the results in hand it was logical to extrapolate to a future in which magnetic resonance could be central to biological research. These high hopes, however, required considerable confidence in extrapolation, because the individual findings were sometimes slight when compared to the exciting cutting edges of biological research.

NMR and Mutagenesis Investigations of a Model Cis: Trans Peptide tsomerization Reaction: Xaa116-Pro117of Staphylococcal Nuclease and its Role in Protein Stability and Folding

The slow rates of peptide bond isomerization in imino acids and the substantial population of the cis peptide bond isomer in Xaa-Pro linkages in peptides were first recognized in NMR studies of proline-containing model compounds (Maia et al., 1971). The important role of this isomerization in protein stability and folding (reviewed by Kim and Baldwin, 1982, 1990; Schmid, 1993) were recognized several years later (Brandts et al., 1975) and the biological relevance of this process was substantiated by the discovery of a ubiquitous enzyme that catalyzes Xaa-Pro peptide bond isomerization (Fischer et al., 1984, 1989; Takahashi et al., 1989). The strict evolutionary conservation of some prolyl residues and the observation that the kinetics of interconversion between alternative functional forms of some systems is consistent with the time scale of proline isomerization suggest that proline isomerization may play a wide role in protein structure and function. Suggestive examples include the sodium pump of Escherichia coli, the disulfide isomerase/thioredoxin class of enzymes, concanavalin A, and bovine prothrornbin fragment I (Brown et al., 1977; Marsh et al, 1979; Dunker, 1982; Brandland Deber, 1986; Langsetmo et al, 1989). NMR spectroscopy is one of the most suitable tools for studying this isomerization reaction. The rates generally are slow on the time scale of NMR chemical shifts but, in favorable cases, are comparable to longitudinal relaxation rates so that the isomerization process can be investigated by chemical exchange spectroscopy. NMR data obtained on calbindin D9k (Chazin et al., 1989), insulin (Higgins et al., 1988), and staphylococcal nuclease (nuclease) as discussed below have shown that each exists in solution under native conditions as a mixture of slowly exchanging conformers. The fact that dynamic molecular heterogeneity in nuclease was first observed in the laboratory of Oleg Jardetzky, as manifested by splitting of the histidyl 1H ε1 resonance from His46 in one-dimensional 1H NMR spectra recorded at 100 MHz (Markley et al., 1970), makes this topic particularly appropriate to a volume celebrating his scientific contributions.

Design of Novel Hemoglobins

Human normal adult hemoglobin (Hb) A, the oxygen carrier of blood, is a tetrameric protein consisting of two α chains of 141 amino acid residues each and two β chains of 146 amino acid residues each. Each Hb chain contains a heme group which is an iron complex of protoporphyrin IX. Under physiological conditions, the heme-iron atoms of Hb remain in the ferrous state. In the absence of oxygen, the four heme-irom atoms in Hb A are in the highspin ferrous state [Fe(II)] with four unpaired electrons each. Each of the four heme-iron atoms in Hb A can combine with an O2 molecule to give oxyhemoglobin (HbO2) in which the iron atom is in a low-spin, diamagnetic ferrous state. The oxygen binding of Hb exhibits sigmoidal behavior, with an overall association constant expression giving a greater than first-power dependence on the concentration of O2. Thus, the oxygenation of Hb is a cooperative process, such that when one O2 is bound, succeeding O2 molecules are bound more readily. Hb is an allosteric protein, i.e., its functional properties are regulated by a number of metabolites [such as hydrogen ions, chloride, carbon dioxide, 2,3-diphosphoglycerate (2,3-DPG)] other than its ligand, O2. It has been used as a model for allosteric proteins, and indeed, hemoglobins of vertebrates are among the most extensively studied allosteric proteins. Their allosteric properties are physiologically important in optimizing O2 transport by erythrocytes. The large number of mutant forms of Hb available provides an array of structural alterations with which to correlate effects on function. For details, see DickersonandGeis (1983), Bunnand Forget (1986), Ho (1992), Ho and Perussi (1994). There are two types of contacts between the α and β subunits of Hb (Perutz, 1970; Dickerson and Geis, 1983). The α1β1 (or α2 β2) contacts, involving B, G, and H helices, and GH corners, are called packing contacts. These contacts remain unchanged and hold the dimer together even when there is a change in the ligation state of the heme.

Simple Insights from the Beginnings of Magnetic Resonance in Molecular Biology

Birthday symposia inevitably provide an opportunity for reflection. Noting that greater minds than mine have offered an apology for their life (St. Augustine, 1853 edition; St. Thomas Aquinas; John Henry cardinal Newman, 1864), I shall attempt to answer the question: What have been the lasting contributions of my generation - the generation that began its work before Richard Ernst’s epoch making development of 2D NMR, and the equally momentous development of high field spectrometers, pioneered by Harry Weaver at Varian, Rex Richards at Oxford and Gunther Laukien at Bruker, revolutionized the technology and put biological applications within everyone’s reach? I offer these insights in the spirit that to fully understand a subject one must understand its history. The essence of scientific endeavor is to see something no one has seen before - or understand something no one had understood before. If there had been such a contribution, it was to understand what biological questions could be asked by NMR and to develop prototype experiments showing how. Difficult as it is to imagine this today when such understanding is taken for granted, the now obvious just wasn’t obvious then. Quite the contrary: well considered expert opinion of the day held the undertaking to be of very dubious merit. Linus Pauling, with whom it was my great fortune to spend my postdoctoral year, was never much interested in nuclear magnetic resonance (and did not think much of its promise for biological applications, as he clearly pointed out at this symposium). But, Linus Pauling firmly believed in giving the young the freedom to explore, and so the first crude interpretation of a protein NMR spectrum, taken a few weeks earlier by Martin Saunders, Arnold Wishnia and J. G. Kirkwoodat Yale, was based on the first amino acid and peptide spectra we had taken at Caltech. When I got my first faculty job at Harvard, and wanted to apply for an NMR spectrometer, it was not quite as easy.