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C R Sanders

Publications and source records attributed to C R Sanders.

At least 37 records · Page 2Linked to original sources
PubMed1995

Reconstitution of membrane proteins into lipid-rich bilayered mixed micelles for NMR studies.

This paper describes a study undertaken to assess the possibility and practical consequences of reconstituting integral and peripheral membrane proteins into bilayered discoidal mixed micelles ("bicelles") composed of dimyristoylphosphatidylcholine and smaller amounts of either CHAPSO or short-chain phosphatidylcholine. The amphiphilic assemblies in these mixtures are uniquely suited for use in NMR structural studies because they can be magnetically oriented with experimentally-tunable system order. The first step of this study was to test about 15 membrane-associating polypeptides and proteins for their ability to interfere with magnetic orientation of the bicellar assemblies. A variety of results were obtained ranging from no perturbation to a complete disruption of orientation. Second, the suitability of bicelles as mimics of natural bilayers was tested by reconstituting diacylglycerol kinase, an integral membrane enzyme. The kinase was observed to be functional and completely stable for at least 24 h when incubated at 38 degrees C in bicelles. Third, the NMR spectra from a number of bicelle-reconstituted proteins were examined. In some cases, 13C NMR resonances from reconstituted proteins were extremely broad and asymmetric. In other cases, resonances from reconstituted proteins were moderately broad, but much less so than resonances from proteins reconstituted into multilayers oriented by mechanical methods. In the cases of two surface-associating proteins (cytochrome c and leucine enkephalin), oriented sample 13C NMR spectra of extremely high resolution were obtained. For these proteins it was also demonstrated that the experimentally variable order of the bicellar assemblies could be exploited to provide a means of screening for detergent-specific structural perturbations, for making spectral assignments, and for measuring chemical shift anisotropies and dipolar couplings. Taken as a whole, these results indicate that bicelles may be uniquely and effectively employed as model membranes to facilitate NMR structural studies of many, but not all, membrane proteins.

Amino Acid Sequence↗
PubMed1994

Structural requirement of the regulatory light chain of smooth muscle myosin as a substrate for myosin light chain kinase.

The substrate structure required for skeletal and smooth muscle myosin light chain kinases (MLC kinase) was studied by using various mutant regulatory light chains of smooth muscle myosin. The deletion of the NH2-terminal 10 residues did not greatly affect the kinetic parameters of smooth MLC kinase; however, deletion of an additional 3 residues, Lys11-Arg13, prevented phosphorylation. In contrast, deletion of Lys11-Arg13 did not completely abolish the phosphorylation for skeletal MLC kinase, and deletion of three additional residues was required for complete inhibition. Substitution of Arg16 with Glu markedly decreased Vmax for both smooth and skeletal MLC kinases. Substitution of Lys11-Arg13 with acidic or noncharged residues decreased Vmax, but these changes were much lower than that occurring on substitution of Arg16. Replacement of Lys11-Arg13 with acidic residues reduced the affinity of the free LC20 but had little effect on the myosin-incorporated LC20. These results were different from those previously obtained with synthetic peptide analogs (Kemp, B. E., Pearson, R. B., and House, C. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7471-7475) and suggest that a cluster of the basic amino acid residues are not fundamentally important for substrate recognition. The structural simulation revealed that the guanidyl group of Arg16 but not the corresponding Glu13 of skeletal light chain resides in close proximity to Ser19, suggesting that the guanidyl group of Arg16 stabilizes the phosphate transfer and that the introduction of Glu at the 16th position would significantly destabilized this reaction.

Amino Acid Sequence↗
PubMed1994

Function of the NH2-terminal domain of the regulatory light chain on the regulation of smooth muscle myosin.

The role of the NH2-terminal domain of the 20,000-dalton light chain on the regulatory function of smooth muscle myosin was studied by exchanging it in myosin with various mutant forms. The 10 S to 6 S conformational transition as well as the thick filament formation were significantly influenced by the deletion or substitution of the amino acid residues at the NH2-terminal side of the phosphorylation site (Ser19). Whereas the deletion of Ser1-Thr10 did not significantly affect these functions, further deletion of Lys11-Arg16 completely abolished the formation of 10 S conformation and induced thick filament formation. Among the residues in this region, Arg13 and Arg16 were most important for these functions since substitution of these residues by Glu or Ala significantly altered these functions. Similar substitutions of Lys11 and Lys12 also stabilized the 6 S conformation and thick filament formation but less effectively. While the 6 S conformation was stabilized, the deletion of NH2-terminal residues did not activate the actin-activated ATPase activity. This suggests that stabilization of the 6 S conformation is not directly coupled with activation of actomyosin ATPase activity but rather a more defined conformational change around the phosphorylation site is necessary for activation. Such a change also influences the 6 S to 10 S conformation and, therefore, the filament formation. To support this notion, substitution of Lys11 and Lys12 by Glu-Glu inhibited the phosphorylation-induced activation of actomyosin ATPase activity.

