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Centromeres are genomic regions that coordinate accurate chromosomal segregation during mitosis and meiosis. Yet, despite their essential function, centromeres evolve rapidly across eukaryotes. Centromeres are often the sites of chromosomal breaks which contribute to genome shuffling and promote speciation by inhibiting gene flow. How centromeres form in strongly host-adapted fungal pathogens has yet to be investigated. Here, we characterized the centromere structures in closely related species of mammalian-specific pathogens of the fungal phylum of Ascomycota. Methods allowing reliable continuous culture ofPneumocystisspecies do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that thePneumocystisCENP-A ortholog is functionally equivalent to CENP-ACnp1ofSchizosaccharomyces pombe. Using organisms from a short-termin vitroculture or infected animal models and ChIP-seq, we identified centromeres in threePneumocystisspecies that diverged ~100 million years ago. Each species has a unique short regional centromere (< 10kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. CENP-C, a scaffold protein that links the inner centromere to the kinetochore appears dispensable in one species, suggesting a kinetochore rewiring. Despite the loss of DNA methyltransferases, 5-methylcytosine DNA methylation occurs in these species, though not related to centromere function. These features suggest an epigenetic specification of centromere function.Short summaryPneumocystisspecies offer a suitable genetic system to study centromere evolution in pathogens during host adaptation because of their unique specificity for mammals, and their phylogenetic proximity with the nonpathogenic yeastSchizosaccharomyces pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after divergence of the two clades ~460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-seq to characterize centromeres in multiplePneumocystisspecies. We show thatPneumocystishave short epigenetic centromeres that function differently from those inS. pombeand exhibit similarities to centromeres in more distantly related host adapted fungal pathogens.

The nucleocapsid (N-)protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a key role in viral assembly and scaffolding of the viral RNA. It promotes liquid-liquid phase separation (LLPS), forming dense droplets that support the assembly of ribonucleoprotein particles with as-of-yet unknown macromolecular architecture. Combining biophysical experiments, molecular dynamics simulations, and analysis of the mutational landscape, we describe a heretofore unknown oligomerization site that contributes to LLPS, is required for the assembly of higher-order protein-nucleic acid complexes, and is coupled to large-scale conformational changes of N-protein upon nucleic acid binding. The self-association interface is located in a leucine-rich sequence of the intrinsically disordered linker between N-protein folded domains and formed by transient helices assembling into trimeric coiled-coils. Critical residues stabilizing hydrophobic and electrostatic interactions between adjacent helices are highly protected against mutations in viable SARS-CoV-2 genomes, and the oligomerization motif is conserved across related coronaviruses, thus presenting a target for antiviral therapeutics.

Computational design of antimicrobial peptides (AMPs) is a promising area of research for developing novel agents against drug-resistant bacteria. AMPs are present naturally in many organisms, from bacteria to humans, a time-tested mechanism that makes them attractive as effective antibiotics. Depending on the environment, AMPs can exhibit α-helical or β-sheet conformations, a mix of both, or lack secondary structure; they can be linear or cyclic. Prediction of their structures is challenging but critical for rational design. Promising AMP leads can be developed using essentially two approaches: traditional modeling of the physicochemical mechanisms that determine peptide behavior in aqueous and membrane environments and knowledge-based, e.g., machine learning (ML) techniques, that exploit ever-growing AMP databases. Here, we explore the conformational landscapes of two recently ML-designed AMPs, characterize the dependence of these landscapes on the medium conditions, and identify features in peptide and membrane landscapes that mediate protein-membrane association. For both peptides, we observe greater conformational diversity in an aqueous solvent than in a less polar solvent, and one peptide is seen to alter its conformation more dramatically than the other upon the change of solvent. Our results support the view that structural rearrangement in response to environmental changes is central to the mechanism of membrane-structure disruption by linear peptides. We expect that the design of AMPs by ML will benefit from the incorporation of peptide conformational substates as quantified here with molecular simulations.

Interleukin (IL)-10 is the primary cytokine driving the modulation of the host response in filarial infections. We performed binding assays with Brugia malayi antigen extracts and human IL-10R1. Bm5539 was the top-binding hit. We identified a short sequence, termed truncated Bm5339, that has structural similarities to the human IL-10 functional dimer. Sequence comparisons revealed that other filarial parasites possess Bm5539 orthologues. Using recombinant Bm5539 in a modified Luciferase Immunoprecipitation System assay, we confirmed that both the truncated and full-length forms of the protein can bind to human IL-10R1. Truncated Bm5539 could inhibit human IL-10-driven phosphorylation of STAT3, thereby demonstrating that Bm5539 acts as an IL-10 antagonist, most likely through competitive binding to the receptor. We provide a structural basis for these observations using computational modeling and simulations. This parasite-encoded cytokine receptor antagonist provides an additional lens through which parasite-induced modulation of the host immune response can be examined.

Seven-transmembrane receptors signal via G-protein- and β-arrestin-dependent pathways. We describe a peripheral CB1R antagonist (MRI-1891) highly biased toward inhibiting CB1R-induced β-arrestin-2 ...

The use of nanoparticles (NPs) in biomedicine has made a gradual transition from proof-of-concept to clinical applications, with several NP types meeting regulatory approval or undergoing clinical trials. A new type of metallic nanostructures called ultrasmall nanoparticles (usNPs) and nanoclusters (NCs), while retaining essential properties of the larger (classical) NPs, have features common to bioactive proteins. This combination expands the potential use of usNPs and NCs to areas of diagnosis and therapy traditionally reserved for small-molecule medicine. Their distinctive physicochemical properties can lead to unique in vivo behaviors, including improved renal clearance and tumor distribution. Both the beneficial and potentially deleterious outcomes (cytotoxicity, inflammation) can, in principle, be controlled through a judicious choice of the nanocore shape and size, as well as the chemical ligands attached to the surface. At present, the ability to control the behavior of usNPs is limited, partly because advances are still needed in nanoengineering and chemical synthesis to manufacture and characterize ultrasmall nanostructures and partly because our understanding of their interactions in biological environments is incomplete. This review addresses the second limitation. We review experimental and computational methods currently available to understand molecular mechanisms, with particular attention to usNP–protein complexation, and highlight areas where further progress is needed. We discuss approaches that we find most promising to provide relevant molecular-level insight for designing usNPs with specific behaviors and pave the way to translational applications., Experimental and computational methods for the study of ultrasmall nanoparticle–protein interactions.

