Hydrodynamic Hunters.

The Gram-negative Bdellovibrio bacteriovorus (BV) is a model bacterial predator that hunts other bacteria and may serve as a living antibiotic. Despite over 50 years since its discovery, it is suggested that BV probably collides into its prey at random. It remains unclear to what degree, if any, BV uses chemical cues to target its prey. The targeted search problem by the predator for its prey in three dimensions is a difficult problem: it requires the predator to sensitively detect prey and forecast its mobile prey's future position on the basis of previously detected signal. Here instead we find that rather than chemically detecting prey, hydrodynamics forces BV into regions high in prey density, thereby improving its odds of a chance collision with prey and ultimately reducing BV's search space for prey. We do so by showing that BV's dynamics are strongly influenced by self-generated hydrodynamic flow fields forcing BV onto surfaces and, for large enough defects on surfaces, forcing BV in orbital motion around these defects. Key experimental controls and calculations recapitulate the hydrodynamic origin of these behaviors. While BV's prey (Escherichia coli) are too small to trap BV in hydrodynamic orbit, the prey are also susceptible to their own hydrodynamic fields, substantially confining them to surfaces and defects where mobile predator and prey density is now dramatically enhanced. Colocalization, driven by hydrodynamics, ultimately reduces BV's search space for prey from three to two dimensions (on surfaces) even down to a single dimension (around defects). We conclude that BV's search for individual prey remains random, as suggested in the literature, but confined, however-by generic hydrodynamic forces-to reduced dimensionality.

Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures.

The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.

Equivalent Isopropanol Concentrations of Aromatic Amino Acids Interactions with Lipid Vesicles.

We show that the interaction of aromatic amino acids with lipid bilayers can be characterized by conventional 1D [Formula: see text]H NMR spectroscopy using reference spectra obtained in isopropanol-d8/D[Formula: see text]O solutions. We demonstrate the utility of this method with three different peptides containing tyrosine, tryptophan, or phenylalanine amino acids in the presence of 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphoserine lipid membranes. In each case, we determine an equivalent isopropanol concentration (EIC) for each hydrogen site of aromatic groups, in essence constructing a map of the chemical environment. These EIC maps provide information on relative affinities of aromatic side chains for either PC or PS bilayers and also inform on amino acid orientation preference when bound to membranes.

Electrodynamics of lipid membrane interactions in the presence of zwitterionic buffers.

Due to thermal motion and molecular polarizability, electrical interactions in biological systems have a dynamic character. Zwitterions are dipolar molecules that typically are highly polarizable and exhibit both a positive and a negative charge depending on the pH of the solution. We use multilamellar structures of common lipids to identify and quantify the effects of zwitterionic buffers that go beyond the control of pH. We use the fact that the repeat spacing of multilamellar lipid bilayers is a sensitive and accurate indicator of the force balance between membranes. We show that common buffers can in fact charge up neutral membranes. However, this electrostatic effect is not immediately recognized because of the concomitant modification of dispersion (van der Waals) forces. We show that although surface charging can be weak, electrostatic forces are significant even at large distances because of reduced ionic screening and reduced van der Waals attraction. The zwitterionic interactions that we identify are expected to be relevant for interfacial biological processes involving lipid bilayers, and for a wide range of biomaterials, including amino acids, detergents, and pharmaceutical drugs. An appreciation of zwitterionic electrodynamic character can lead to a better understanding of molecular interactions in biological systems and in soft materials in general.

Productive versus unproductive nucleotide binding in yeast guanylate kinase mutants: comparison of R41M with K14M by proton two dimensional transferred NOESY.

