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Bacterial resistance to antibiotics has been long recognized as a priority to address for human health. Among all micro-organisms, the so-called multi-drug resistant (MDR) bacteria, which are resistant to most, if not all drugs in our current arsenal, are particularly worrisome. The World Health Organization has prioritized the ESKAPE ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa and Enterobacter species) pathogens, which include four Gram-negative bacterial species. In these bacteria, active extrusion of antimicrobial compounds out of the cell by means of ‘molecular guns’ known as efflux pumps is a main determinant of MDR phenotypes. The resistance-nodulation-cell division (RND) superfamily of efflux pumps connecting the inner and outer membrane in Gram-negative bacteria is crucial to the onset of MDR and virulence, as well as biofilm formation. Thus, understanding the molecular basis of the interaction of antibiotics and inhibitors with these pumps is key to the design of more effective therapeutics. With the aim to contribute to this challenge, and complement and inspire experimental research, in silico studies on RND efflux pumps have flourished in recent decades. Here, we review a selection of such investigations addressing the main determinants behind the polyspecificity of these pumps, the mechanisms of substrate recognition, transport and inhibition, as well as the relevance of their assembly for proper functioning, and the role of protein–lipid interactions. The journey will end with a perspective on the role of computer simulations in addressing the challenges posed by these beautifully complex machineries and in supporting the fight against the spread of MDR bacteria.

Over the last two decades, an increasing number of studies has been devoted to a deeper understanding of the molecular process involved in the binding of various agonists and antagonists to active and inactive conformations of β2-adrenergic receptor (β2 AR). The 3.2 A X-ray crystal structure of human β2 AR active-state in combination with the endogenous low affinity agonist adrenaline offers an ideal starting structure for studying the binding of various catecholamines to adrenergic receptors. We show that molecular docking of L-DOPA and Droxidopa into rigid and flexible β2 AR models leads for both ligands to binding anchor sites comparable to those experimentally reported for adrenaline, namely D113/N312 and S203/S204/S207 side chains. Both ligands have a hydrogen bond network that is extremely similar to those of noradrenaline and dopamine. Interestingly, re-docking neutral and protonated versions of adrenaline to rigid and flexible β2 AR models results in binding poses that are more energetically stable and distinct from the X-ray crystal structure. Similarly, lowest energy conformations of noradrenaline and dopamine generated by docking into flexible β2 AR models had binding free energies lower than those of best poses in rigid receptor models. Furthermore, our findings show that L-DOPA and Droxidopa molecules have binding affinities comparable to those predicted for adrenaline, noradrenaline, and dopamine, which are consistent with previous experimental and computational findings and supported by the MD simulations of β2AR-ligand complexes performed here. SIGNIFICANCE The β2-adrenergic receptor (β2 AR), which belongs to the vast family of Guanine nucleotide-binding protein coupled receptors (GPCRs), is a transmembrane protein that is activated by the catecholamines norepinephrine (noradrenaline) and epinephrine (adrenaline). Using molecular docking methods, we discovered that the exogenous ligands L-DOPA and Droxidopa have the same hydrogen bond binding sites as endogenous agonists such as adrenaline, noradrenaline, and dopamine. Furthermore, all of the catecholamines studied had different hydrophobic binding sites in the receptor. Our findings might help researchers to better understand how existing and new drugs chemically similar to Droxidopa, which is used to treat Parkinson's disease, interact with β2 AR.

An integrated theoretical/experimental strategy has been applied to the study of environmental effects on the spectroscopic parameters of 4-(diphenylamino)phtalonitrile (DPAP), a fluorescent molecular rotor. The computational part starts from the development of an effective force field for the first excited electronic state of DPAP and proceeds through molecular dynamics simulations in solvents of different polarities toward the evaluation of Stokes shifts by quantum mechanics/molecular mechanics (QM/MM) approaches. The trends of the computed results closely parallel the available experimental results thus giving confidence to the interpretation of new experimental studies of the photophysics of DPAP in lipid bilayers. In this context, results show unambiguously that both flexible dihedral angles and global rotations are significantly retarded in a cholesterol/DPPC lipid matrix with respect to the DOPC matrix, thus confirming the sensitivity of DPAP to probe different environments and, therefore, its applicability as a probe for detecting different structures and levels of plasma membrane organization.

