Three-dimensional lipophilicity characterization of molecular pores and channel-like cavities

Molecular lipophilicity is a useful property for assessing molecular similarity or complementarity within the context of computer-aided drug design. As well, local contributions to solvent affinity help us to understand both dynamics and conformational stability in biomolecules. In this work, we discuss an approach to characterize the local contributions to hydrophobicity by using one- and two-dimensional representations of molecular channel-like cavities. The method monitors how a phenomenological lipophilicity potential (based on fragmental atom contributions) changes over a continuum of “molecular tubes” used for modeling channels and pores. Our results convey a relatively detailed picture of the spatial distribution of water affinity. The procedure can then be used as a complement to the hydrophobicity scales based on averaging contributions from single amino acids. In addition, we can study how the water affinity changes for inner and outer regious of the pores. As an application, we compute the 3D distribution of lipophilicity in the “pore conformation” of gramicidin A. The qualitative trends indicated by our results are broadly consistent with computer simulations of the gramicidin channel in the presence of hydrated ions. The behavior revealed by the simulations can then be incorporated to produce an improved, simple 2D model for water-channel interactions.     

The structure of glutamate transporters shows channel-like features

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large family of secondary transporters, which includes transporters from a variety of bacterial, archaeal and eukaryotic organisms. The transporters consist of eight membrane-spanning alpha-helices and two pore-loop structures, which are unique among secondary transporters but may resemble pore-loops found in ion channels. Another distinctive structural feature is the presence of a highly amphipathic membrane-spanning alpha-helix that provides a hydrophilic path through the membrane. The unusual structural features of the transporters are discussed in relation to their function.     

Water and bacteriorhodopsin: structure, dynamics, and function

A wealth of information has been gathered during the past decades that water molecules do play an important role in the structure, dynamics, and function of bacteriorhodopsin (bR) and purple membrane. Light-induced structural alterations in bR as detected by X-ray and neutron diffraction at low and high resolution are discussed in relationship to the mechanism of proton pumping. The analysis of high resolution intermediate structures revealed photon-induced rearrangements of water molecules and hydrogen bonds concomitant with conformational changes in the chromophore and the protein. These observations led to an understanding of key features of the pumping mechanism, especially the vectoriality and the different modes of proton translocation in the proton release and uptake domain of bR. In addition, water molecules influence the function of bR via equilibrium fluctuations, which must occur with adequate amplitude so that energy barriers between conformational states can be overcome.     

Molecular dynamics simulation of sperm whale myoglobin: effects of mutations and trapped CO on the structure and dynamics of cavities

The results of extended (80-ns) molecular dynamics simulations of wild-type and YQR triple mutant of sperm whale deoxy myoglobin in water are reported and compared with the results of the simulation of the intermediate(s) obtained by photodissociation of CO in the wild-type protein. The opening/closure of pathways between preexistent cavities is different in the three systems. For the photodissociated state, we previously reported a clear-cut correlation between the opening probability and the presence of the photolyzed CO in the proximity of the passage; here we show that in wild-type deoxy myoglobin, opening is almost random. In wild-type deoxy myoglobin, the passage between the distal pocket and the solvent is strictly correlated to the presence/absence of a water molecule that simultaneously interacts with the distal histidine side chain and the heme iron; conversely, in the photodissociated myoglobin, the connection with the bulk solvent is always open when CO is in the vicinity of the A pyrrole ring. In YQR deoxy myoglobin, the mutated Gln(E7)64 is stably H-bonded with the mutated Tyr(B10)29. The essential dynamics analysis unveils a different behavior for the three systems. The motion amplitude is progressively restricted in going from wild-type to YQR deoxy myoglobin and to wild-type myoglobin photoproduct. In all cases, the principal motions involve mainly the same regions, but their directions are different. Analysis of the dynamics of the preexisting cavities indicates large fluctuations and frequent connections with the solvent, in agreement with the earlier hypothesis that some of the ligand may escape from the protein through these pathways.     

Water structure and hydration.