Actins↗
PubMed1994

Qualitative comparison of the bilayer-associated structures of diacylglycerol and a fluorinated analog based upon oriented sample NMR data.

sn-1,2-Dimyristoylglycerol (DMDAG) and sn-1,2-dimyristoyl-3-fluoropropanediol (DMFPD) were synthesized in carbonyl 13C-labeled and acyl chain perdeuterated forms. These compounds were reconstituted at low levels into both randomly dispersed dimyristoylphosphatidylcholine (DMPC) bilayers and magnetically orientable DMPC media. Samples were subjected to NMR analysis, leading to a substantial body of 2H quadrupolar splitting, 13C-13C and 13C-19F dipolar coupling and 13C chemical shift anisotropy data for both compounds. A number of measurements were also made for DMPC. The data acquired from magnetically oriented samples were found to be undesirably affected by the presence of artifacts related to the experimental use of the oriented lipid media. However, it was possible to draw a number of qualitative conclusions from the data and to correct the data so that they may ultimately prove useful in quantitative structural analyses of bilayer-associated DMDAG and DMFPD. Comparison of the DMDAG data with corresponding measurements of DMPC suggests a high degree of similarity between the two compounds within bilayers composed primarily of L alpha phase phosphatidylcholine, consistent with previous work (S.O. Smith et al. (1992) Biochemistry 31, 11660-11664). Comparison of the data for DMDAG and DMFPD suggests that the hydroxyl proton of DMDAG is involved in hydrogen bonding interactions, which appear to be largely responsible for maintaining the orientational inequivalence of its two acyl chains. Bilayer-associated DMFPD also appears to exhibit a higher degree of whole-molecule disorder than DMDAG, again suggestive of an important structural role for the hydroxyl proton of DMDAG. Finally, comparison of 13C-NMR data for oriented DMPC in the presence and absence of low levels of DMDAG and DMFPD indicated that neither compound induces a significant change in the averaged conformational state of DMPC comprising the bilayer matrix of the oriented samples.

Carbon Isotopes↗
PubMed1994

Membrane topology of Escherichia coli diacylglycerol kinase.

The topology of Escherichia coli diacylglycerol kinase (DAGK) within the cytoplasmic membrane was elucidated by a combined approach involving both multiple aligned sequence analysis and fusion protein experiments. Hydropathy plots of the five prokaryotic DAGK sequences available were uniform in their prediction of three transmembrane segments. The hydropathy predictions were experimentally tested genetically by fusing C-terminal deletion derivatives of DAGK to beta-lactamase and beta-galactosidase. Following expression, the enzymatic activities of the chimeric proteins were measured and used to determine the cellular location of the fusion junction. These studies confirmed the hydropathy predictions for DAGK with respect to the number and approximate sequence locations of the transmembrane segments. Further analysis of the aligned DAGK sequences detected probable alpha-helical N-terminal capping motifs and two amphipathic alpha-helices within the enzyme. The combined fusion and sequence data indicate that DAGK is a polytopic integral membrane protein with three transmembrane segments with the N terminus of the protein in the cytoplasm, the C terminus in the periplasmic space, and two amphipathic helices near the cytoplasmic surface.

Amino Acid Sequence↗
PubMed1993

Synthesis and characterization of a 13C-labeled alpha-mannosyl glycolipid analog from [13C]glucose.

A method is described for the synthesis of a [13C]alpha-mannosyl glycolipid analog from [13C]glucose. After acetylation and reduction of glucose (U-13C6, 30%) to tri-O-acetyl-D-glucal (U-13C6, 30%), addition of the nucleophile 2-[2-[2-[2-(tetradecyloxy)ethoxy]ethoxy]ethoxy]- ethanol (tetra-decyltetraglycol) yields the rearrangement product alpha-tetradecyltetraglycol 2,3-dideoxyl-4,6-di-O-acetyl-D-gluco- pyranoside (U-13C6, 30%). The rearrangement product is oxidized with osmium tetroxide to produce tetradecyltetraglycol alpha-mannoside (U-13C6, 30%). The interaction of the glycolipid with the plant lectin concanavalin A is characterized by a vesicle agglutination assay.

Agglutination↗
PubMed1993

An approximate model and empirical energy function for solute interactions with a water-phosphatidylcholine interface.