The R41M and K14M mutant enzymes of yeast guanylate kinase (GKy) were studied to investigate the effects of these site-directed mutations on bound-substrate conformations. Published X-ray crystal structures of yeast guanylate kinase indicate that K14 is part of the "P" loop involved in ATP and ADP binding, while R41 is suggested as a hydrogen bonding partner for the phosphoryl moiety of GMP. Both of these residues might be involved in transition state stabilization. Adenosine conformations of ATP and ADP and guanosine conformations of GMP bound to R41M and K14M mutant yeast guanylate kinase in the complexes GKy.MgATP, GKy.MgADP, and GKy.MgADP.[u-(13)C]GMP were determined by two-dimensional transferred nuclear Overhauser effect (TRNOESY) measurements combined with molecular dynamics simulations, and these conformations were compared with previously published conformations for the wild type. In the fully constrained, two substrate complexes, GKy.MgADP.[u-(13)C]GMP, the guanyl glycosidic torsion angle, chi, is 51 +/- 5 degrees for R41M and 47 +/- 5 degrees for K14M. Both are similar to the published 50 +/- 5 degrees published for wild type. For R41M with adenyl nucleotides, the glycosidic torsion angle, chi, was 55 +/- 5 degrees with MgATP, and 47 +/- 5 degrees with MgADP, which compares well to the 54 +/- 5 degrees published for wild type. However, for K14M with adenyl nucleotides, the glycosidic torsion angle was 30 +/- 5 degrees with MgATP and 28 +/- 5 degrees with MgADP. The results indicate that bound adenyl-nucleotides have significantly different conformations in the wild-type and K14M mutant enzymes, suggesting that K14 plays an important role in orienting the triphosphate of MgATP for catalysis.

Direct affinity of dopamine to lipid membranes investigated by Nuclear Magnetic Resonance spectroscopy.

Dopamine, a naturally occurring neurotransmitter, plays an important role in the brain's reward system and acts on sensory receptors in the brain. Neurotransmitters are contained in lipid membraned vesicles and are released by exocytosis. All neurotransmitters interact with transport and receptor proteins in glial cells, on neuronal dendrites, and at the axonal button, and also must interact with membrane lipids. However, the extent of direct interaction between lipid membranes in the absence of receptors and transport proteins has not been extensively investigated. In this report, we use UV and NMR spectroscopy to determine the affinity and the orientation of dopamine interacting with lipid vesicles made of either phosphatidylcholine (PC) or phosphatidylserine (PS) lipids which are primary lipid components of synaptic vesicles. We quantify the interaction of dopamine's aromatic ring with lipid membranes using our newly developed method that involves reference spectra in hydrophobic environments. Our measurements show that dopamine interacts with lipid membranes primarily through the aromatic side opposite to the hydroxyl groups, with this aromatic side penetrating deeper into the hydrophobic region of the membrane. Since dopamine's activity involves its release into extracellular space, we have used our method to also investigate dopamine's release from lipid vesicles. We find that dopamine trapped inside PC and PS vesicles is released into the external solution despite its affinity to membranes. This result suggests that dopamine's interaction with lipid membranes is complex and involves both binding as well as permeation through lipid bilayers, a combination that could be an effective trigger for apoptosis of dopamine-generating cells.

Conformations of nucleotides bound to wild type and Y78F mutant yeast guanylate kinase: proton two-dimensional transferred NOESY measurements.

Wild type and Y78F mutant yeast guanylate kinase (GKy) were studied to investigate the effects of a site-directed mutation on bound substrate conformations. Previously published work showed that Y78 is involved in GMP binding and that the Y78F mutant has 30-fold weaker GMP binding and 2 orders of magnitude less activity, than the wild type. Adenosine conformations of adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP) and guanosine conformations of guanosine 5'-monophosphate (GMP) bound to wild type and Y78F mutant yeast guanylate kinase in the complexes GKy x Mg(II)ATP, GKy x Mg(II)ADP, GKy x GMP, and GKy x Mg(II)ADP x [U-13C]GMP were determined by two-dimensional transferred nuclear Overhauser effect (TRNOESY) measurements combined with molecular dynamics simulations. For adenyl nucleotides in wild type complexes, all glycosidic torsion angles, chi, were 54 +/- 5 degrees. In Y78F mutant complexes, adenyl nucleotide glycosidic torsion angles were 55 +/- 5 degrees (GKy x MgATP) and 49 +/- 5 degrees (GKy x MgADP). Thus, the adenyl nucleotides bind similarly for both the wild type and Y78F mutant complexes. However, in the fully constrained, two-substrate complexes, GKy x Mg(II)ADP x [U-13C]GMP, the guanyl glycosidic torsion angle, chi, is 50 +/- 5 degrees with the wild type and 83 +/- 5 degrees with the Y78F mutant. This difference suggests that an unfavorable torsion may be a large part of the mechanism for significantly weaker GMP binding to reaction complexes of the Y78F mutant.