Worldwide SARS-CoV-2 sequencing efforts track emerging mutations in its spike protein, as well as characteristic mutations in other viral proteins. Besides their epidemiological importance, the observed SARS-CoV-2 sequences present an ensemble of viable protein variants, and thereby a source of information on viral protein structure and function. Charting the mutational landscape of the nucleocapsid (N) protein that facilitates viral assembly, we observe variability exceeding that of the spike protein, with more than 86% of residues that can be substituted, on average by three to four different amino acids. However, mutations exhibit an uneven distribution that tracks known structural features but also reveals highly protected stretches of unknown function. One of these conserved regions is in the central disordered linker proximal to the N-G215C mutation that has become dominant in the Delta variant, outcompeting G215 variants without further spike or N-protein substitutions. Structural models suggest that the G215C mutation stabilizes conserved transient helices in the disordered linker serving as protein–protein interaction interfaces. Comparing Delta variant N-protein to its ancestral version in biophysical experiments, we find a significantly more compact and less disordered structure. N-G215C exhibits substantially stronger self-association, shifting the unliganded protein from a dimeric to a tetrameric oligomeric state, which leads to enhanced coassembly with nucleic acids. This suggests that the sequence variability of N-protein is mirrored by high plasticity of N-protein biophysical properties, which we hypothesize can be exploited by SARS-CoV-2 to achieve greater efficiency of viral assembly, and thereby enhanced infectivity.

Gonadotropin-regulated testicular RNA helicase (GRTH)/DDX25 is a DEAD-box RNA helicase essential for the completion of spermatogenesis. Our previous studies indicated that blocking the GRTH phospho-site or perturbing the GRTH/protein kinase A (PKA) interface could provide an avenue for developing a nonhormonal male contraceptive. In this study, cyclic peptides were rationally designed and synthesized as promising therapeutic agents. The peptides showed effective delivery into COS-1 and germ cells and a dose-dependent inhibitory effect on GRTH phosphorylation. The peptides inhibit GRTH phosphorylation in the presence of PKA, and binding to the helicase resulted in thermal stabilization of non-phospho GRTH. Increased efficiency in fluorescence resonance energy transfer (FRET) assay revealed their interaction with GRTH. Cyclic peptide exposure of cultures from mice seminiferous tubules resulted in significant inhibition of phospho GRTH. These peptides did not exhibit toxicity. Effective delivery and targeted decrease of in vitro expression of phospho GRTH by cyclic peptides provide a promising angle to develop effective compounds as a nonhormonal male contraceptive.

Protein-protein interaction (PPI) networks are known to be valuable targets for therapeutic intervention; yet the development of PPI modulators as next-generation drugs to target specific vertices, edges, and hubs has been impeded by the lack of structural information of many of the proteins and complexes involved. Building on recent advancements in cross-linking mass spectrometry (XL-MS), we describe an effective approach to obtain relevant structural data on R7BP, a master regulator of itch sensation, and its interfaces with other proteins in its network. This approach integrates XL-MS with a variety of modeling techniques to successfully develop antibody inhibitors of the R7BP and RGS7/Gβ5 duplex interaction. Binding and inhibitory efficiency are studied by surface plasmon resonance spectroscopy and through an R7BP-derived dominant negative construct. This approach may have broader applications as a tool to facilitate the development of PPI modulators in the absence of crystal structures or when structural information is limited., Adikaram et al perform protein cross-linking coupled with mass spectrometry and structural modelling to obtain a structure of R7BP, a master regulator of itch. They identify domains interacting with R7BP partners RGS7 and Gβ5 and develop R7BP antibodies that obstruct its binding to the RGS7/Gβ5 duplex.

Gonadotropin Regulated Testicular Helicase (GRTH/DDX25), expressed in the male gonad, is essential for the completion of spermatogenesis. Our early studies revealed a missense mutation (R242H) of GRTH in 5.8% of Japanese patient population with azoospermia. Transfection of the mutant GRTH construct in COS-1 cells leads to loss of the 61 kDa cytoplasmic phospho-species. Mice with knock-in of the human GRTH mutation are sterile and lack sperm with normal androgen and mating behavior. These findings provide an avenue for the development of a non-hormonal male contraceptive. Using site directed mutagenesis and a site-specific phospho-antibody, we have identified T239, structurally adjacent to the patient’s mutant site as the GRTH phospho-site. Molecular modelling provided structural basis for the role of R242 and other critical solvent-exposed residues at the GRTH/PKA interface (E165/K240/D237), on the control of GRTH phosphorylation at T239. Single or double mutations of these residues caused marked reduction or abolition of the phospho-form. These effects can be ascribed to critical disruptions of intramolecular H-bonds at the GRTH/PKA interface, which leads to modest but consequential structural changes that can affect PKA catalytic efficiency. Inhibition of phosphorylation may be achieved by small, drug-like molecules that bind to GRTH and reconfigure the GRTH/PKA interface.

GATA2 deficiency is an inherited or sporadic genetic disorder characterized by distinct cellular deficiency, bone marrow failure, various infections, lymphedema, pulmonary alveolar proteinosis, and predisposition to myeloid malignancies resulting from heterozygous loss-of-function mutations in the GATA2 gene. How heterozygous GATA2 mutations affect human hematopoietic development or cause characteristic cellular deficiency and eventual hypoplastic myelodysplastic syndrome or leukemia is not fully understood. We used induced pluripotent stem cells (iPSCs) to study hematopoietic development in the setting of GATA2 deficiency. We performed hematopoietic differentiation using iPSC derived from patients with GATA2 deficiency and examined their ability to commit to mesoderm, hemogenic endothelial precursors (HEPs), hematopoietic stem progenitor cells, and natural killer (NK) cells. Patient-derived iPSC, either derived from fibroblasts/marrow stromal cells or peripheral blood mononuclear cells, did not show significant defects in committing to mesoderm, HEP, hematopoietic stem progenitor, or NK cells. However, HEP derived from GATA2-mutant iPSC showed impaired maturation toward hematopoietic lineages. Hematopoietic differentiation was nearly abolished from homozygous GATA2 knockout (KO) iPSC lines and markedly reduced in heterozygous KO lines compared with isogenic controls. On the other hand, correction of the mutated GATA2 allele in patient-specific iPSC did not alter hematopoietic development consistently in our model. GATA2 deficiency usually manifests within the first decade of life. Newborn and infant hematopoiesis appears to be grossly intact; therefore, our iPSC model indeed may resemble the disease phenotype, suggesting that other genetic, epigenetic, or environmental factors may contribute to bone marrow failure in these patients following birth. However, heterogeneity of PSC-based models and limitations of in vitro differentiation protocol may limit the possibility to detect subtle cellular phenotypes.

Seven-transmembrane receptors signal via G-protein- and β-arrestin-dependent pathways. We describe a peripheral CB

Safe and efficient use of ultrasmall nanoparticles (NPs) in biomedicine requires numerous independent conditions to be met, including colloidal stability, selectivity for proteins and membranes, binding specificity, and low affinity for plasma proteins. The ability of a NP to satisfy one or more of these requirements depends on its physicochemical characteristics, such as size, shape, and surface chemistry. Multiscale and pattern recognition techniques are here integrated to guide the design of NPs with preferential nano-bio behaviors. Data systematically collected from simulations (or experiments, if available) are first used to train one or more artificial neural networks, each optimized for a specific kind of nano-bio interaction; the trained networks are then interconnected in suitable arrays to obtain the NP core morphology and layer composition that best satisfy all the nano-bio interactions underlying more complex behaviors. This reverse engineering approach is illustrated in the case of NP-membrane interactions, using binding modes and affinities and early stage membrane penetrations as training data. Adaptations for designing NPs with preferential nano-protein interactions and for optimizing solution conditions in the test tube are discussed.

