Determination of substrate specificity of carboxylases by nuclear magnetic resonance

Determination of whether CO2 or HCO3- is the substrate for an enzymatic carboxylation has generally been accomplished by taking advantage of the fact that equilibration of these two compounds requires more than a minute at temperatures below 15 degrees C; thus different kinetics of carboxylation are obtained depending on whether CO2 or HCO3- is used to initiate the reaction. We report a new method using 13C18O2 as substrate for determining the CO2/HCO3- specificity of carboxylases. If CO2 is the substrate, then the 18O content of the 13C-containing product is the same as that of the 13CO2 used, whereas if HCO3- is the substrate, the 18O content is 2/3 that of the starting material. The method is independent of the detailed kinetics of the CO2/HCO3- interconversion and independent of the presence of contaminating unlabeled CO2 or HCO3-. Isotopic analysis is accomplished by 13C NMR. The method has been used to confirm that HCO3- is the substrate for phosphoenolpyruvate carboxylase. Studies of oxygen-18 isotope shifts in phosphorus NMR spectra have permitted confirmation of the observation that label is transferred from HC18O3- into Pi during the carboxylation of phosphoenolpyruvate.     

Ribosome-chloramphenicol interactions: a nuclear magnetic resonance study

Proton magnetic resonance line broadening of chloramphenicol resonances has been employed to study the binding of this inhibitor of protein synthesis to the Escherichia coli 70 S ribosome. Temperature dependence measurements of the resonance line widths show that chloramphenicol is in fast exchange with the ribosome. Differential broadening of the various drug resonances suggests that it binds in its receptor site in essentially the same conformation that exists free in solution. Thus, even though the drug possesses a fair degree of structural flexibility, this is not necessary for its interaction with the ribosome. The recognition is most likely of the classic lock and key type, with the ribosomal site being essentially an open gate for the fitting of the drug. From the proton line-width measurements and ¹⁹F spectra of a derivative, it has been possible to propose a model for the geometry of chloramphenicol when it resides on the ribosome which is consistent with established structure-activity relationships for the drug.     

Mapping of the auto-inhibitory interactions of protein kinase R by nuclear magnetic resonance

The dsRNA-dependent protein kinase (PKR) is a key mediator of the anti-viral and anti-proliferative effects of interferon. Unphosphorylated PKR is characterized by inhibitory interactions between the kinase and RNA binding domains (RBDs), but the structural details of the latent state and its unraveling during activation are not well understood. To study PKR regulation by NMR we assigned a large portion of the backbone resonances of the catalytically inactive K296R kinase domain, and performed (15)N-heteronuclear single quantum coherence (HSQC) titrations of this kinase domain with the RBDs. Chemical shift perturbations in the kinase indicate that RBD2 binds to the substrate eIF2alpha docking site in the kinase C-lobe. Consistent with these results, a mutation in the eIF2alpha docking site, F495A, displays weaker interactions with the RBD. The full-length RBD1+2 binds more strongly to the kinase domain than RBD2 alone. The observed chemical shift changes extend from the eIF2alpha binding site into the kinase N-lobe and inside the active site, consistent with weak interactions between the N-terminal part of the RBD and the kinase.     

Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions

Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.     

Determination of the secondary structure of interleukin-8 by nuclear magnetic resonance spectroscopy

The solution conformation of interleukin-8 (IL-8), a small protein of 72 residues with a wide range of proinflammatory activities, has been investigated by two-dimensional NMR spectroscopy. The 1H-NMR spectrum of IL-8 is assigned in a sequential manner and regular elements of secondary structure are identified on the basis of a qualitative interpretation of the nuclear Overhauser, coupling constant and amide exchange data. The IL-8 monomer contains a triple stranded anti-parallel beta-sheet arranged in a Greek key and a long C-terminal helix (residues 57-72). It is shown that IL-8 is a dimer in solution in which the interface is principally formed by six backbone hydrogen bonds between residues 25, 27, and 29 of one monomer and residues 29, 27, and 25, respectively, of the other. As a result, the two units of the dimer form a contiguous six-stranded anti-parallel beta-sheet. The secondary structure of IL-8 is similar to that found in the crystal structure of the sequence related protein platelet factor 4.     

