Biomedical subjects

J P Schwonek

Publications and source records attributed to J P Schwonek.

6 recordsLinked to original sources

PubMed1995

Structural basis for actomyosin chemomechanical transduction by non-nucleoside triphosphate analogues.

Methylation of 2-[(2,4-dinitrophenyl)amino]ethyl triphosphate (dNOTP) was found to abolish its ability to support actin sliding in the in vitro motility assay. A comparative study of the interaction of myosin subfragment 1 (S1) and actoS1 with methylated (MdNOTP) and non-methylated dNOTP was undertaken. Both analogues were shown to be substrates for S1 NTPase in the presence of K+/EDTA, Ca2+, or Mg2+, although their rates of hydrolysis in the presence of the divalent cations were significantly greater than that occurring for ATP. However, actin had only a marginal effect on the rate of hydrolysis of MdNOTP, in sharp contrast to its effect on the hydrolysis of dNOTP and ATP which were quite similar. Moreover, while dNODP is able to form stable ternary S1 complexes with orthovanadate (Vi) or berylium fluoride (BeFx), whose formation results in increased thermal stability of S1, the methylated diphosphate analogue was unable to do so. These differences can be related to methylation-induced changes in the conformation of dNOTP indicated by molecular-modeling approaches. These studies suggest that methylation prevents the specific interaction of the aryl ring of dNOTP with S1 in the adenine binding region necessary for the formation of the force-producing intermediate (M. D. P*) during the S1 Mg(2+)-NTPase cycle.

Acid Anhydride Hydrolases↗

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↗

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↗

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↗