A series of compounds have been synthesized with a variety of substituents based on a three-carbon chain at the C9-position of 3-hydroxy-N-phenethyl-5-phenylmorphan (3-(2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol). Three of these were found to be μ-opioid receptor agonists in the inhibition of forskolin-induced cAMP accumulation assay and they did not recruit β-arrestin at all in the PathHunter assay and in the Tango assay. Compound 12 (3-((1S,5R,9R)-2-phenethyl-9-propyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), 13 (3-((1S,5R,9R)-9-((E)-3-hydroxyprop-1-en-1-yl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), and 15a (3-((1S,5R,9R)-9-(2-hydroxypropyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol) were partial μ-agonists. Two of them had moderate efficacies (E(MAX)ca. 65%) and one had lower efficacy, and they were ca. 5, 3, and 4 times more potent, respectively, than morphine in vitro. Computer simulations were carried out to provide a molecular basis for the high bias ratios of the C9-substituted 5-phenylmorphans toward G-protein activation.

(&minus, )-N-Phenethyl analogs of optically pure N-norhydromorphone were synthesized and pharmacologically evaluated in several in vitro assays (opioid receptor binding, stimulation of [35S]GTP&gamma, S binding, forskolin-induced cAMP accumulation assay, and MOR-mediated &beta, arrestin recruitment assays). &ldquo, Body&rdquo, and &ldquo, tail&rdquo, interactions with opioid receptors (a subset of Portoghese&rsquo, s message-address theory) were used for molecular modeling and simulations, where the &ldquo, address&rdquo, can be considered the &ldquo, body&rdquo, of the hydromorphone molecule and the &ldquo, message&rdquo, delivered by the substituent (tail) on the aromatic ring of the N-phenethyl moiety. One compound, N-p-chloro-phenethynorhydromorphone ((7aR,12bS)-3-(4-chlorophenethyl)-9-hydroxy-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one, 2i), was found to have nanomolar binding affinity at MOR and DOR. It was a potent partial agonist at MOR and a full potent agonist at DOR with a &delta, /&mu, potency ratio of 1.2 in the ([35S]GTP&gamma, S) assay. Bifunctional opioids that interact with MOR and DOR, the latter as agonists or antagonists, have been reported to have fewer side-effects than MOR agonists. The p-chlorophenethyl compound 2i was evaluated for its effect on respiration in both mice and squirrel monkeys. Compound 2i did not depress respiration (using normal air) in mice or squirrel monkeys. However, under conditions of hypercapnia (using air mixed with 5% CO2), respiration was depressed in squirrel monkeys.

Synthetic ultrasmall nanoparticles (NPs) can be designed to interact with biologically active proteins in a controlled manner. However, the rational design of NPs requires a clear understanding of their interactions with proteins and the precise molecular mechanisms that lead to association/dissociation in biological media. Although much effort has been devoted to the study of the kinetics mechanism of protein corona formation on large NPs, the nature of NP-protein interactions in the ultrasmall regime is radically different and poorly understood. Using a combination of experimental and computational approaches, we studied the interactions of a model protein, CrataBL, with ultrasmall gold NPs passivated with p-mercaptobenzoic acid (AuMBA) and glutathione (AuGSH). We have identified this system as an ideal in vitro platform to understand the dependence of binding affinity and kinetics on NP surface chemistry. We found that the structural and chemical complexity of the passivating NP layer leads to quite different association kinetics, from slow and reaction-limited (AuGSH) to fast and diffusion-limited (AuMBA). We also found that the otherwise weak and slow AuGSH-protein interactions measured in buffer solution are enhanced in macromolecular crowded solutions. These findings advance our mechanistic understanding of biomimetic NP-protein interactions in the ultrasmall regime and have implications for the design and use of NPs in the crowded conditions common to all biological media.

A method is described for the efficient simulation of multiprotein systems in crowded aqueous solutions.

Gonadotropin Regulated Testicular Helicase (GRTH/DDX25), expressed in the male gonad, is essential for the completion of spermatogenesis. Our early studies revealed a missense mutation (R242H) of GRTH in 5.8 % of a patient population with azoospermia. Transfection of the mutant protein in COS-1 cells showed loss of the 61 kDa cytoplasmic phospho-species with preservation of the nuclear non-phospho form of GRTH (1). Mice with knock-in of the human GRTH mutation, lack the phospho-form of GRTH, are sterile due to lack of sperm, and have normal testosterone levels and mating behavior (2). These findings provide an avenue for the development of a non-hormonal male contraceptive. Using site directed mutagenesis and a site-specific phospho-antibody, we have identified T239, structurally adjacent to the patient’s mutant site, as the GRTH-phospho-site. Molecular modelling of the phospho-site based on the crystal structure of DDX19 (64% a.a. identity), provided structural basis for the role of R242 and other critical solvent-exposed residues at the GRTH/PKA interface (E165/K240/D237), on the control of GRTH phosphorylation at T239. Single or double mutations of these residues caused marked reduction or abolition of the phospho-form. These effects can be ascribed to critical disruptions of intramolecular H-bonds at the GRTH/PKA-interface, which leads to modest but consequential structural changes that can affect PKA binding or its catalytic efficiency. Inhibition of phosphorylation may be achieved by small molecules that bind to GRTH and reconfigure the GRTH/PKA-interface. These studies based on the abolition of the phospho-form of GRTH observed in vivo and in vitro provide the basis for drug design and virtual and throughput screening for the discovery of a non-toxic specific, reversible chemical inhibitor for use as a male contraceptive. (1) Tsai-Morris et al., Mol Hum Reprod. 2017; 13: 887-892. (2) Kavarthapu et al., Endo Reviews 2018 Vol 39, Issue 2 Suppl.

Recent in vivo studies have established ultrasmall (< 3 nm) gold nanoparticles coated with glutathione (AuGSH) as a promising platform for applications in nanomedicine. However, systematic in vitro investigations to gain a more fundamental understanding of the particles’ biointeractions are still lacking. Herein we examined the behavior of ultrasmall AuGSH in vitro, focusing on their ability to resist aggregation and adsorption from serum proteins. Despite having net negative charge, AuGSH particles were colloidally stable in biological media and able to resist binding from serum proteins, in agreement with the favorable bioresponses reported for AuGSH in vivo. However, our results revealed disparate behaviors depending on nanoparticle size: particles between 2 and 3 nm in core diameter were found to readily aggregate in biological media, whereas those strictly under 2 nm were exceptionally stable. Molecular dynamics simulations provided microscopic insight into interparticle interactions leading to aggregation and their sensitivity to the solution composition and particle size. These results have important implications, in that seemingly small variations in size can impact the biointeractions of ultrasmall AuGSH, and potentially of other ultrasmall nanoparticles as well.