Conformational analysis of the cholecystokinin C-terminal octapeptide: a nuclear magnetic resonance and computer-simulation approach

The C-terminal octapeptide portion of cholecystokinin (CCK8) has well-defined biological properties which include action as a neurotransmitter and induction of gall-bladder contraction and pancreatic enzyme secretion. Many analogues of CCK8 have been prepared and tested for potency, making this an ideal model system in which to initiate evaluation of structure-function relationships. The present study uses high-resolution proton nuclear magnetic resonance (NMR) spectroscopy and energy minimization techniques to evaluate the solution (DMSO) and in vacuo conformation(s) of CCK8. The NMR results provide amide and C alpha H alpha chemical shift temperature dependencies and all phi dihedral angles and chi 1 rotamer populations. The energy minimization data located deep potential energy wells, for which all torsion angles are reported. Collectively, the data support models for CCK8 where the structures are characterized by a high degree of folding. These conformations are characterized by sharp turns, possibly stabilized by hydrogen-bonds. Taken together with pharmacologic data and somewhat similar folded structures implied from fragments of CCK8, it is suggested that both electrostatic and steric effects are needed for full biological potency.     

Flavodoxin-cytochrome c interactions: circular dichroism and nuclear magnetic resonance studies

Circular dichroism and 1H and 31P nuclear magnetic resonance spectroscopy have been used to investigate complex formation between cytochrome c and the flavodoxins from Azotobacter vinelandii and Clostridium pasteurianum. Such complexes are known to be involved in the mechanism of electron transfer between these two redox proteins. A large increase in ellipticity in the Soret band of the cytochrome heme was observed upon formation of the Clostridium flavodoxin complex, whereas much smaller changes were found for the complexes with either Azotobacter flavodoxin or an 8 alpha-imidazolyl-FMN-substituted Clostridium flavodoxin analogue. Similarly, the magnitudes of the perturbations of the contact-shifted heme proton resonances obtained upon complexation of cytochrome c by Azotobacter flavodoxin were much smaller than those previously shown for Clostridium flavodoxin [Hazzard, J. T., & Tollin, G. (1985) Biochem. Biophys. Res. Commun. 130, 1281-1286]. 31P nuclear magnetic resonance measurements were also consistent with differences in the interactions between the components in the complexes of the two flavodoxins with cytochrome c. It is suggested that these spectral changes are due to a loosening or opening of the heme crevice upon Clostridium flavodoxin binding, which allows closer contact between the heme and flavin prosthetic groups and results in a faster rate of electron transfer. The implications of these observations for biological oxidation-reduction processes are considered.     

Rate constants determined by nuclear magnetic resonance

Fast kinetic methods are used to measure reactions that take place in less time than required to mix the reagents manually and to measure the reaction by usual methods, like UV-visible spectrophotometry and fluorescence. The best known of them are rapid-mixing and relaxation methods, which are used for reactions with half-times in the millisecond and microsecond ranges, respectively. The picosecond range is usually measured with electrical field and ultrasonic waves (A. Cornish-Bowden, 1976, Principles of Enzyme Kinetics, pp. 164-167, Butterworths, London). Normally these very fast rates occur when a ligand binds to or dissociates from a protein. When the binding is mediated only by the diffusion, the lower limit of the association rate constant (k(on)) should not exceed the value of a diffusion-controlled reaction (around 10(10) M(-1) s(-1)). Therefore, the values most frequently found for these rate constants, for example, in the association of a substrate with an enzyme, are in the range 10(6) to 10(9) M(-1) s(-1) (M. Eigen and G. G. Hammes, 1963, Adv. Enzymol. 25, 1-38). The values for the dissociation rate constants (k(off)) for these reactions, which depend on the equilibrium constant for the enzyme-substrate complex interaction, are in the range 10(1) to 10(5) s(-1), most often between 10(3) and 10(4) s(-1) (A. Fersht, 1999, Structure and Mechanism in Protein Science, pp. 164-165, Freeman, New York). If the equilibrium constant is known, and the value of koff is determined by nuclear magnetic resonance (NMR), as described in this chapter, the value of k(on) can be calculated; this should not exceed the value of diffusion rate in the media in which the reaction is performed.     

Probing protein dynamics by nuclear magnetic resonance

Proteins are dynamic molecules that often undergo conformational changes while performing their specific functions, such as target recognition, ligand binding and catalysis. NMR spectroscopy is uniquely suited to study protein dynamics, because site-specific information can be obtained for motions that span a broad range of time scales. The information obtained from NMR dynamics experiments has provided insights into specific structural changes or conformational energetics associated with molecular function. In the last decade, a number of new advancements in NMR methodologies have further extended our ability to characterize protein dynamics. Here, we present an overview of current NMR technology that is used to monitor the dynamic properties of proteins.     