An empirical model of a liquid crystalline (L alpha phase) phosphatidylcholine (PC) bilayer interface is presented along with a function which calculates the position-dependent energy of associated solutes. The model approximates the interface as a gradual two-step transition, the first step being from an aqueous phase to a phase of reduced polarity, but which maintains a high enough concentration of water and/or polar head group moieties to satisfy the hydrogen bond-forming potential of the solute. The second transition is from the hydrogen bonding/low polarity region to an effectively anhydrous hydrocarbon phase. The "interfacial energies" of solutes within this variable medium are calculated based upon atomic positions and atomic parameters describing general polarity and hydrogen bond donor/acceptor propensities. This function was tested for its ability to reproduce experimental water-solvent partitioning energies and water-bilayer partitioning data. In both cases, the experimental data was reproduced fairly well. Energy minimizations carried out on beta-hexyl glucopyranoside led to identification of a global minimum for the interface-associated glycolipid which exhibited glycosidic torsion angles in agreement with prior results (Hare, B.J., K.P. Howard, and J.H. Prestegard. 1993. Biophys. J. 64:392-398). Molecular dynamics simulations carried out upon this same molecule within the simulated interface led to results which were consistent with a number of experimentally based conclusions from previous work, but failed to quantitatively reproduce an available NMR quadrupolar/dipolar coupling data set (Sanders, C.R., and J.H. Prestegard. 1991. J. Am. Chem. Soc. 113:1987-1996). The proposed model and functions are readily incorporated into computational energy modeling algorithms and may prove useful in future studies of membrane-associated molecules.

Biophysical Phenomena↗
PubMed1993

Simulation of NMR data from oriented membrane proteins: practical information for experimental design.

Several hundred solid state NMR dipolar couplings and chemical shift anisotropies were simulated for the polytopic membrane protein, bacteriorhodopsin, and for an idealized transmembrane peptide conforming to several different secondary structures (alpha- and 3(10)-helices and parallel and antiparallel beta-sheets), each at several tilt angles with respect to the bilayer normal. The use of macroscopically oriented samples was assumed. The results of these simulations suggest: (i) Because of the r-3 dependence of dipolar coupling, it is likely to prove difficult to successfully execute uniform isotopic enrichment strategies to generate large numbers of quantitatively interpretable structural measurements in oriented sample NMR studies of membrane proteins. (ii) There are a number of readily implementable specific isotopic labeling schemes which can yield data patterns sufficient to identify local secondary structure for transmembrane segments of idealized proteins which are tilted by < 10 degrees with respect to the bilayer normal. (iii) The measurement of dipolar coupling constants between 13C-, 19F-, and/or 3H-labeled side chains of proximal residues may prove effective as routes to long range tertiary structural data constraints.

Amino Acid Sequence↗
PubMed1993

Solid state 13C NMR of unlabeled phosphatidylcholine bilayers: spectral assignments and measurement of carbon-phosphorus dipolar couplings and 13C chemical shift anisotropies.

The direct measurement of 13C chemical shift anisotropies (CSA) and 31P-13C dipolar splitting in random dispersions of unlabeled L alpha-phase phosphatidylcholine (PC) has traditionally been difficult because of extreme spectral boradening due to anisotropy. In this study, mixtures of dimyristoyl phosphatidylcholine (DMPC) with three different detergents known to promote the magnetic orientation of DMPC were employed to eliminate the powder-pattern nature of signals without totally averaging out spectral anisotropy. The detergents utilized were CHAPSO, Triton X-100, and dihexanoylphosphatidylcholine (DHPC). Using such mixtures, many of the individual 13C resonances from DMPC were resolved and a number of 13C-31P dipolar couplings were evident. In addition, differing line widths were observed for the components of some dipolar doublets, suggestive of dipolar/chemical shift anisotropy (CSA) relaxation interference effects. Oriented sample resonance assignments were made by varying the CHAPSO or DHPC to DMPC ratio to systematically scale overall bilayer order towards the isotropic limit. In this manner, peaks could be identified based upon extrapolation to their isotropic positions, for which assignments have previously been made (Lee, C.W.B., and R.G. Griffin. 1989. Biophys. J. 55:355-358; Forbes, J., J. Bowers, X. Shan, L. Moran, E. Oldfield, and M.A. Moscarello. 1988. J. Chem. Soc., Faraday, Trans. 1 84:3821-3849). It was observed that the plots of CSA or dipolar coupling versus overall bilayer order obtained from DHPC and CHAPSO titrations were linear. Estimates of the intrinsic dipolar couplings and chemical shift anisotropies for pure DMPC bilayers were made by extrapolating shifts and couplings from the detergent titrations to zero detergent. Both detergent titrations led to similar "intrinsic" CSAs and dipolar couplings. Results extracted from an oriented Triton-DMPC mixture also led to similar estimates for the detergent-free DMPC shifts and couplings. The results from these experiments were found to compare favorably with limited measurements made from pure L alpha PC. This detergent-based method for assigning spectra and for determining dipolar couplings and CSA in detergent-free systems should be extendable to other lipid systems. The resulting data set from this study may prove useful in future modeling of the structure and dynamics of DMPC bilayers. In addition, the fact that experiments utilizing each of the three detergents led to similar estimates for the spectral parameters of pure DMPC, and the fact that spectral parameter versus bilayer order plots were linear, indicate that the averaged conformation and dynamics of DMPC in the presence of the three detergents are very similar to those of pure L alpha DMPC.

