Interaction of phloretin with lipid monolayers: relationship between structural changes and dipole potential change.

Phloretin is known to adsorb to lipid surfaces and alters the dipole potential of lipid monolayers and bilayers. Its adsorption to biological and artificial membranes results in a change of the membrane permeability for a variety of charged and neutral compounds. In this respect phloretin represents a model substance to study the effect of dipole potentials on membrane permeability. In this investigation we studied the interaction of phloretin with monolayers formed of different lipids in the liquid-expanded and the condensed state. Phloretin integrated into the monolayers as a function of the aqueous concentration of its neutral form, indicated by an increase of the surface pressure in the presence of phloretin. Simultaneous recording of the surface potential of the monolayers allowed us to correlate the degree of phloretin integration and the phloretin-induced dipole potential change. Increasing the surface pressure decreased the phloretin-induced shift of the isotherms, but did not influence the phloretin-induced surface potential change. This means that phloretin adsorption to the lipid surface can occur without affecting the lipid packing. The surface potential effect of phloretin is accompanied by a change of the lipid dipole moment vector dependent on the lipid packing. This means that the relation between the surface potential change and the lipid packing cannot be described by a static model alone. Taking into account the deviations of the surface potential change versus molecular area isotherms of the experimental data to the theoretically predicted course, we propose a model that relates the area change to the dipole moment in a dynamic manner. By using this model the experimental data can be described much better than with a static model.     

Pitfalls in the interpretation of structural changes in mutant proteins from crystal structures

PpcA is a small protein with 71 residues that contains three covalently bound hemes. The structures of single mutants at residue 58 have shown larger deviations in another part of the protein molecule than at the site of the mutation. Closer examination of the crystal packing has revealed the origin of this unexpected structural change. The site of mutation is within Van der Waals distance from another protein molecule related by a crystallographic twofold axis within the crystal. The structural changes occurred at or near the mutation site have led to a slight adjustment of the surface residues in contact. The observed deviations between the native and the mutant molecular structures are derived from the new crystal packing even though the two crystals are essentially isomorphous. Without careful consideration of the crystal lattice a non-expert looking at only the coordinates deposited in the Protein Data Bank could draw erroneous conclusion that mutation in one part of the molecule affected the structure of the protein in a distant part of the molecule.     

Evaluation and ranking of enzyme designs

In 2008, a successful computational design procedure was reported that yielded active enzyme catalysts for the Kemp elimination. Here, we studied these proteins together with a set of previously unpublished inactive designs to determine the sources of activity or lack thereof, and to predict which of the designed structures are most likely to be catalytic. Methods that range from quantum mechanics (QM) on truncated model systems to the treatment of the full protein with ONIOM QM/MM and AMBER molecular dynamics (MD) were explored. The most effective procedure involved molecular dynamics, and a general MD protocol was established. Substantial deviations from the ideal catalytic geometries were observed for a number of designs. Penetration of water into the catalytic site and insufficient residue‐packing around the active site are the main factors that can cause enzyme designs to be inactive. Where in the past, computational evaluations of designed enzymes were too time‐extensive for practical considerations, it has now become feasible to rank and refine candidates computationally prior to and in conjunction with experimentation, thus markedly increasing the efficiency of the enzyme design process.     

Packing of human erythrocytes in a centrifugal field

The rate of packing of human erythrocytes in whole blood and washed ones in aqueous suspension was investigated in a centrifugal field of 250 g. The Voigt-Kelvin rheological model was found to be well suited to describe the packing process. The ratio of the elastic modulus to viscosity was evaluated from this model. Its value suggests that the flexibility of the cell plays a minor role compared to other viscosity factors. Also the model suggests that the rate of packing is a complicated function of various viscoelastic factors. Empirical parameters describing the rate of packing are sensitive to drastic changes in cell flexibility, such as caused by formaldehyde treatment, whereas no fluidizing effect of procaine on cell membrane was detected. The rate of packing is not affected by decreasing the pH from 7.4 to 6.5. The method of mild centrifugation could be of some use for rapid evaluation of substantial flexibility changes in washed blood cells.     