Nanoparticle (NP) aggregation can lead to prolonged retention in tissues or embolism, among other adverse effects. Successful use in biomedicine thus requires the capability to make NPs with limited aggregative : potential. Rational design is presently a challenge due to incomplete knowledge of their interactions in biofluids. Recently, ultrasmall gold NPs passivated with endogenous antioxidant glutathione have shown promise for use in vivo. Computer simulations are here conducted to identify the forces underlying aggregation (or lack thereof) of these NPs in a cell culture. Electrostatic interactions are insufficient to induce association, but the van der Waals forces exerted by cations, anions, and net-neutral polar species can promote the formation of stable dimers. The entropic effects of depletion are negligible, but the combined effect of depletion and macromolecular crowding at physiological concentrations can stabilize aggregates containing just a few NPs. Interparticle interactions are controlled by modest changes in both the structure and dynamic of the interfacial liquid. The molecular origin of these effects and their dependence on NP size are described. The liquid is shown to be highly structured, with large and long-lived hydrogen-bonded water clusters developing often in the interparticle space; their potential role as transient, long-range proton wires connecting and enveloping neighboring NPs is discussed. The basis for a parsimonious theory of ultrasmall NPs in complex fluids is established.

A model is proposed for the evaluation of dispersive forces in a continuum solvent representation for use in large-scale computer simulations. The model captures the short- and long-range effects of water-exclusion in conditions of partial and anisotropic hydration. The model introduces three parameters, one of which represents the degree of hydration (water occupancy) at any point in the system, which depends on the solute conformation, and two that represent the strength of water-water and water-solute dispersive interactions. The model is optimized for proteins, using hydration data of a suboptimally hydrated binding site and results from dynamics simulations in explicit water. The model is applied to a series of aliphatic-alcohol/protein complexes and a set of binary and ternary complexes of various sizes. Implications for weak and ultra-weak protein-protein association and for simulation in crowded media are discussed.

The enantiomers of a variety of N-alkyl-, N-aralkyl-, and N-cyclopropylalkyl-9β-hydroxy-5-(3-hydroxyphenyl)morphans were synthesized employing cyanogen bromide and K2CO3 to improve the original N-demethylation procedure. Their binding affinity to the μ-, δ-, and κ-opioid receptors (ORs) was determined and functional (GTPγ35S) assays were carried out on those with reasonable affinity. The 1R,5R,9S-enantiomers (1R,5R,9S)-(−)-5-(3-hydroxyphenyl)-2-(4-nitrophenethyl)-2-azabicyclo[3.3.1]nonan-9-ol (1R,5R,9S-16), (1R,5R,9S)-(−) 2-cinnamyl-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ol (1R,5R,9S-20), and (1R,5R,9S)-(−)-5-(3-hydroxyphenyl)-2-(4-(trifluoromethyl)phenethyl)-2-azabicyclo[3.3.1]nonan-9-ol (1R,5R,9S-15), had high affinity for the μ-opioid receptor (e.g., 1R,5R,9S-16: Ki = 0.073, 0.74, and 1.99 nM, respectively). The 1R,5R,9S-16 and 1R,5R,9S-15 were full, high efficacy μ-agonists (EC50 = 0.74 and 18.5 nM, respectively) and the former was found to be a partial agonist at δ-OR and an antagonist at κ-OR, while the latter was a partial agonist at δ-OR and κ-OR in the GTPγ35S assay. The enantiomer of 1R,5R,9S-16, (+)-1S,5S,9R-16 was unusual, it had good affinity for the μ-OR (Ki = 26.5 nM) and was an efficacious μ-antagonist (Ke = 29.1 nM). Molecular dynamics simulations of the μ-OR were carried out with the 1R,5R,9S-16 μ-agonist and the previously synthesized (1R,5R,9S)-(−)-5-(9-hydroxy-5-(3-hydroxyphenyl-2-phenylethyl)-2-azabicyclo[3.3.1]nonane (1R,5R,9S-(−)-NIH 11289) to provide a structural basis for the observed high affinities and efficacies. The critical roles of both the 9β-OH and the p-nitro group are elucidated, with the latter forming direct, persistent hydrogen bonds with residues deep in the binding cavity, and the former interacting with specific residues via highly structured water bridges.

The interactions between nanoparticles (NPs) and proteins, cells, and tissues, broadly known as nano-bio interactions, depend on the NP size and shape and on the characteristics of the NP coating layer, such as density, thickness, and chemical makeup. The dependence of nano-membrane interactions on the design parameters of ultrasmall nanostructures is studied by computer simulations. Considered here are spheres, plates, rings, rods, tubes, and helices made up of either bare magnetite or passivated gold, interacting with charged or zwitterionic membranes. The analysis reveals a strong dependence on shape, size, and layer composition of various quantities that characterize the nano-bio behavior, including binding modes and affinities. This sensitivity can be exploited to design nanostructures that bind preferentially to membranes or that stabilize or disrupt membrane structural integrity. The method used here is general and not limited to the ultrasmall regime, so it can be adopted to study other nano-bio interactions systematically. The implications for the distribution of NPs in cells and tissues (biodistribution) and for passive and active transmembrane transport are discussed, both important processes in biomedicine.

Weak and ultraweak protein-protein association play a role in molecular recognition and can drive spontaneous self-assembly and aggregation. Such interactions are difficult to detect experimentally, and are a challenge to the force field and sampling technique. A method is proposed to identify low-population protein-protein binding modes in aqueous solution. The method is designed to identify preferential first-encounter complexes from which the final complex(es) at equilibrium evolve. A continuum model is used to represent the effects of the solvent, which accounts for short- and long-range effects of water exclusion and for liquid-structure forces at protein/liquid interfaces. These effects control the behavior of proteins in close proximity and are optimized on the basis of binding enthalpy data and simulations. An algorithm is described to construct a biasing function for self-adaptive configurational-bias Monte Carlo of a set of interacting proteins. The function allows mixing large and local changes in the spatial distribution of proteins, thereby enhancing sampling of relevant microstates. The method is applied to three binary systems. Generalization to multiprotein complexes is discussed.

The hyperactivity of the cyclic dependent kinase 5 (CDK5) induced by the activator protein p25 has been linked to a number of pathologies of the brain. The CDK5-p25 complex has thus emerged as a major therapeutic target for Alzheimer's disease (AD) and other neurodegenerative conditions. Experiments have shown that the peptide p5 reduces the CDK5-p25 activity without affecting the endogenous CDK5-p35 activity, whereas the peptide TFP5, obtained from p5, elicits similar inhibition, crosses the blood-brain barrier, and exhibits behavioral rescue of AD mice models with no toxic side effects. The molecular basis of the kinase inhibition is not currently known, and is here investigated by computer simulations. It is shown that p5 binds the kinase at the same CDK5/p25 and CDK5/p35 interfaces, and is thus a non-selective competitor of both activators, in agreement with available experimental data in vitro. Binding of p5 is enthalpically driven with an affinity estimated in the low µM range. A quantitative description of the binding site and pharmacophore is presented, and options are discussed to increase the binding affinity and selectivity in the design of drug-like compounds against AD.