Fluorescent probes are widely employed to label lipids for the investigation of structural and dynamic properties of model and cell membranes through optical microscopy techniques. Although the effect of tagging a lipid with an organic dye is generally assumed to be negligible, optically modified lipids can nonetheless affect the local lipid structure and, in turn, the lipid lateral mobility. To better assess this potential issue, all-atom (MD) molecular dynamics simulations have been performed to study structural and dynamic effects in a model DOPC membrane in the presence of a standard Rhodamine B-labeled DOPE lipid (RHB) as a function of temperature, i.e., 293K, 303K, and 320K. As the temperature is increased, we observe similar changes in the structural properties of both pure DOPC and RHB-DOPC lipid bilayers: an increase of the area per lipid, a reduction of the membrane thickness and a decrease of lipid order parameters. The partial density profile of the RHB headgroups and their orientation within the lipid bilayer confirm the amphiphilic nature of the RHB fluorescent moiety, which mainly partitions in the DOPC glycerol backbone region at each temperature. Moreover, at all temperatures, our results on lipid lateral diffusion support a non-neutral role of the dye with respect to the unlabeled lipid mobility, thus suggesting important implications for optical microscopy studies of lipid membranes.

Over the last two decades, an increasing number of studies has been devoted to a deeper understanding of the molecular process involved in the binding of various agonists and antagonists to active and inactive conformations of β(2)-adrenergic receptor (β(2)AR). The 3.2 Å x-ray crystal structure of human β(2)AR active state in combination with the endogenous low affinity agonist adrenaline offers an ideal starting structure for studying the binding of various catecholamines to adrenergic receptors. We show that molecular docking of levodopa (L-DOPA) and droxidopa into rigid and flexible β(2)AR models leads for both ligands to binding anchor sites comparable to those experimentally reported for adrenaline, namely D113/N312 and S203/S204/S207 side chains. Both ligands have a hydrogen bond network that is extremely similar to those of noradrenaline and dopamine. Interestingly, redocking neutral and protonated versions of adrenaline to rigid and flexible β(2)AR models results in binding poses that are more energetically stable and distinct from the x-ray crystal structure. Similarly, lowest energy conformations of noradrenaline and dopamine generated by docking into flexible β(2)AR models had binding free energies lower than those of best poses in rigid receptor models. Furthermore, our findings show that L-DOPA and droxidopa molecules have binding affinities comparable to those predicted for adrenaline, noradrenaline, and dopamine, which are consistent with previous experimental and computational findings and supported by the molecular dynamics simulations of β(2)AR-ligand complexes performed here.

Lipid lateral diffusion in membrane bilayers is a fundamental process exploited by cells to enable complex protein structural and dynamic reorganizations. For its importance, lipid mobility in both cellular and model bilayers has been extensively investigated in recent years, especially through the application of time-resolved, fluorescence-based, optical microscopy techniques. However, one caveat of fluorescence techniques is the need to use dye-labeled variants of the lipid of interest, thus potentially perturbing the structural and dynamic properties of the native species. Generally, the effect of the dye/tracer molecule is implicitly assumed to be negligible. Nevertheless, in view of the widespread use of optically modified lipids for studying lipid bilayer dynamics, it is highly desirable to well assess this point. Here, fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations have been combined together to uncover subtle structural and dynamic effects in DOPC planar membranes enriched with a standard Rhodamine-labeled lipid. Our findings support a non-neutral role of the dye-labeled lipids in diffusion experiments, quantitatively estimating a decrease in lipid mobility of up to 20% with respect to the unlabeled species. Moreover, results highlight the existing interplay between dye concentration, lipid lateral diffusion and membrane permeability, thus suggesting possible implications for future optical microscopy studies of biophysical processes occurring at the membrane level.