The role of atomic level steric effects and attractive forces in protein folding

Protein folding into tertiary structures is controlled by an interplay of attractive contact interactions and steric effects. We investigate the balance between these contributions using structure‐based models using an all‐atom representation of the structure combined with a coarse‐grained contact potential. Tertiary contact interactions between atoms are collected into a single broad attractive well between the C^β atoms between each residue pair in a native contact. Through the width of these contact potentials we control their tolerance for deviations from the ideal structure and the spatial range of attractive interactions. In the compact native state dominant packing constraints limit the effects of a coarse‐grained contact potential. During folding, however, the broad attractive potentials allow an early collapse that starts before the native local structure is completely adopted. As a consequence the folding transition is broadened and the free energy barrier is decreased. Eventually two‐state folding behavior is lost completely for systems with very broad attractive potentials. The stabilization of native‐like residue interactions in non‐perfect geometries early in the folding process frequently leads to structural traps. Global mirror images are a notable example. These traps are penalized by the details of the repulsive interactions only after further collapse. Successful folding to the native state requires simultaneous guidance from both attractive and repulsive interactions. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Cover Image,Volume 88, Issue 9

There have been several studies suggesting that protein structures solved by NMR spectroscopy and X-ray crystallography show significant differences. To understand the origin of these differences, we assembled a database of high-quality protein structures solved by both methods. We also find significant differences between NMR and crystal structures—in the root-mean-square deviations of the C _α atomic positions, identities of core amino acids, backbone, and side-chain dihedral angles, and packing fraction of core residues. In contrast to prior studies, we identify the physical basis for these differences by modeling protein cores as jammed packings of amino acid-shaped particles. We find that we can tune the jammed packing fraction by varying the degree of thermalization used to generate the packings. For an athermal protocol, we find that the average jammed packing fraction is identical to that observed in the cores of protein structures solved by X-ray crystallography. In contrast, highly thermalized packing-generation protocols yield jammed packing fractions that are even higher than those observed in NMR structures. These results indicate that thermalized systems can pack more densely than athermal systems, which suggests a physical basis for the structural differences between protein structures solved by NMR and X-ray crystallography.     

Non-randomness in side-chain packing: the distribution of interplanar angles

We analyze the distributions of interplanar angles between interacting side chains with well-defined planar regions, to see whether these distributions correspond to random packing or alternatively show orientational preferences. We use a non-homologous set of 79 high-resolution protein chain structures to show that the observed distributions are significantly different from the sinusoidal one expected for random packing. Overall, we see a relative excess of small angles and a paucity of large interplanar angles; the difference between the expected and observed distributions can be described as a shift of 5% of the interplanar angles from large (≥60°) to small (<30°) values. By grouping the residue pairs into categories based on chemical similarity, we find that some categories have very non-sinusoidal interplanar angle distributions, whereas other categories have distributions that are close to sinusoidal. For a few categories, observed deviations from a sinusoidal distribution can be explained by the electrostatic anisotropy of the isolated pair potential energy. In other cases, the observed distributions reflect the longer range effects of different possible interaction geometries. In particular, geometries that disrupt external hydrogen bonding are disfavored. Proteins 29:370–380, 1997. © 1997 Wiley-Liss, Inc.     

A radiochemical study of irreversible adsorption of proteins on reversed-phase chromatographic packing materials

A radiochemical study of the irreversible adsorption of proteins on commercial reversed-phase HPLC packing materials is reported. The conditions of study are similar to those used in HPLC separation of protein. The effects of the amount and contact time of two proteins, ovalbumin and cytochrome c, are reported. Additional results include the effect of column pretreatment with protein, silanophilic mobile-phase blocking agent, and type of packing material on the extent of irreversible adsorption. The loss process is shown to be at least biphasic and the mechanisms of loss distinct for different proteins.     