A continuum model of solvation is proposed to describe (i) long-range electrostatic effects of water exclusion resulting from incomplete and anisotropic hydration in crowded environments and (ii) short-range effects of liquid-structure forces on the hydrogen-bond interactions at solute/water interfaces. The model is an extension of the phenomenological screened coulomb potential-based implicit model of solvation. The developments reported here allow a more realistic representation of highly crowded and spatially heterogeneous environments, such as those in the interior of a living cell. Only the solvent is treated as a continuum medium. It is shown that the electrostatic effects of long-range water-exclusion can strongly affect protein-protein binding energies and are then related to the thermodynamics of complex formation. Hydrogen-bond interactions modulated by the liquid structure at interfaces are calibrated based on systematic calculations of potentials of mean force in explicit water. The electrostatic component of the model is parametrized for monovalent, divalent and trivalent ions. The conceptual and practical aspects of the model are discussed based on simulations of protein complexation and peptide folding. The current implementation is ~1.5 times slower than the gas-phase force field and exhibits good parallel performance.

A method is described for the efficient simulation of multiprotein systems in crowded environments. It is based on an adaptive, reversible structural coarsening algorithm that preserves relevant physical features of the proteins across scales. Water is treated implicitly whereas all the other components of the aqueous solution, such as ions, cosolutes, or osmolytes, are treated in atomic detail. The focus is on the analytical adaptation of the solvent model to different levels of molecular resolutions, which allows continuous, on-the-fly transitions between scales. This permits the analytical calculation of forces during dynamics and preserves detailed balance in Monte Carlo simulations. A major computational speedup can be achieved in systems containing hundreds of proteins without cutting off the long-range interactions. The method can be combined with a self-adaptive configurational-bias sampling technique described previously, designed to detect strong, weak, or ultra-weak protein associations and shown to improve sampling efficiency and convergence. The implementation aims to simulate early stages of multimeric complexation, aggregation, or self-assembly. The method can be adopted as the basis for a more general algorithm to identify vertices, edges, and hubs in protein interaction networks or to predict critical steps in signal transduction pathways.

The prolactin receptor (PRLR) belongs to the class I cytokine receptor superfamily (4, 5). It binds the pituitary hormone prolactin (PRL) with high affinity and triggers intracellular responses that participate in diverse biological functions in target tissues, including the mammary gland, organs of the reproductive system, the central nervous system, pituitary, and adrenal. At least nine variants are generated by alternative splicing of the human PRLR (hPRLR) gene (16-18, 21, 35); they differ in the lengths and compositions of their cytoplasmic domains and/or extracellular (EC) domains (http://atlasgeneticsoncology.org/Genes/PRLRID42891ch5p14.html). The full-length receptor, or long form (LF), is composed of a ligand binding EC domain, a single transmembrane domain, and a cytoplasmic domain required for signal transduction (5, 20). PRL acts through the LF to stimulate cell proliferation and differentiation. The short forms (SFs) of the receptor, S1a and S1b, are derived from alternative splicing of exons 10 and 11 and contain unique cytoplasmic sequences (16). S1a is a 376-amino-acid (aa) receptor that contains partial exon 10 sequences and a unique 39-aa C terminus derived from exon 11. S1b is a 288-aa variant that lacks the entire exon 10 and contains 3 aa derived from exon 11 at the C terminus. Unlike the LF, neither of the SF receptors can mediate activation of the β-casein gene promoter induced by PRL, and both SFs are inhibitory of the transcriptional activation induced by PRL through the LF (16). S1b is more effective than S1a in inhibiting LF actions due to its higher stability (16). The reduced expression of S1a and S1b relative to the LF in breast cancer tissues and cell lines, compared to adjacent normal tissues/cells, suggests that the relatively reduced expression of SFs in cancer could lead to gradations of unopposed PRL-induced LF stimulatory function and contribute to breast tumor development and progression (28). The initiation of signal transduction associated with members of the cytokine receptor family depends on the interaction of cognate ligand preformed receptor dimers (9, 13, 24, 32). In the case of the PRLR, immunoprecipitation studies (12, 31) and bioluminescence resonance energy transfer using coelenterazine (BRET1) analysis (31) demonstrated that hetero- and homodimerization of hPRLR can occur independently of ligand binding. This indicated that PRL is a conformational modifier that induces activation of the JAK2/STAT5 pathway through the LF and possibly through other JAK2-dependent pathways via the SFs. The dominant-negative effect of the SFs results from their heterodimerization with the LF, with the consequent functional inactivation due to the absence of cytoplasmic sequences of the dimerized LF partner (SF) required for downstream JAK2/STAT signaling (16, 31). Compared to the LF, S1b does not contain the conserved cytoplasmic structural motif beyond the Box-1 JAK2 docking site. In the homodimerized LF, the ligand-induced activation of JAK2 phosphorylates the receptor, preferentially at Y580 (rat) or Y587 (human) in its C terminus (2, 22). This induces phosphorylation, dimerization, and nuclear translocation of STAT5, which causes transcriptional activation of PRL-responsive genes, including the β-casein gene (8). Alternatively, Src has been demonstrated to participate in PRL-induced LF-mediated biological function by recruiting either Fak, Erk1/2, or P13K independently of the classic JAK2/STAT5 pathway (1, 5). These additional pathways have been proposed to operate in SF-mediated action in rodent species (10). Our studies of the mutagenesis of unpaired conserved cysteines adjacent to and within the transmembrane region of the PRLR (S1b) showed no significant changes in receptor homo- or heterodimerization (31). In the present study, we evaluated the relevance of the four conserved extracellular Cys residues that form two intramolecular disulfide (S-S) bonds in subdomain 1 (D1) of the EC domain to the formation of homo- and/or heterodimers and their relevance to the inhibitory function of the SF on the LF. These residues are known to be crucial for ligand binding (33) and hence for PRL-induced activation of the JAK2/STAT5 pathway through the PRLR LF and also presumably in the hormonal activation of S1a and S1b. These intramolecular S-S bonds might provide strong spatial constraints for tertiary folding. Moreover, these bonds might control the quaternary arrangement of the receptor, affecting the formation of homo- and/or heterodimers and the inhibitory function of the SF on the LF. We observed that these residues are essential for maintaining the conformation that favors SF/LF heterodimerization and are also important for the constitutive activity of JAK2 bound to the SF. These studies further revealed that the intramolecular S-S bonds are relevant to the inhibitory action of the SF on the LF function. In addition, molecular modeling of the PRLR wild type and Cys mutants in the EC domain provides insights into the molecular mechanism by which the structure impacts homodimer/heterodimer formation, JAK2 association, and the PRL-induced PRLR action.