Possible structures adopted by bulk water are discussed with special reference to the possible presence of monomeric water and the detection of 'free' -OH groups. The way in which water tends to accommodate small hydrophobic molecules is considered, with particular reference to the clathrate theory and the phenomenon of 'structure making'. Cage-pairing and cage-sharing processes are described. Consideration of the way water solvates cations and anions is followed by a discussion of the way these solvated ions interact with the bulk medium. Large symmetrical alkylammonium ions probably encourage clathrate cage formation, at least at low temperatures. Particular reference is made to the use of infrared, Raman, ultraviolet, n.m.r. and e.s.r. spectroscopic techniques to the study of water and aqueous solutions.     

Transmembrane helix structure, dynamics, and interactions: multi-nanosecond molecular dynamics simulations.

To probe the fundamentals of membrane/protein interactions, all-atom multi-nanosecond molecular dynamics simulations were conducted on a single transmembrane poly(32)alanine helix in a fully solvated dimyristoyphosphatidylcholine (DMPC) bilayer. The central 12 residues, which interact only with the lipid hydrocarbon chains, maintained a very stable helical structure. Helical regions extended beyond these central 12 residues, but interactions with the lipid fatty-acyl ester linkages, the lipid headgroups, and water molecules made the helix less stable in this region. The C and N termini, exposed largely to water, existed as random coils. As a whole, the helix tilted substantially, from perpendicular to the bilayer plane (0 degree) to a 30 degrees tilt. The helix experienced a bend at its middle, and the two halves of the helix at times assumed substantially different tilts. Frequent hydrogen bonding, of up to 0.7 ns in duration, occurred between peptide and lipid molecules. This resulted in correlated translational diffusion between the helix and a few lipid molecules. Because of the large variation in lipid conformation, the lipid environment of the peptide was not well defined in terms of "annular" lipids and on average consisted of 18 lipid molecules. When compared with a "neat" bilayer without peptide, no significant difference was seen in the bilayer thickness, lipid conformations or diffusion, or headgroup orientation. However, the lipid hydrocarbon chain order parameters showed a significant decrease in order, especially in those methylene groups closest to the headgroup.     

Focal adhesions: structure and dynamics

Interactions of cells with the extracellular matrix are essential for the control of tissue remodelling, cell migration, and embryogenesis. At the cell-extracellular matrix contact points, specialized structures are formed and termed focal adhesions, where transmembrane adhesion receptors provide a structural link between the actin cytoskeleton and the extracellular matrix components. Numerous structural and regulatory proteins assemble at the cytoplasmic face of focal adhesions in a Rho-dependent fashion.     

Chromatin structure and dynamics: state-of-the-art

A meeting entitled "Chromatin Structure and Dynamics: State-of-the-Art" organized by Jordanka Zlatanova and Sanford Leuba was held at the NIH from May 8-10, 2002. It was a timely meeting and addressed our current understanding of chromatin structure, dynamics, and function.     

Kissper, a kiwi fruit peptide with channel-like activity: structural and functional features.

Kissper is a 39-residue peptide isolated from kiwi fruit (Actinidia deliciosa). Its primary structure, elucidated by direct protein sequencing, is identical to the N-terminal region of kiwellin, a recently reported kiwi fruit allergenic protein, suggesting that kissper derives from the in vivo processing of kiwellin. The peptide does not show high sequence identity with any other polypeptide of known function. However, it displays a pattern of cysteines similar, but not identical, to those observed in some plant and animal proteins, including toxins involved in defence mechanisms. A number of these proteins are also active on mammalian cells. Functional characterization of kissper showed pH-dependent and voltage-gated pore-forming activity, together with anion selectivity and channeling in model synthetic PLMs, made up of POPC and of DOPS:DOPE:POPC. A 2DNMR analysis indicates that in aqueous solution kissper has only short regions of regular secondary structure, without any evident similarity with other bioactive peptides. Comparative analysis of the structural and functional features suggests that kissper is a member of a new class of pore-forming peptides with potential effects on human health.     

The plasma membrane of Entamoeba histolytica: structure and dynamics.