vGNM: a better model for understanding the dynamics of proteins in crystals

The dynamics of proteins are important for understanding their functions. In recent years, the simple coarse-grained Gaussian Network Model (GNM) has been fairly successful in interpreting crystallographic B-factors. However, the model clearly ignores the contribution of the rigid body motions and the effect of crystal packing. The model cannot explain the fact that the same protein may have significantly different B-factors under different crystal packing conditions. In this work, we propose a new GNM, called vGNM, which takes into account both the contribution of the rigid body motions and the effect of crystal packing, by allowing the amplitude of the internal modes to be variables. It hypothesizes that the effect of crystal packing should cause some modes to be amplified and others to become less important. In doing so, vGNM is able to resolve the apparent discrepancy in experimental B-factors among structures of the same protein but with different crystal packing conditions, which GNM cannot explain. With a small number of parameters, vGNM is able to reproduce experimental B-factors for a large set of proteins with significantly better correlations (having a mean value of 0.81 as compared to 0.59 by GNM). The results of applying vGNM also show that the rigid body motions account for nearly 60% of the total fluctuations, in good agreement with previous findings.     

Packing interface energetics in different crystal forms of the λ Cro dimer

Variation among crystal structures of the λ Cro dimer highlights conformational flexibility. The structures range from a wild type closed to a mutant fully open conformation, but it is unclear if each represents a stable solution state or if one may be the result of crystal packing. Here we use molecular dynamics (MD) simulation to investigate the energetics of crystal packing interfaces and the influence of site‐directed mutagenesis on them in order to examine the effect of crystal packing on wild type and mutant Cro dimer conformation. Replica exchange MD of mutant Cro in solution shows that the observed conformational differences between the wild type and mutant protein are not the direct consequence of mutation. Instead, simulation of Cro in different crystal environments reveals that mutation affects the stability of crystal forms. Molecular Mechanics Poisson‐Boltzmann Surface Area binding energy calculations reveal the detailed energetics of packing interfaces. Packing interfaces can have diverse properties in strength, energetic components, and some are stronger than the biological dimer interface. Further analysis shows that mutation can strengthen packing interfaces by as much as ～5 kcal/mol in either crystal environment. Thus, in the case of Cro, mutation provides an additional energetic contribution during crystal formation that may stabilize a fully open higher energy state. Moreover, the effect of mutation in the lattice can extend to packing interfaces not involving mutation sites. Our results provide insight into possible models for the effect of crystallization on Cro conformational dynamics and emphasize careful consideration of protein crystal structures. Proteins 2014; 82:1128–1141. © 2013 Wiley Periodicals, Inc.     

Variability of conformations at crystal contacts in BPTI represent true low-energy structures: correspondence among lattice packing and molecular dynamics structures.

The structures of five basic pancreatic trypsin inhibitor (BPTI) molecules are compared to establish the extent and nature of the conformational variability resulting from crystal packing effects. BPTI is an ideal system to evaluate such factors because of the availability of high resolution X-ray models of five different BPTI structures, each in a different crystal packing environment. Differences observed among the structures are found to be distributed throughout the molecule, although the regions that display most variability are associated with the loop structures (residues 14-17 and 24-29). The regions of structure that show the largest rms deviations from the mean of the five packing motifs correlate well with the presence of intermolecular contacts in the crystal lattice. For most of the molecules there is also a correspondence between a larger number of intermolecular contacts and systematically higher B-factors, although it is not apparent whether this is induced by the crystal contact or results from the fact that the contacts are made predominantly through surface loops. The conformational differences seen among the X-ray models constitute more than local shifts at the lattice contact surfaces, and in fact involve in some cases the making and breaking of intramolecular H-bonds. The magnitudes of the differences among packing models are significantly larger than those usually associated with changes induced by mutagenesis; for instance; the structural differences at the site of mutation observed on removing an internal disulfide from the molecule are significantly less than those associated with lattice contact effects. The crystal packing conformations are compared to representative structures of BPTI generated during a 96-psec molecular dynamics (MD) simulation. This comparison shows a high level of correspondence between the protein flexibility indicated by the X-ray and MD analyses, and specifically between those regions that are most variable. This suggests that the regions that show most variability among the crystal packing models are not artifacts of crystallization, but rather represent true low-energy conformers that have been preferentially selected by crystallization factors.     