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the N-acetylation of serotonin, the penultimate step in the synthesis of melatonin. Pineal AANAT activity increases at night in all vertebrates, resulting in increased melatonin production. This increases circulating levels of melatonin, thereby providing a hormonal signal of darkness. Kinetic and structural analysis of AANAT has determined that one element is floppy. This element, termed Loop 1, is one of three loops that comprise the arylalkylamine binding pocket. During the course of chordate evolution, Loop 1 acquired the tripeptide CPL, and the enzyme became highly active. Here we focused on the functional importance of the CPL tripeptide and found that activity was markedly reduced when it was absent. Moreover, increasing the local flexibility of this tripeptide region by P64G and P64A mutations had the counterintuitive effect of reducing activity and reducing the overall movement of Loop 1, as estimated from Langevin dynamics simulations. Binding studies indicate that these mutations increased the off-rate constant of a model substrate without altering the dissociation constant. The structural kink and local rigidity imposed by Pro-64 may enhance activity by favoring configurations of Loop 1 that facilitate catalysis and do not become immobilized by intramolecular interactions.

With the help of the crystal structure of rhodopsin an ab initio method has been developed to calculate the three-dimensional structure of the loops that connect the transmembrane helices (TMHs). The goal of this procedure is to calculate the loop structures in other G-protein coupled receptors (GPCRs) for which only model coordinates of the TMHs are available. To mimic this situation a construct of rhodopsin was used that only includes the experimental coordinates of the TMHs while the rest of the structure, including the terminal domains, has been removed. To calculate the structure of the loops a method was designed based on Monte Carlo (MC) simulations which use a temperature annealing protocol, and a scaled collective variables (SCV) technique with proper structural constraints. Because only part of the protein is used in the calculations the usual approach of modeling loops, which consists of finding a single, lowest energy conformation of the system, is abandoned because such a single structure may not be a representative member of the native ensemble. Instead, the method was designed to generate structural ensembles from which the single lowest free energy ensemble is identified as representative of the native folding of the loop. To find the native ensemble a successive series of SCV-MC simulations are carried out to allow the loops to undergo structural changes in a controlled manner. To increase the chances of finding the native funnel for the loop, some of the SCV-MC simulations are carried out at elevated temperatures. The native ensemble can be identified by an MC search starting from any conformation already in the native funnel. The hypothesis is that native structures are trapped in the conformational space because of the high-energy barriers that surround the native funnel. The existence of such ensembles is demonstrated by generating multiple copies of the loops from their crystal structures in rhodopsin and carrying out an extended SCV-MC search. For the extracellular loops e1 and e3, and the intracellular loop i1 that were used in this work, the procedure resulted in dense clusters of structures with Calpha-RMSD approximately 0.5 angstroms. To test the predictive power of the method the crystal structure of each loop was replaced by its extended conformations. For e1 and i1 the procedure identifies native clusters with Calpha-RMSD approximately 0.5 angstroms and good structural overlap of the side chains; for e3, two clusters were found with Calpha-RMSD approximately 1.1 angstroms each, but with poor overlap of the side chains. Further searching led to a single cluster with lower Calpha-RMSD but higher energy than the two previous clusters. This discrepancy was found to be due to the missing elements in the constructs available from experiment for use in the calculations. Because this problem will likely appear whenever parts of the structural information are missing, possible solutions are discussed.

Long dynamics simulations were carried out on the B1 immunoglobulin-binding domain of streptococcal protein G (ProtG) and bovine pancreatic trypsin inhibitor (BPTI) using atomistic descriptions of the proteins and a continuum representation of solvent effects. To mimic frictional and random collision effects, Langevin dynamics (LD) were used. The main goal of the calculations was to explore the stability of tens-of-nanosecond trajectories as generated by this molecular mechanics approximation and to analyze in detail structural and dynamical properties. Conformational fluctuations, order parameters, cross correlation matrices, residue solvent accessibilities, pKa values of titratable groups, and hydrogen-bonding (HB) patterns were calculated from all of the trajectories and compared with available experimental data. The simulations comprised over 40 ns per trajectory for ProtG and over 30 ns per trajectory for BPTI. For comparison, explicit water molecular dynamics simulations (EW/MD) of 3 ns and 4 ns, respectively, were also carried out. Two continuum simulations were performed on each protein using the CHARMM program, one with the all-atom PAR22 representation of the protein force field (here referred to as PAR22/LD simulations) and the other with the modifications introduced by the recently developed CMAP potential (CMAP/LD simulations). The explicit solvent simulations were performed with PAR22 only. Solvent effects are described by a continuum model based on screened Coulomb potentials (SCP) reported earlier, i.e., the SCP-based implicit solvent model (SCP-ISM). For ProtG, both the PAR22/LD and the CMAP/LD 40-ns trajectories were stable, yielding C(alpha) root mean square deviations (RMSD) of about 1.0 and 0.8 A respectively along the entire simulation time, compared to 0.8 A for the EW/MD simulation. For BPTI, only the CMAP/LD trajectory was stable for the entire 30-ns simulation, with a C(alpha) RMSD of approximately 1.4 A, while the PAR22/LD trajectory became unstable early in the simulation, reaching a C(alpha) RMSD of about 2.7 A and remaining at this value until the end of the simulation; the C(alpha) RMSD of the EW/MD simulation was about 1.5 A. The source of the instabilities of the BPTI trajectories in the PAR22/LD simulations was explored by an analysis of the backbone torsion angles. To further validate the findings from this analysis of BPTI, a 35-ns SCP-ISM simulation of Ubiquitin (Ubq) was carried out. For this protein, the CMAP/LD simulation was stable for the entire simulation time (C(alpha) RMSD of approximately 1.0 A), while the PAR22/LD trajectory showed a trend similar to that in BPTI, reaching a C(alpha) RMSD of approximately 1.5 A at 7 ns. All the calculated properties were found to be in agreement with the corresponding experimental values, although local deviations were also observed. HB patterns were also well reproduced by all the continuum solvent simulations with the exception of solvent-exposed side chain-side chain (sc-sc) HB in ProtG, where several of the HB interactions observed in the crystal structure and in the EW/MD simulation were lost. The overall analysis reported in this work suggests that the combination of an atomistic representation of a protein with a CMAP/CHARMM force field and a continuum representation of solvent effects such as the SCP-ISM provides a good description of structural and dynamic properties obtained from long computer simulations. Although the SCP-ISM simulations (CMAP/LD) reported here were shown to be stable and the properties well reproduced, further refinement is needed to attain a level of accuracy suitable for more challenging biological applications, particularly the study of protein-protein interactions.