Developing therapeutic proteins by engineering ligand-receptor interactions

Ligand-receptor interactions govern myriad cell signaling pathways that regulate homeostasis and ensure that cells respond properly to stimuli. Growth factors, cytokines and other regulatory elements use these interactions to mediate cell responses, including proliferation, migration, angiogenesis, immune responses and cell death. Proteins that inhibit these processes have potential as therapeutics for cancer and autoimmune disorders, whereas proteins that stimulate these processes offer promise in regenerative medicine. Although much of the focus in this area over the past decade has been on monoclonal antibodies, recently there has been increased interest in the use of non-antibody proteins as therapeutic agents. Here, we review recent advances and accomplishments in the use of rational and combinatorial protein engineering approaches to developing ligands and receptors as agonists and antagonists against clinically important targets.     

Phospholipid-protein interactions in human low density lipoprotein detected by 31P nuclear magnetic resonance.

31P nuclear magnetic resonance (NMR) spectra of human low density lipoprotein (LDL) has been obtained and the major phospholipid components identified. Analysis of the spectra revealed two phospholipid environments: one occupied by 4/5 of the phospholipid with high resolution resonances possessing properties similar to phospholipids in vesicles, and a second occupied by 1/5 of the phospholipid with broad lines indicative of immobilization. Limited trypsin treatment of the particle cleaved all of the B peptide into smaller molecular weight peptides which remained with the particle. Trypsin-treated LDL eluted from a Sepharose CL-6B column similarly to native LDL so that the modified particle remained intact. 31P NMR spectra of trypsin-treated LDL showed little or no immobilized phospholipid. The immobilization in the native LDL particle is attributed to lipid-protein interactions between 1/5 of the phospholipid and the B peptide.     

A nuclear magnetic resonance study of the interactions of antimalarial drugs with porphyrins

Haematins (hydroxyferriprotoporphyrin IX) constitute a possible receptor for antimalarial drugs such as chloroquine or quinine. This paper reports the study of the interactions of these two molecules with two tetrapyrrole (haematin and uroporphyrin I) by 1H-NMR spectroscopy. This method provided us with the geometry of the interactions in aqueous medium. The interaction consists of a close stacking of the porphyrin ring and the quinoleine moiety of the drugs. Using a porphyrin ring current model it was possible to reach the spatial relationships of the interacting species. It was concluded that hydrophobic forces play a key role in the interaction. The porphyrin plane can accommodate wide structural variations of the interacting species, leading to a weak specificity. The consequences on the mode of action of antimalarial drugs are discussed.     

Conformational analysis by nuclear magnetic resonance: insulin

High-resolution 270-MHz proton nuclear magnetic resonance (NMR) spectra of the native two-zinc insulin hexamer at pH 9 have been obtained, and assignments of key resonances have been made. Spectra of zinc-free insulin titrated with Zn2+ are unchanged after the addition of 1 equiv of zinc per insulin hexamer, indicating that the conformation of the hexamer is fixed at this point and that the second zinc ion does not significantly change the conformation. Titration of the two-zinc insulin hexamer with anions high on the Hofmeister series such as SCN- causes marked changes in the NMR spectra which are interpreted as the result of major conformational changes to a new hexameric form of insulin having a twofold axis perpendicular to the threefold axis. Analysis of difference spectra indicates that this new hexamer (which should be capable of binding six zinc ions) binds 2 equiv of SCN- at two sites which are assumed to be identical and independent (K1 = 10(3), K2 = 2.5 X 10(2) M-1).     

Accurate nucleic acid concentrations by nuclear magnetic resonance

Determination of the concentration of biochemical samples often yields values with uncertainties of 10-20% or more. This paper details a protocol for use with 500- to 600-MHz NMR spectrometers to measure approximately 1mM concentrations within +/-1-3% accuracy. With suitable precautions, all compounds have equal NMR "absorption coefficients" for protons. About 2mg of sample are needed for proteins and nucleic acids with MW=5000, although less accurate determinations could be made with smaller amounts. The technique utilizes standardized internal reference reagent compounds, cacodylic acid or 3-(trimethylsilyl)propionic-2,2,3,3-d(4) acid sodium salt. Spectra were signal-averaged using long interpulse delays so that integrals of nonexchangeable protons could be quantified relative to the reference standard. Accurate determinations require careful optimization of the homogeneity of the magnetic field and meticulous attention to sample preparation and spectral processing. The main source of error is usually the accuracy of micropipets. If sample preparation errors could be eliminated, the lower limit of accuracy with the current generation of NMR spectrometers is probably near 0.4%. However, this would require >99.5% sample purity. Methods are described to establish the concentration of the standards, and applications are illustrated with DNA mono- and oligonucleotides. Similar procedures should apply to proteins, polysaccharides, and other biomolecules, with about the same accuracy and precision.