Langevin dynamics simulations of genome packing in bacteriophage

We use Langevin dynamics simulations to study the process by which a coarse-grained DNA chain is packaged within an icosahedral container. We focus our inquiry on three areas of interest in viral packing: the evolving structure of the packaged DNA condensate; the packing velocity; and the internal buildup of energy and resultant forces. Each of these areas has been studied experimentally, and we find that we can qualitatively reproduce experimental results. However, our findings also suggest that the phage genome packing process is fundamentally different than that suggested by the inverse spool model. We suggest that packing in general does not proceed in the deterministic fashion of the inverse-spool model, but rather is stochastic in character. As the chain configuration becomes compressed within the capsid, the structure, energy, and packing velocity all become dependent upon polymer dynamics. That many observed features of the packing process are rooted in condensed-phase polymer dynamics suggests that statistical mechanics, rather than mechanics, should serve as the proper theoretical basis for genome packing. Finally we suggest that, as a result of an internal protein unique to bacteriophage T7, the T7 genome may be significantly more ordered than is true for bacteriophage in general.     

General architecture of the alpha-helical globule

A model is presented for the arrangement of alpha-helices in globular proteins. In the model, helices are placed on certain ribs of "quasi-spherical" polyhedra. The polyhedra are chosen so as to allow the close packing of helices around a hydrophobic core and to stress the collective interactions of the individual helices. The model predicts a small set of stable architectures for alpha-helices in globular proteins and describes the geometries of the helix packings. Some of the predicted helix arrangements have already been observed in known protein structures; others are new. An analysis of the three-dimensional structures of all proteins for which co-ordinates are available shows that the model closely approximates the arrangements and packing of helices actually observed. The average deviations of the real helix axes from those in the model polyhedra is +/- 20 degrees in orientation and +/- 2 A in position (1 A = 0.1 nm). We also show that for proteins that are not homologous, but whose helix arrangements are described by the same polyhedron, the root-mean-square difference in the position of the C alpha atoms in the helices is 1.6 to 3.0 A.     

Is counterion delocalization responsible for collapse in RNA folding?

Although energetic and phylogenetic methods have been very successful for prediction of nucleic acid secondary structures, arrangement of these secondary structure elements into tertiary structure has remained a difficult problem. Here we explore the packing arrangements of DNA, RNA, and DNA/RNA hybrid molecules in crystals. In the conventional view, the highly charged double helix will be pushed toward isolation by favorable solvation effects; interactions with other like-charged stacks would be strongly disfavored. Contrary to this expectation, we find that most of the cases analyzed ( approximately 80%) exhibit specific, preferential packing between elements of secondary structure, which falls into three categories: (i) interlocking of major grooves of two helices, (ii) side-by-side parallel packing of helices, and (iii) placement of the ribose-phosphate backbone ridge of one helix into the major groove of another. The preponderance of parallel packing motifs is especially surprising. This category is expected to be maximally disfavored by charge repulsion. Instead, it comprises in excess of 50% of all packing interactions in crystals of A-form RNA and has also been observed in crystal structures of large RNA molecules. To explain this puzzle, we introduce a novel model for RNA folding. A simple calculation suggests that the entropy gained by a cloud of condensed cations surrounding the helices more than offsets the Coulombic repulsion of parallel arrangements. We propose that these condensed counterions are responsible for entropy-driven RNA collapse, analogous to the role of the hydrophobic effect in protein folding.     

Nanomechanical behaviors of microcantilever-based single-stranded DNA chips induced by counterion osmotic effects

Experiments show that deflections of microcantilever-DNA chip can be induced by many factors, such as grafting density, hybridization efficiency, concentration, length and sequence of DNA molecules, buffer salt concentration, time, and temperature variation. However, there are few theoretical works on microcantilever-DNA chips. The present paper is aimed to study the influence of counterion effects of single-stranded DNA (ssDNA) polyelectrolyte solution on the nanomechanical behaviors of microcantilever-based ssDNA chips during packing process. First, the effect of osmotic pressure induced by ingress of counterions into DNA brush structures is studied with Hagan’s model for a cylindrical polyelectrolyte brush system on the basis of Poisson-Boltzmann distribution hypothesis. Second, Zhang’s two-variable method for a laminated cantilever is used to formulate a four-layer energy model for ssDNA chips with weak interactions. Third, the influence of grafting density, ssDNA chain length, and salt concentration on packing deflection is investigated using the principle of minimum energy. The predictive tendency is qualitatively similar to those observed in some related ssDNA chip experiments. The difference between the four-layer model and the simplified two-layer model for ssDNA chips is also discussed.     