Biological macromolecules and other polymers belong to the class of mesoscopic systems, with characteristic length scale of the order of a nanometer. Although microscopic models would be the preferred choice in theoretical calculations, their use in computer simulations becomes prohibitive for large systems or long simulation times. On the other hand, the use of purely macroscopic models in the mesoscopic domain may introduce artifacts, with effects that are difficult to assess and that may compromise the reliability of the calculations. Here is proposed an approach with the aim of minimizing the empirical nature of continuum approximations of solvent effects within the scope of molecular mechanics (MM) approximations in mesoscopic systems. Using quantum chemical methods, the potential generated by the molecular electron density is first decomposed in a multicenter-multipole expansion around predetermined centers. The monopole and dipole terms of the expansion at each site create electric fields that polarize the surrounding aqueous medium whose dielectric properties can be described by the classical theory of polar liquids. Debye's theory allows a derivation of the dielectric profiles created around isolated point charges and dipoles that can incorporate Onsager reaction field corrections. A superposition of screened Coulomb potentials obtained from this theory makes possible a simple derivation of a formal expression for the total electrostatic energy and the polar component of the solvation energy of the system. A discussion is presented on the physical meaning of the model parameters, their transferability, and their convergence to calculable quantities in the limit of simple systems. The performance of this continuum approximation in computer calculations of amino acids in the context of an atomistic force field is discussed. Applications of a continuum model based on screened Coulomb potentials in multinanosecond simulations of peptides and proteins are briefly reviewed. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005

A systematic study of the potentials of mean force (PMF) of hydrogen-bonded amino acid side chains in water is reported. Hydrogen-bonding (HB) partners are classified according to the hybridization state of their donor and acceptor atoms, as well as the net charge of the interacting pairs. This classification leads to a total of 42 classes of representative HB interactions. Constrained molecular dynamics simulations are carried out to calculate the intermolecular mean force (MF) of the solute molecules in an explicit solvent composed of nonpolarizable TIP3P water. Long-range forces are calculated using particle mesh Ewald (PME) summation in a cubic lattice with periodic boundary conditions. The intermolecular PMF are obtained by integrating the MF along a specified reaction path. MF autocorrelation functions and correlation times are calculated for each HB class. Statistical errors in the MF and PMF are estimated and reported. The results are compared with those reported in the literature for simpler syst...

A continuum electrostatics approach for molecular dynamics (MD) simulations of macromolecules is presented and analyzed for its performance on a peptide and a globular protein. The approach incorporates the screened Coulomb potential (SCP) continuum model of electrostatics, which was reported earlier. The model was validated in a broad set of tests some of which were based on Monte Carlo simulations that included single amino acids, peptides, and proteins. The implementation for large-scale MD simulations presented in this article is based on a pairwise potential that makes the electrostatic model suitable for fast analytical calculation of forces. To assess the suitability of the approach, a preliminary validation is conducted, which consists of (i) a 3-ns MD simulation of the immunoglobulin-binding domain of streptococcal protein G, a 56-residue globular protein and (ii) a 3-ns simulation of Dynorphin, a biological peptide of 17 amino acids. In both cases, the results are compared with those obtained from MD simulations using explicit water (EW) molecules in an all-atom representation. The initial structure of Dynorphin was assumed to be an alpha-helix between residues 1 and 9 as suggested from NMR measurements in micelles. The results obtained in the MD simulations show that the helical structure collapses early in the simulation, a behavior observed in the EW simulation and consistent with spectroscopic data that suggest that the peptide may adopt mainly an extended conformation in water. The dynamics of protein G calculated with the SCP implicit solvent model (SCP-ISM) reveals a stable structure that conserves all the elements of secondary structure throughout the entire simulation time. The average structures calculated from the trajectories with the implicit and explicit solvent models had a cRMSD of 1.1 A, whereas each average structure had a cRMSD of about 0.8A with respect to the X-ray structure. The main conformational differences of the average structures with respect to the crystal structure occur in the loop involving residues 8-14. Despite the overall similarity of the simulated dynamics with EW and SCP models, fluctuations of side-chains are larger when the implicit solvent is used, especially in solvent exposed side-chains. The MD simulation of Dynorphin was extended to 40 ns to study its behavior in an aqueous environment. This long simulation showed that the peptide has a tendency to form an alpha-helical structure in water, but the stabilization free energy is too weak, resulting in frequent interconversions between random and helical conformations during the simulation time. The results reported here suggest that the SCP implicit solvent model is adequate to describe electrostatic effects in MD simulation of both peptides and proteins using the same set of parameters. It is suggested that the present approach could form the basis for the development of a reliable and general continuum approach for use in molecular biology, and directions are outlined for attaining this long-term goal.

An analysis of the screened Coulomb potential–implicit solvent model (SCP–ISM) is presented showing that general equations for both the electrostatic and solvation free energy can be derived in a continuum approach, using statistical averaging of the polarization field created by the solvent around the molecule. The derivation clearly shows how the concept of boundary, usually found in macroscopic approaches, is eliminated when the continuum model is obtained from a microscopic treatment using appropriate averaging techniques. The model is used to study the alanine dipeptide in aqueous solution, as well as the discrimination of native protein structures from misfolded conformations. For the alanine dipeptide the free energy surface in the ϕ–ψ space is calculated and compared with recently reported results of a detailed molecular dynamics simulation using an explicit representation of the solvent, and with other available data. The study showed that the results obtained using the SCP–ISM are comparable to those of the explicit water calculation and compares favorably to the FDPB approach. Both transition states and energy minima show a high correlation (r > 0.98) with the results obtained in the explicit water analysis. The study of the misfolded structures of proteins comprised the analysis of three standard decoy sets, namely, the EMBL, Park and Levitt, and Baker's CASP3 sets. In all cases the SCP–ISM discriminated well the native structures of the proteins, and the best-predicted structures were always near-native (cRMSD ∼2 A). Proteins 2002;47:45–61. © 2002 Wiley-Liss, Inc.

Some key concerns raised by molecular modeling and computational simulation of functional mechanisms for membrane proteins are discussed and illustrated for members of the family of G protein coupled receptors (GPCRs). Of particular importance are issues related to the modeling and computational treatment of loop regions. These are demonstrated here with results from different levels of computational simulations applied to the structures of rhodopsin and a model of the 5-HT2A serotonin receptor, 5-HT2AR. First, comparative Molecular Dynamics (MD) simulations are reported for rhodopsin in vacuum and embedded in an explicit representation of the membrane and water environment. It is shown that in spite of a partial accounting of solvent screening effects by neutralization of charged side chains, vacuum MD simulations can lead to severe distortions of the loop structures. The primary source of the distortion appears to be formation of artifactual H-bonds, as has been repeatedly observed in vacuum simulations. To address such shortcomings, a recently proposed approach that has been developed for calculating the structure of segments that connect elements of secondary structure with known coordinates, is applied to 5-HT2AR to obtain an initial representation of the loops connecting the transmembrane (TM) helices. The approach consists of a simulated annealing combined with biased scaled collective variables Monte Carlo technique, and is applied to loops connecting the TM segments on both the extra-cellular and the cytoplasmic sides of the receptor. Although this initial calculation treats the loops as independent structural entities, the final structure exhibits a number of interloop interactions that may have functional significance. Finally, it is shown here that in the case where a given loop from two different GPCRs (here rhodopsin and 5-HT2AR) has approximately the same length and some degree of sequence identity, the fold adopted by the loops can be similar. Thus, in such special cases homology modeling might be used to obtain initial structures of these loops. Notably, however, all other loops in these two receptors appear to be very different in sequence and structure, so that their conformations can be found reliably only by ab initio, energy based methods and not by homology modeling.