The plasma membrane components of the parasitic protozoan Entamoeba histolytica, the causative agent of human invasive amebiasis, have been biochemically and immunologically characterized during the last decade. In addition, genes coding for certain surface proteins have been cloned. In spite of these advances, a unified characterization of plasma membrane antigenic components of the parasite is still required for badly needed advancements in the design of useful diagnostic, epidemiologic, and immunoprophylactic tools. Here we review current knowledge on this issue and address the problem of the considerable variation in the electrophoretic profiles of plasma membrane proteins obtained by different groups. In addition, the differences in the degree of recognition of reported membrane antigens with human immune sera, and the diverse interpretations concerning the possible functions of the surface molecules characterized are discussed. A comparative analysis of plasma membrane proteins of E histolytica trophozoites using three different isolation methods revealed that it is possible to select for specific membrane proteins, depending on the lysis conditions. In our view, the method of Calderón and Avila preserves more proteins than other methods tested. Using sera from recent cases of invasive amebiasis studied by several laboratories in various geographical areas, a basic antigenic pattern of 11 principal proteins with molecular weights of 220, 170, 150, 125, 97, 80, 60, 45, 20 and 9 kDA was established for the pathogenic E histolytica strain HM1:IMSS, used by most research groups.     

Structural dynamics of myoglobin: effect of internal cavities on ligand migration and binding

Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation. Photoproduct intermediates, characterized by CO in different locations, were selectively enhanced by laser illumination at specific temperatures. Measurements were performed on the wild-type protein and a series of mutants (L104W, I107W, I28F, and I28W) in which bulky amino acid side chains were introduced to block passageways between cavities or to fill these sites. Binding of xenon was also employed as an alternative means of filling cavities. In all samples, photolyzed CO ligands were observed to initially bind at primary docking site B in the vicinity of the heme iron, from where they migrate to the secondary docking sites, the Xe4 and/or Xe1 cavities. To examine the relevance of these internal docking sites for physiological ligand binding, we have performed room-temperature flash photolysis on the entire set of proteins in the CO- and O(2)-bound form. Together with the cryospectroscopic results, these data provide a clear picture of the role of the internal sites for ligand escape from and binding to myoglobin.     

A molecular dynamics study of thermodynamic and structural aspects of the hydration of cavities in proteins.

The structure and activity of a protein molecule are strongly influenced by the extent of hydration of its cavities. This is, in turn, related to the free energy change on transfer of a water molecule from bulk solvent into a cavity. Such free energy changes have been calculated for two cavities in a sulfate-binding protein. One of these cavities contains a crystallographically observed water molecule while the other does not. Thermodynamic integration and perturbation methods were used to calculate free energies of hydration for each of the cavities from molecular dynamics simulations of two separate events: the removal of a water molecule from pure water, and the introduction of a water molecule into each protein cavity. From the simulations for the pure water system, the excess chemical potential of water was computed to be -6.4 +/- 0.4 kcal/mol, in accord with experiment and with other recent theoretical calculations. For the protein cavity containing an experimentally observed water molecule, the free energy change on hydrating it with one water molecule was calculated as -10.0 +/- 1.3 kcal/mol, indicating the high probability that this cavity is occupied by a water molecule. By contrast, for the cavity in which no water molecules were experimentally observed, the free energy change on hydrating it with one water molecule was calculated as 0.2 +/- 1.5 kcal/mol, indicating its low occupancy by water. The agreement of these results with experiment suggests that thermodynamic simulation methods may become useful for the prediction and analysis of internal hydration in proteins.     

Solution structure and dynamics of ribonuclease Sa.

We have used NMR methods to characterize the structure and dynamics of ribonuclease Sa in solution. The solution structure of RNase Sa was obtained using the distance constraints provided by 2,276 NOEs and the C6-C96 disulfide bond. The 40 resulting structures are well determined; their mean pairwise RMSD is 0.76 A (backbone) and 1.26 A (heavy atoms). The solution structures are similar to previously determined crystal structures, especially in the secondary structure, but exhibit new features: the loop composed of Pro 45 to Ser 48 adopts distinct conformations and the rings of tyrosines 51, 52, and 55 have reduced flipping rates. Amide protons with greatly reduced exchange rates are found predominantly in interior beta-strands and the alpha-helix, but also in the external 3/10 helix and edge beta-strand linked by the disulfide bond. Analysis of (15)N relaxation experiments (R1, R2, and NOE) at 600 MHz revealed five segments, consisting of residues 1-5, 28-31, 46-50, 60-65, 74-77, retaining flexibility in solution. The change in conformation entropy for RNase SA folding is smaller than previously believed, since the native protein is more flexible in solution than in a crystal.     