Combining supercritical fluid extraction of soil herbicides with enzyme immunoassay analysis

Supercritical fluid extraction (SFE) of soil herbicides followed by enzyme immunoassay analysis (EIA) is explained in a step-by-step process. Extracted herbicides, include 2,4-D, simazine, atrazine, and alachlor. The herbicide, trifluralin was not successfully analyzed by EIA because of crossreacting metabolites. Problems with SFE, including uneven packing of cells, leaks, uneven flow and clogging, can largely be eliminated as the method parameters are optimized. It was necessary to add modifiers including methanol or acetone to the SF CO₂ to increase the solubility of the analytes. Detection limits of 2.5 ng/g soil for atrazine and alachlor and 15 ng/g soil for simazine and 2,4-D without concentration of the sample were achieved. Recoveries above 80% and relative standard deviations (RSDs) less than 15% for 2,4-D simazine, atrazine and alachlor were achieved. Atrazine and alachlor recoveries were above 90% with RSDs below 10%. Forty soil samples could be extracted and analyzed in an 8-h day.     

Structure and conformation of linear peptides. III. Structure of glycyl-glycyl-L-valine

The tripeptide, glycyl-glycyl-L-valine, crystallizes as a dihydrate in the monoclinic space group P2(1), with a = 5.786(1), b = 7.954(2), c = 14.420(3)A, beta = 93.85(2) degrees, Z = 2. The structure was solved by direct methods and refined to an R-value of 0.040 for 876 observed reflections. The molecule exists as a zwitterion in the crystal. The peptide planes show significant deviations from planarity. The chain conformation resembles a reverse turn if the orientation of the carboxyl group is also taken into account. An intramolecular water bridge links the amino and carboxyl ends of the molecule. The crystal packing involves spatial segregation of polar and nonpolar moieties.     

Mechanism of membrane curvature sensing by amphipathic helix containing proteins

There are several examples of membrane-associated protein domains that target curved membranes. This behavior is believed to have functional significance in a number of essential pathways, such as clathrin-mediated endocytosis, which involve dramatic membrane remodeling and require the recruitment of various cofactors at different stages of the process. This work is motivated in part by recent experiments that demonstrated that the amphipathic N-terminal helix of endophilin (H0) targets curved membranes by binding to hydrophobic lipid bilayer packing defects which increase in number with increasing membrane curvature. Here we use state-of-the-art atomistic simulation to explore the packing defect structure of curved membranes, and the effect of this structure on the folding of H0. We find that not only are packing defects increased in number with increasing membrane curvature, but also that their size distribution depends nontrivially on the curvature, falling off exponentially with a decay constant that depends on the curvature, and crucially that even on highly curved membranes defects large enough to accommodate the hydrophobic face of H0 are never observed. We furthermore find that a percolation model for the defects explains the defect size distribution, which implies that larger defects are formed by coalescence of noninteracting smaller defects. We also use the recently developed metadynamics algorithm to study in detail the effect of such defects on H0 folding. It is found that the comparatively larger defects found on a convex membrane promote H0 folding by several kcal/mol, while the smaller defects found on flat and concave membrane surfaces inhibit folding by kinetically trapping the peptide. Together, these observations suggest H0 folding is a cooperative process in which the folding peptide changes the defect structure relative to an unperturbed membrane.     

Simple growth laws and selection consequences

It is commonly thought that various types of population growth can be satisfactorily modelled as deviations from an inherently exponential (malthusian) growth law. Consideration of kinetic results from research on the origin of life, laser physics and more-conventional population dynamics makes it clear, however, that in certain cases the simplest and mechanistically most satisfactory assumption is either a basic subexponential or a hyperbolic growth law. Although these simple growth laws cannot be used instead of more-complicated models of density-dependent population growth when exact quantities are important, the insight gained by thinking them over can be substantial. Ideas about species packing, for example, await reconsideration.