Quantitation of movement of the phosphoryl group during catalytic transfer in the arginine kinase reaction: 31P relaxation measurements on enzyme-bound equilibrium mixtures.

31P nuclear spin relaxation measurements have been made on enzyme-bound equilibrium mixtures of lobster-muscle arginine kinase in the presence of substituent activating paramagnetic cation Co(II) (in place of Mg(II)), i.e., on samples in which the reaction, E.CoATP.arginine <=> E.CoADP.P-arginine, is in progress. The results have been analyzed on the basis of a previously published theory (Nageswara Rao, B.D. (1995) J. Magn. Reson., B108, 289-293) to determine the structural changes in the reaction complex accompanying phosphoryl transfer. The analysis enables the determination of the change in the Co(II)-31P (gamma-P(ATP)) vector as the transferable phosphoryl group moves over and attaches to arginine to form P-arginine. It is shown that the Co(II)-31P distance of approximately 3.0 A, representing direct coordination of Co(II) to gamma-P(ATP), changes to approximately 4.0 A when P-arginine is formed in the enzyme-bound reaction complex. This elongation of the Co(II)-31P vector implies an excursion of at least 1.0 A for the itinerant phosphoryl group on the surface of the enzyme.

Changes in the 31P NMR spectrum of rabbit muscle myosin subfragment 1. MgADP with temperature.

In pioneering studies on the 31P NMR spectra of MgADP bound to the "molecular motor" myosin subfragment 1 (S1) in the temperature range of 0 to 25 degrees C, Shriver and Sykes [Biochemistry 20 (1981) 2004-2012/6357-6362; Biochemistry 21 (1982) 3022-3028], proposed that MgADP binds to myosin S1 as a mixture of two interconvertible conformers with different chemical shifts for the beta-P resonance of the S1-bound MgADP and that the concentrations of these conformers are related by an equilibrium constant K(T). Their model implied that the weighted average of the chemical shifts of the beta-P(MgADP) for S1-bound MgADP asymptotically approaches a high temperature limit. Here, and in our earlier paper [K. Konno, K. Ue, M. Khoroshev, H., Martinez, B.D. Ray, M.F. Morales, Proc. Natl. Acad. Sci. USA 97 (2000) 1461-1466], we report experimental similarities to Shriver and Sykes, but diverge from them (especially at 0 degrees C) in not finding two distinct peaks and in finding that the average chemical shift does not change with temperature. Our observations can be explained by chemical exchange of beta-P(MgADP) of S1-bound MgADP between two nearly energetically equivalent environments.

Ni(II).Arg-Gly-His-DNA interactions: investigation into the basis for minor-groove binding and recognition.

A study of the minor-groove recognition of A/T-rich DNA sites by Ni(II).L-Arg-Gly-His and Ni(II).D-Arg-Gly-His was carried out with a fluorescence-based binding assay, one- and two-dimensional (1D and 2D) NMR methodologies, and molecular simulations. Fluorescence displacement titrations revealed that Ni(II).L-Arg-Gly-His binds to A/T-rich sequences better than the D-Arg diastereomer, while NMR investigations revealed that both metallopeptides bind to the minor groove of an AATT core region as evidenced by an intermolecular nuclear Overhauser effect (NOE) between each metallopeptide His imidazole C4 proton and the C2 proton of adenine. Results from molecular dynamics simulations of these systems were consistent with the experimental data and indicated that the His imidazole N-H, the N-terminal peptide amine, and Arg side chains of each metallopeptide are major determinants of minor-groove recognition by functioning as H-bond donors to the O2 of thymine residues or N3 of adenine residues.