Glutamate transporters combine transporter- and channel-like features

Glutamate transporters in the mammalian central nervous system have a unique position among secondary transport proteins as they exhibit glutamate-gated chloride-channel activity in addition to glutamate-transport activity. In this article, the available data on the structure of the glutamate transporters are compared with high-resolution crystal structures of channel proteins. In addition, binding-site properties of glutamate transporters, and the ligand-binding site of an ionotropic glutamate receptor of which the crystal structure is known, are compared. Possible structural solutions for the combination of channel and transporter activity in one membrane protein are proposed.     

传粉网络的研究进展:网络的结构和动态

植物与传粉者之间相互作用,构成了复杂的传粉网络。近年来,社会网络分析技术的发展使得复杂生态网络的研究成为可能。从群落水平上研究植物与传粉者之间的互惠关系,为理解群落的结构和动态以及花部特征的演化提供了全新的视角。传粉网络的嵌套结构说明自然界的传粉服务存在冗余,而且是相对泛化的物种主导了传粉。在多年或者多季度的传粉网络中,虽然有很高的物种替换率,但是其网络结构仍然保持相对稳定,说明传粉网络对干扰有很强的抗性。尽管有关网络结构和动态的研究逐渐增多,但传粉网络维持的机制仍不清楚。网络结构可以部分由花部特征与传粉者的匹配来解释,也受到系统发生的制约,影响因素还包括群落构建的时间和物种多样性,以及物种在群落中的位置。开展大尺度群落动态的研究,为探索不同时间尺度、不同物种多样性水平上的传粉网络的生态学意义提供了条件。但已有的研究仍存在不足,比如基于访问观察的网络无法准确衡量传粉者的访问效率和植物间的花粉流动,以及结果受到调查精度区域研究不平衡的制约等。目前的研究只深入到传粉者携带花粉构成成分的水平,传粉者访问植物的网络不能代表植物的整个传粉过程。因此,研究应当更多地深入到物种之间关系对有性生殖的切实影响上。     

Internal cavities and ligand passageways in human hemoglobin characterized by molecular dynamics simulations

Molecular dynamics simulations of the unliganded T state of human hemoglobin showed the existence of a spontaneous, very wide cavity on the distal side of the alpha subunit. This cavity consists of three tunnels spreading from the vicinity of the iron atom (the ligand binding site) to the surface of the subunit, constituting possible passageways for the entrance of the ligand. A fourth passageway was characterized due to the trajectory of water molecules entering or leaving the heme pocket. Analogous passages were observed in the beta subunits. They all appear and disappear dynamically, although some parts of them are more persistent along the trajectories. The most persistent regions within these tunnels correspond to all the xenon docking sites of human cytoglobin and to some of those of sperm whale and horse heart myoglobins and group I truncated hemoglobins.     

Protein structure and dynamics in nonaqueous solvents: insights from molecular dynamics simulation studies

Protein structure and dynamics in nonaqueous solvents are here investigated using molecular dynamics simulation studies, by considering two model proteins (ubiquitin and cutinase) in hexane, under varying hydration conditions. Ionization of the protein groups is treated assuming "pH memory," i.e., using the ionization states characteristic of aqueous solution. Neutralization of charged groups by counterions is done by considering a counterion for each charged group that cannot be made neutral by establishing a salt bridge with another charged group; this treatment is more physically reasonable for the nonaqueous situation, contrasting with the usual procedures. Our studies show that hydration has a profound effect on protein stability and flexibility in nonaqueous solvents. The structure becomes more nativelike with increasing values of hydration, up to a certain point, when further increases render it unstable and unfolding starts to occur. There is an optimal amount of water, approximately 10% (w/w), where the protein structure and flexibility are closer to the ones found in aqueous solution. This behavior can explain the experimentally known bell-shaped dependence of enzyme catalysis on hydration, and the molecular reasons for it are examined here. Water and counterions play a fundamental and dynamic role on protein stabilization, but they also seem to be important for protein unfolding at high percentages of bound water.