The voltage-gated potassium channel Kv4.3 plays a vital role in shaping the timing, frequency, and backpropagation of electrical signals in the brain and heart by generating fast transient currents at subthreshold membrane potentials in repetitive firing neurons. To achieve its physiological function, Kv4.3 is assisted by auxiliary β-subunits that become integral parts of the native A-type potassium channels, among which there are the Kv channel-interacting proteins (KChIPs). KChIPs are a family of cytosolic proteins that, when coexpressed with Kv4, lead to higher current density, modulation of channel inactivation and faster recovery from inactivation, while the loss of KChIP function may lead to severe pathological states. Recently, the structural basis of the KChIP1-Kv4.3 interaction was reported by using two similar X-ray crystallographic structures, which supported a crucial role for KChIP1 in enhancing the stability of the Kv4.3 tetrameric assembly, thus helping the trafficking of the channel to the plasma membrane. Here, we investigate through fully atomistic simulations the structure and stability of the human Kv4.3 tetramerization (T1) domain in complex with KChIP1 upon specific mutations located in the first and second interfaces of the complex, as compared to the wild-type (WT). Our results nicely complement the available structural and biophysical information collected so far on these complex variants. In particular, the degree of structural deviations and energetic instability, from small to substantial, observed in these variants with respect to the WT model seems to parallel well the level of channel dysfunction known from electrophysiology data. Our simulations provide an octameric structure of the WT KChIP1-Kv4.3 assembly very similar to the known crystal structures, and, at the same time, highlight the importance of a previously overlooked site of interaction between KChIP1 and the Kv4.3 T1 domain.

Of the many biophysical techniques now being brought to bear on studies of membranes, electron paramagnetic resonance (EPR) of nitroxide spin probes was the first to provide information about both mobility and ordering in lipid membranes. Here, we report the first prediction of variable temperature EPR spectra of model lipid bilayers in the presence and absence of cholesterol from the results of large scale fully atomistic molecular dynamics (MD) simulations. Three types of structurally different spin probes were employed in order to study different parts of the bilayer. Our results demonstrate very good agreement with experiment and thus confirm the accuracy of the latest lipid force fields. The atomic resolution of the simulations allows the interpretation of the molecular motions and interactions in terms of their impact on the sensitive EPR line shapes. Direct versus indirect effects of cholesterol on the dynamics of spin probes are analysed. Given the complexity of structural organisation in lipid bilayers, the advantage of using a combined MD‐EPR simulation approach is two‐fold. Firstly, prediction of EPR line shapes directly from MD trajectories of actual phospholipid structures allows unambiguous interpretation of EPR spectra of biological membranes in terms of complex motions. Secondly, such an approach provides an ultimate test bed for the up‐to‐date MD simulation models employed in the studies of biological membranes, an area that currently attracts great attention.

Antimicrobial peptides (AMPs) are small cationic proteins that are able to destabilize a lipid bilayer structure through one or more modes of action. In this study, we investigate the processes of peptide aggregation and pore formation in lipid bilayers and vesicles by the highly cationic AMP, Chrysophsin-3 (chrys-3), using coarse-grained molecular dynamics (CG-MD) simulations and potential of mean force calculations. We study long 50 μs simulations of chrys-3 at different concentrations, both at the surface of dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylcholine (POPC) bilayers, and also interacting within the interior of the lipid membrane. We show that aggregation of peptides at the surface, leads to pronounced deformation of lipid bilayers, leading in turn to lipid protrusions for peptide : ligand ratios > 1 : 12. In addition, aggregation of chrys-3 peptides within the centre of a lipid bilayer leads to spontaneous formation of pores and aggregates. Both mechanisms of interaction are consistent with previously reported experimental data for chrys-3. Similar results are observed also in POPC vesicles and mixed lipid bilayers composed of the zwitterionic lipid palmitoyloleoylphosphatidylethanolamine (POPE) and the negatively charged lipid palmitoyloleoylphosphatidylglycerol (POPG). The latter are employed as models of the bacterial membrane of Escherichia coli.