A method is proposed to study protein-ligand binding in a system governed by specific and non-specific interactions. Strong associations lead to narrow distributions in the proteins configuration space; weak and ultra-weak associations lead instead to broader distributions, a manifestation of non-specific, sparsely-populated binding modes with multiple interfaces. The method is based on the notion that a discrete set of preferential first-encounter modes are metastable states from which stable (pre-relaxation) complexes at equilibrium evolve. The method can be used to explore alternative pathways of complexation with statistical significance and can be integrated into a general algorithm to study protein interaction networks. The method is applied to a peptide-protein complex. The peptide adopts several low-population conformers and binds in a variety of modes with a broad range of affinities. The system is thus well suited to analyze general features of binding, including conformational selection, multiplicity of binding modes, and nonspecific interactions, and to illustrate how the method can be applied to study these problems systematically. The equilibrium distributions can be used to generate biasing functions for simulations of multiprotein systems from which bulk thermodynamic quantities can be calculated.

The screened Coulomb potential based implicit solvent model (SCP-ISM) was recently proposed to describe bulk solvent effects in proteins. In this work it is combined with the Monte Carlo approach of conformational memories (CM) and the PAR22 force field of CHARMM to carry out sequence to structure calculations on peptides that have been shown to adopt α-helix and β-hairpin conformations. Simulations at 270 K and 300 K on the peptide, CH3CO-(AAQAA)3-NHCH3, result in α-helix containing populations of 60% and 53% at these two temperatures, respectively. The residue helicities were found to change dramatically between these two temperatures, and at the lower temperature are in good agreement with the experimental helicities estimated at 274 K. CM simulations on the second peptide, CH3CO-V5DPGV5-NH2, were carried out to study the β-hairpin forming propensity of DPro. The calculations gave a population of about 40% β-hairpin conformations at 300 K with the residues DPro-Gly forming all or part of the loop. Four main subfamilies of different β-hairpin/β-turn structures were found. It was observed that hydrophobic interactions between specific side chains play a fundamental role in the formation and stabilization of the different types of β-turns. For both peptides, populations of about 40% random coil structures were found. Comparison with available theoretical and experimental results is presented and discussed. © 2001 John Wiley & Sons, Inc. Int J Quant Chem 83: 193–202, 2001

A simple approach for calculating hydrogen bonding (H-bonding) effects in molecular mechanics simulations of peptides and proteins is presented for use in the framework of the continuum solvent model described in the previous paper. In this approach, the solvated macromolecule is treated as a three-component dielectric system consisting of the solvent, bulk protein, and proton acceptor media. The hydrogen bond (H-bond) interaction is identified from the interpenetration of the van der Waals spheres of the polar hydrogen and the proton acceptor. The H-bond geometry is characterized by the ideal orientation of the electron lone pairs in the acceptor atom and the directionality of the proton donor bond, as observed in experimental and ab initio studies, and classified according to the hybridization state of the acceptor atom. The algorithm was implemented into CHARMM using the PAR22 force field. By introducing the concept of a Born radius of a polar hydrogen immersed in an acceptor environment, the stabiliza...

A new implicit solvent model for Monte Carlo simulations of peptides and proteins is presented. The important features of the model are: electrostatics of polar solvation described by nonlinear, distance-dependent screened Coulomb potentials that are formally connected to classical dielectric theory of polar solvation; the self-energy is derived from the integral form of the Born equation; and the introduction of the Born radius of an atom embedded in a protein modeled as a dielectric continuum. The model was parametrized in the context of the CHARMM PAR22 force field by fitting to experimental solvation free energies of amino acid side chain analogues. Introduction of an intrinsic vacuum screening constant for each amino acid was required to obtain the correct screening behavior in solution. Solvation energies of several peptides in different conformations were calculated and compared with the corresponding quantities calculated with the Poisson−Boltzmann equation. The comparative results were highly co...

We analyze in detail the nonlinear kinetics of a carrier system in a photoinjected plasma in semiconductors under the action of constant illumination with ultraviolet light. We show that the spatially homogeneous steady-state becomes unstable, and a charge density wave emerges after a critical intensity of the incident radiation is achieved. It is shown that this instability can only follow in doped p-type materials. In bulk systems the critical intensity was found to be too high making the phenomenon not observable under realistic experimental conditions. However, a more efficient electron excitation can be obtained in low dimensional p-type systems, like some molecular and biological polymers, where the interaction may follow by chemical interaction with the medium. We show that for intensities beyond the critical threshold an increasing number of modes provide further contributions (subharmonics) to the space inhomogeneity. It is conjectured that this process could lead the system to display chaotic-like behavior.

We consider the steady-state photoinjected two-component plasma in quasi–one-dimensional semiconductor materials, generated by c.w. UV-light illumination. We analyze the two types of carrier's collective elementary excitations in this far-from-equilibrium dissipative system. It is predicted that at a critical level of excitation there follows a phenomenon consisting in a drastic change of behavior in the propagation of the so-called upper plasma waves.

We study the eigenvalue spectrum of the informational-statistical-entropy operator, a quantity that plays a fundamental role in the nonequilibrium statistical operator method. We obtain explicit expressions for the informational entropy for inhomogeneous nonequilibrium (dissipative) systems, relevant for the study of its nonclassical nonlinear hydrodynamics. Expressions for the single-particle dynamical matrix and, in particular, for the distribution functions of quasi-particles, in conditions arbitrarily away from equilibrium, are also derived. We apply the results considering some aspects of the hydrodynamics of a Fermion system in weak interaction with a thermal bath of Bosons.

We describe the production and the optical properties of the non-equilibrium photoinjected plasma in semiconductor quantum wires under continuous UV-light illumination. The wavenumber-dependent dynamic dielectric function of this system is derived and the Raman scattering cross-section calculated. From the latter we identify the contributions from different types of elementary excitations. They consist of, besides the single-particle excitations, two types of collective oscillations: an upper one, consisting of an intrasubband-like plasmon and a lower one, identified as an acoustic-like plasmon. The dependence of both on the non-equilibrium (dissipative) macroscopic state of the system is evidenced and discussed.

An analysis of the nonequilibrium thermodynamics of a dissipative system, dealth with at the mechano-statistical level provided by informational-statistical thermodynamics is presented. We consider inp particular the case of a highly excited photoinjected plasma in a polar semiconductor. Under continuous illumination, a steady state sets in, whose macroscopic state is characterized, the main mechanisms for pumping and dissipation that are involved are analyzed, and the characteristics of the evolution towards the steady state and its stability are discussed. It is shown that such evolution displays a phenomenon of quasitemperature overshoot.