Biophysical Phenomena↗
PubMed1993

Orientational behavior of phosphatidylcholine bilayers in the presence of aromatic amphiphiles and a magnetic field.

A number of aromatic-containing additives which can influence the orientation of fragments of lipid bilayer membranes by a magnetic field have been investigated. Two properties of these additives prove important: (1) sufficient detergency to facilitate reorganization of bilayer components and (2), sufficient anisotropy in magnetic susceptibility the preferred direction of fragment orientation. Triton X-100 is identified as effective in terms of facilitating magnetic field ordering of bilayer fragments but does not alter the preferred direction of orientation. A combination of the detergent CHAPSO (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate) and the aromatic alcohol 1-naphthol facilitates both ordering and alters the preferred direction of bilayer orientation. As mixtures of dimyristoylphosphatidylcholine (DMPC) and CHAPSO, which orient with bilayer normals perpendicular to the magnetic field, were titrated with 1-naphthol, the assemblies underwent transitions, first to random orientation, and then to an orientation with bilayer normals parallel to the field. Based on temperature-induced phase transitions and the extent of motional averaging of the 31P shielding tensor of the DMPC headgroup, the DMPC in these oriented samples appears to maintain a bilayer morphology during transitions. The insight provided in this study regarding factors which influence fragment stability and orientation lays the groundwork for the design of improved field-oriented media for spectroscopic investigation of membrane components.

Biophysical Phenomena↗
PubMed1992

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.

Circular Dichroism↗
PubMed1992

Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR.

Mixtures of long-chain and short-chain phosphatidylcholine (PC) were characterized by multinuclear (13C, 31P, 2H) solid-state nuclear magnetic resonance. This work complements and extends previous characterization of such mixtures by focusing on concentrated mixtures at temperatures above the gel to liquid crystalline phase transition temperature (Tm) of the long-chain PC component. Above Tm it was observed that highly oriented, bilayer-like assemblies could be formed of mixtures of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) in molar ratios ranging from approximately 1:3.5 to 1:2 (DHPC:DMPC) over a considerable range of lipid concentrations (at least 3-40% w/v total lipid, for a 1:2.5 sample). Orientation was observed to occur only in an L alpha-like phase. The NMR data can be accounted for by a general model of the DHPC-DMPC aggregates in which DHPC can be found in two distinct populations (one highly ordered, one not). The averaged conformations of the glycerol backbone/headgroup regions of the long- and short-chain PC composing the assemblies were judged by solid-state 13C NMR to be similar to each other. The information gleaned about these mixtures and the quality of the oriented NMR spectra obtained suggest that DHPC-DMPC mixtures may prove to be useful as model membrane media in solid-state NMR studies of biomembranes.

Dimyristoylphosphatidylcholine↗
PubMed1990

Magnetically orientable phospholipid bilayers containing small amounts of a bile salt analogue, CHAPSO.

Buffered mixtures of the detergent 3-(cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO) and dimyristoylphosphatidylcholine (DMPC) orient in the presence of a strong magnetic field over a wide range of water contents (at least 65-85%) and CHAPSO:DMPC molar ratios (typically 1:10-1:3). 31P NMR studies show that the phospholipid in such mixtures is oriented with its director axis perpendicular to the magnetic field. 31P and 2H NMR results also suggest that the structure and dynamics of the DMPC molecules are similar to that of pure phospholipids existing in the liquid crystalline (L alpha) bilayer phase. The ability of 1:5 CHAPSO:DMPC samples to orient is highly tolerant of large changes in temperature, pH, and ionic strength, as well as to the addition of substantial amounts of charged amphiphiles or soluble protein. However, 2H NMR studies of deuterated beta-dodecyl melibiose (DD-MB) solubilized in the system indicate the head group conformation and/or dynamics of this glycolipid analogue is dependent upon the CHAPSO concentration. Despite the latter results, the orientational versatility of the system, together with the nondenaturing properties of CHAPSO, makes this system useful in spectroscopic studies of membrane-associated phenomena.