Antimicrobial peptides (AMPs) are small cationic proteins that are able to destabilize a lipid bilayer structure through one or more modes of action. In this study, we investigate the processes of peptide aggregation and pore formation in lipid bilayers and vesicles by the highly cationic AMP, Chrysophsin-3 (chrys-3), using coarse-grained molecular dynamics (CG-MD) simulations and potential of mean force calculations. We study long 50 μs simulations of chrys-3 at different concentrations, both at the surface of dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylcholine (POPC) bilayers, and also interacting within the interior of the lipid membrane. We show that aggregation of peptides at the surface, leads to pronounced deformation of lipid bilayers, leading in turn to lipid protrusions for peptide : ligand ratios1 : 12. In addition, aggregation of chrys-3 peptides within the centre of a lipid bilayer leads to spontaneous formation of pores and aggregates. Both mechanisms of interaction are consistent with previously reported experimental data for chrys-3. Similar results are observed also in POPC vesicles and mixed lipid bilayers composed of the zwitterionic lipid palmitoyloleoylphosphatidylethanolamine (POPE) and the negatively charged lipid palmitoyloleoylphosphatidylglycerol (POPG). The latter are employed as models of the bacterial membrane of Escherichia coli.

Summary Biogenesis of high-density lipoproteins (HDL) is coupled to the transmembrane protein, ATP-binding cassette transporter A1 (ABCA1), which transports phospholipid (PL) from the inner to the outer membrane monolayer. Using a combination of computational and experimental approaches, we show that increased outer lipid monolayer surface density, driven by excess PL or membrane insertion of amphipathic helices, results in pleating of the outer monolayer to form membrane-attached discoidal bilayers. Apolipoprotein (apo)A-I accelerates and stabilizes the pleats. In the absence of apoA-I, pleats collapse to form vesicles. These results mimic cells overexpressing ABCA1 that, in the absence of apoA-I, form and release vesicles. We conclude that the basic driving force for nascent discoidal HDL assembly is a PL pump-induced surface density increase that produces lipid monolayer pleating. We then argue that ABCA1 forms an extracellular reservoir containing an isolated pressurized lipid monolayer decoupled from the transbilayer density buffering of cholesterol.

Apolipoprotein (apo) A-I-containing lipoproteins in the form of high-density lipoproteins (HDL) are inversely correlated with atherosclerosis. Because HDL is a soft form of condensed matter easily deformable by thermal fluctuations, the molecular mechanisms for HDL remodeling are not well understood. A promising approach to understanding HDL structure and dynamics is molecular dynamics (MD). In the present study, two computational strategies, MD simulated annealing (MDSA) and MD temperature jump, were combined with experimental particle reconstitution to explore molecular mechanisms for phospholipid- (PL-) rich HDL particle remodeling. The N-terminal domains of full-length apoA-I were shown to be "sticky", acting as a molecular latch largely driven by salt bridges, until, at a critical threshold of particle size, the associated domains released to expose extensive hydrocarbon regions of the PL to solvent. The "sticky" N-termini also associate with other apoA-I domains, perhaps being involved in N-terminal loops suggested by other laboratories. Alternatively, the overlapping helix 10 C-terminal domains of apoA-I were observed to be extremely mobile or "promiscuous", transiently exposing limited hydrocarbon regions of PL. Based upon these models and reconstitution studies, we propose that separation of the N-terminal domains, as particles exceed a critical size, triggers fusion between particles or between particles and membranes, while the C-terminal domains of apoA-I drive the exchange of polar lipids down concentration gradients between particles. This hypothesis has significant biological relevance since lipid exchange and particle remodeling are critically important processes during metabolism of HDL particles at every step in the antiatherogenic process of reverse cholesterol transport.

For several decades, the standard model for high density lipoprotein (HDL) particles reconstituted from apolipoprotein A-I (apoA-I) and phospholipid (apoA-I/HDL) has been a discoidal particle ∼100 A in diameter and the thickness of a phospholipid bilayer. Recently, Wu et al. (Wu, Z., Gogonea, V., Lee, X., Wagner, M. A., Li, X. M., Huang, Y., Undurti, A., May, R. P., Haertlein, M., Moulin, M., Gutsche, I., Zaccai, G., Didonato, J. A., and Hazen, S. L. (2009) J. Biol. Chem. 284, 36605-36619) used small angle neutron scattering to develop a new model they termed double superhelix (DSH) apoA-I that is dramatically different from the standard model. Their model possesses an open helical shape that wraps around a prolate ellipsoidal type I hexagonal lyotropic liquid crystalline phase. Here, we used three independent approaches, molecular dynamics, EM tomography, and fluorescence resonance energy transfer spectroscopy (FRET) to assess the validity of the DSH model. (i) By using molecular dynamics, two different approaches, all-atom simulated annealing and coarse-grained simulation, show that initial ellipsoidal DSH particles rapidly collapse to discoidal bilayer structures. These results suggest that, compatible with current knowledge of lipid phase diagrams, apoA-I cannot stabilize hexagonal I phase particles of phospholipid. (ii) By using EM, two different approaches, negative stain and cryo-EM tomography, show that reconstituted apoA-I/HDL particles are discoidal in shape. (iii) By using FRET, reconstituted apoA-I/HDL particles show a 28-34-A intermolecular separation between terminal domain residues 40 and 240, a distance that is incompatible with the dimensions of the DSH model. Therefore, we suggest that, although novel, the DSH model is energetically unfavorable and not likely to be correct. Rather, we conclude that all evidence supports the likelihood that reconstituted apoA-I/HDL particles, in general, are discoidal in shape.

Conversion of discoidal phospholipid (PL)-rich high density lipoprotein (HDL) to spheroidal cholesteryl ester-rich HDL is a central step in reverse cholesterol transport. A detailed understanding of this process and the atheroprotective role of apolipoprotein A-I (apoA-I) requires knowledge of the structure and dynamics of these various particles. This study, combining computation with experimentation, illuminates structural features of apoA-I allowing it to incorporate varying amounts of PL. Molecular dynamics simulated annealing of PL-rich HDL models containing unesterified cholesterol results in double belt structures with the same general saddle-shaped conformation of both our previous molecular dynamics simulations at 310 K and the x-ray structure of lipid-free apoA-I. Conversion from a discoidal to a saddle-shaped particle involves loss of helicity and formation of loops in opposing antiparallel parts of the double belt. During surface expansion caused by the temperature-jump step, the curved palmitoyloleoylphosphatidylcholine bilayer surfaces approach planarity. Relaxation back into saddle-shaped structures after cool down and equilibration further supports the saddle-shaped particle model. Our kinetic analyses of reconstituted particles demonstrate that PL-rich particles exist in discrete sizes corresponding to local energetic minima. Agreement of experimental and computational determinations of particle size/shape and apoA-I helicity provide additional support for the saddle-shaped particle model. Truncation experiments combined with simulations suggest that the N-terminal proline-rich domain of apoA-I influences the stability of PL-rich HDL particles. We propose that apoA-I incorporates increasing PL in the form of minimal surface bilayers through the incremental unwinding of an initially twisted saddle-shaped apoA-I double belt structure.

The product of transesterification of phospholipid acyl chains and unesterified cholesterol (UC) by the enzyme lecithin:cholesterol acyltransferase (LCAT) is cholesteryl ester (CE). Activation of LCAT by apolipoprotein (apo) A-I on nascent (discoidal) high-density lipoproteins (HDL) is essential for formation of mature (spheroidal) HDL during the antiatherogenic process of reverse cholesterol transport. Here we report all-atom and coarse-grained (CG) molecular dynamics (MD) simulations of HDL particles that have major implications for mechanisms of LCAT activation. Both the all-atom and CG simulations provide support for a model in which the helix 5/5 domains of apoA-I create an amphipathic "presentation tunnel" that exposes methyl ends of acyl chains at the bilayer center to solvent. Further, CG simulations show that UC also becomes inserted with high efficiency into the amphipathic presentation tunnel with its hydroxyl moiety (UC-OH) exposed to solvent; these results are consistent with trajectory analyses of the all-atom simulations showing that UC is being concentrated in the vicinity of the presentation tunnel. Finally, consistent with known product inhibition of CE-rich HDL by CE, CG simulations of CE-rich spheroidal HDL indicate partial blockage of the amphipathic presentation tunnel by CE. These results lead us to propose the following working hypothesis. After attachment of LCAT to discoidal HDL, the helix 5/5 domains in apoA-I form amphipathic presentation tunnels for migration of hydrophobic acyl chains and amphipathic UC from the bilayer to the phospholipase A2-like and esterification active sites of LCAT, respectively. This hypothesis is currently being tested by site-directed mutagenesis.