Cholic Acids↗
PubMed1989

Mechanism of adenylate kinase. Is there a relationship between local substrate dynamics, local binding energy, and the catalytic mechanism?

Adenylyl (beta,gamma-methylene)diphosphonic acid (AMPPCP) labeled with deuterium at the adenine ring ([8-2H]AMPPCP) and at the beta,gamma-methylene group (AMPPCD2P), as well as adenosine 5'-monophosphate labeled at the adenine ring ([8-2H]AMP), was synthesized and used for deuterium nuclear magnetic resonance (NMR) determination of effective correlation times (tau c) of the free nucleotide and the complexes with adenylate kinase (AK). Extensive and rigorous control experiments and theoretical analysis were performed to justify the validity of the experimental approaches, particularly the fast exchange condition, and the reliability of the tau c values obtained. For the free nucleotide, the results suggest that the phosphonate group of free AMPPCP possesses appreciable local mobility relative to the adenine ring and that complexation with Mg2+ greatly reduced such a local mobility. For the complexes with AK, effective tau c values of 7, 15, 28, 28, and 27 ns were obtained for AMPPCD2P, MgAMPPCD2P, [8-2H]AMPPCP, Mg[8-2H]AMPPCP, and [8-2H]AMP, respectively. These results suggest that the adenine ring of substrates is rigidly bound in all cases, that the phosphonate chain of AMPPCP possesses considerable local mobility, and that Mg2+ reduces such local mobility but does not totally immobilize it. The local dynamics of the analogues bound to AK was correlated with local binding energies for the binding of MgAMPPCP and MgATP to AK estimated from the binding studies by proton NMR and other techniques, in conjunction with the binding theory of Jencks [Jencks, W. P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4046-4050]. The results suggest that no general correlation exists between the local rigidity of portions of a bound substrate and the corresponding (ground state) local binding energy contributed by these portions. In particular, the adenosine moiety contributes little to the binding energy despite the fact that the adenine ring is rigidly bound; the triphosphate (PPPi) moiety behaves oppositely; Mg2+ immobilizes the triphosphate chain but does not enhance binding. Finally, isomers of the substitution-inert beta,gamma-bidentate Cr(III) complexes of adenosine 5'-triphosphate (CrATP) were used to probe two unresolved catalytic problems implicitly related to the local mobility of the phosphonate chain of AMPPCP in the AK-MgAMPPCP complex. The first problem concerns the result of electron paramagnetic resonance (EPR) studies that (Rp)- but not (Sp)-[beta-17O]ATP caused a line broadening in the Mn(II) EPR spectrum of the AK-MnATP complex [Kalbitzer, H. R., Marquetant, R., Connolly, B. A., & Goody, R. S. (1983) Eur. J. Biochem. 133, 221-227].(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine↗
PubMed1988

Mechanism of adenylate kinase. Histidine-36 is not directly involved in catalysis, but protects cysteine-25 and stabilizes the tertiary structure.

Several previous reports on muscle adenylate kinase (AK) have suggested that histidine-36 (His-36) is located in the binding site of adenosine 5'-triphosphate (ATP) and is involved in catalysis. We have tested the role of His-36 using site-specific mutagenesis on chicken muscle AK expressed in Escherichia coli. Three mutant proteins (H36Q, H36N, and H36G) were obtained by substituting His-36 with glutamine, asparagine, and glycine, respectively. Steady-state kinetic studies showed that the mutants have similar kinetic properties to those of the wild-type (WT) AK, which suggested that His-36 is not directly involved in catalysis. However, His-36 is likely to interact with or protect cysteine-25 (Cys-25) on the basis of the following evidence: The crystal structure of porcine muscle AK revealed a close proximity between His-36 and Cys-25; the mutants were unstable during purification (the order of stability was WT greater than H36Q greater than H36N greater than H36G); the H36G mutant readily dimerized; the sulfhydryl groups of mutants became more reactive (WT less than H36Q less than H36N) toward 5,5'-dithiobis(2-nitrobenzoic acid). Furthermore, His-36 was found to stabilize the tertiary structure of AK on the basis of guanidine hydrochloride induced denaturation studies, which showed that the conformational stability decreases in the order WT greater than H36Q greater than H36N.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Kinase↗