Apolipoprotein (apo) A-I is an unusually flexible protein whose lipid-associated structure is poorly understood. Thermal denaturation, which is used to measure the global helix stability of high-density lipoprotein (HDL)-associated apoA-I, provides no information about local helix stability. Here we report the use of temperature jump molecular dynamics (MD) simulations to scan the per-residue helix stability of apoA-I in phospholipid-rich HDL. When three 20 ns MD simulations were performed at 500 K on each of two particles created by MD simulations at 310 K, bilayers remained intact but expanded by 40%, and total apoA-I helicity decreased from 95% to 72%. Of significance, the conformations of the overlapping N- and C-terminal domains of apoA-I in the particles were unusually mobile, exposing hydrocarbon regions of the phospholipid to solvent; a lack of buried interhelical salt bridges in the terminal domains correlated with increased mobility. Nondenaturing gradient gels show that 40% expansion of the phospholipid surface of 100:2 particles by addition of palmitoyloleoylphosphatidylcholine exceeds the threshold of particle stability. As a unifying hypothesis, we propose that the terminal domains of apoA-I are phospholipid concentration-sensitive molecular triggers for fusion/remodeling of HDL particles. Since HDL remodeling is necessary for cholesterol transport, our model for remodeling has substantial biomedical implications.

Spheroidal high-density lipoprotein (HDL) particles circulating in the blood are formed through an enzymatic process activated by apoA-I, leading to the esterification of cholesterol, which creates a hydrophobic core of cholesteryl ester molecules in the middle of the discoidal phospholipid bilayer. In this study, we investigated the conformation of apoA-I in model spheroidal HDL (ms-HDL) particles using both atomistic and coarse-grained molecular dynamics simulations, which are found to provide consistent results for all HDL properties we studied. The observed small contribution of cholesteryl oleate molecules to the solvent-accessible surface area of the entire ms-HDL particle indicates that palmitoyloleoylphosphatidylcholines and apoA-I molecules cover the hydrophobic core comprised of cholesteryl esters particularly well. The ms-HDL particles are found to form a prolate ellipsoidal shape, with sizes consistent with experimental results. Large rigid domains and low mobility of the protein are seen in all the simulations. Additionally, the average number of contacts of cholesteryl ester molecules with apoA-I residues indicates that cholesteryl esters interact with protein residues mainly through their cholesterol moiety. We propose that the interaction of annular cholesteryl oleate molecules contributes to apoA-I rigidity stabilizing and regulating the structure and function of the ms-HDL particle.

Despite the vast amount of experimental and theoretical studies on the binding affinity of cations - especially the biologically relevant Na(+) and Ca(2+) - for phospholipid bilayers, there is no consensus in the literature. Here we show that by interpreting changes in the choline headgroup order parameters according to the 'molecular electrometer' concept [Seelig et al., Biochemistry, 1987, 26, 7535], one can directly compare the ion binding affinities between simulations and experiments. Our findings strongly support the view that in contrast to Ca(2+) and other multivalent ions, Na(+) and other monovalent ions (except Li(+)) do not specifically bind to phosphatidylcholine lipid bilayers at sub-molar concentrations. However, the Na(+) binding affinity was overestimated by several molecular dynamics simulation models, resulting in artificially positively charged bilayers and exaggerated structural effects in the lipid headgroups. While qualitatively correct headgroup order parameter response was observed with Ca(2+) binding in all the tested models, no model had sufficient quantitative accuracy to interpret the Ca(2+):lipid stoichiometry or the induced atomistic resolution structural changes. All scientific contributions to this open collaboration work were made publicly, using nmrlipids.blogspot.fi as the main communication platform. acceptedVersion

LCAT is activated by apoA-I to form cholesteryl ester. We combined two structures, phospholipase A2(PLA2) that hydrolyzes the ester bond at the sn-2 position of oxidized (short) acyl chains of phospholipid, and bacteriophage tubulin PhuZ, as C- and N-terminal templates, respectively, to create a novel homology model for human LCAT. The juxtaposition of multiple structural motifs matching experimental data is compelling evidence for the general correctness of many features of the model: i ) The N-terminal 10 residues of the model, required for LCAT activity, extend the hydrophobic binding trough for the sn-2 chain 15-20 Å relative to PLA2. ii ) The topography of the trough places the ester bond of the sn-2 chain less than 5 Å from the hydroxyl of the catalytic nucleophile, S181. iii ) Aβ -hairpin resembling a lipase lid separates S181 from solvent. iv ) S181 interacts with three functionally critical residues: E149, that regulates sn-2 chain specifi city, and K128 and R147, whose mutations cause LCAT defi ciency. Because the model provides a novel explanation for the complicated thermodynamic problem of the transfer of hydrophobic substrates from HDL to the catalytic triad of LCAT, it is an important step toward understanding the antiatherogenic role of HDL in reverse cholesterol transport. -Segrest, J. P., M. K. Jones, A. Catte, and S. P. Thirumuruganandham. A robust all-atom model for LCAT generated by homology modeling.

A combined use of (31)P, (23)Na, (2)H and (17)O NMR spectroscopies and polarized light microscopy has been employed to investigate the effect of the ethidium bromide (EB) binding on the liquid crystalline phase of concentrated double stranded DNA solutions. The optical textures and the (31)P and (23)Na NMR spectra of the DNA anisotropic solutions show that the intercalation of EB induces significant modifications either in the arrangements of the DNA rods and the surrounding ionic atmosphere. On the contrary, no indication of significant changes of the orientational order of the water molecules around DNA emerges from the water (2)H and (17)O NMR spectra.

The interaction of the divalent metal ions Mg(2+), Cd(2+), and Ni(2+) with liquid crystalline NaDNA solutions (molar ratios Me(2+)/DNA-phosphate/=0.050) was investigated by polarized light microscopy and multinuclear (31)P, (2)H, and (23)Na NMR. Our findings show that the state of the cholesteric NaDNA phase at equal MgCl(2), CdCl(2), or NiCl(2) concentration is affected in a different way and to a different extent by the nature of the divalent cation. Indeed, we found that the occurrence of an isotropic phase is markedly favored by Mg(2+), and to a lower extent by Cd(2+), and that the pitch of the cholesteric phase decreases in the presence of Cd(2+) or Ni(2+). (23)Na NMR spectroscopy also shows differences between the binding behavior of Mg(2+) and the transition metal ions in the counterion atmosphere around DNA. The results are discussed in terms of different binding modes of the metal ions.

ApoA-I is a uniquely flexible lipid-scavenging protein capable of incorporating phospholipids into stable particles. Here we report molecular dynamics simulations on a series of progressively smaller discoidal high density lipoprotein particles produced by incremental removal of palmitoyloleoylphosphatidylcholine via four different pathways. The starting model contained 160 palmitoyloleoylphosphatidylcholines and a belt of two antiparallel amphipathic helical lipid-associating domains of apolipoprotein (apo) A-I. The results are particularly compelling. After a few nanoseconds of molecular dynamics simulation, independent of the starting particle and method of size reduction, all simulated double belts of the four lipidated apoA-I particles have helical domains that impressively approximate the x-ray crystal structure of lipid-free apoA-I, particularly between residues 88 and 186. These results provide atomic resolution models for two of the particles produced by in vitro reconstitution of nascent high density lipoprotein particles. These particles, measuring 95 angstroms and 78 angstroms by nondenaturing gradient gel electrophoresis, correspond in composition and in size/shape (by negative stain electron microscopy) to the simulated particles with molar ratios of 100:2 and 50:2, respectively. The lipids of the 100:2 particle family form minimal surfaces at their monolayer-monolayer interface, whereas the 50:2 particle family displays a lipid pocket capable of binding a dynamic range of phospholipid molecules.