CHARMM (Chemistry at HARvard Molecular Mechanics) is a highly versatile and widely used molecular simulation program. It has been developed over the last three decades with a primary focus on molecules of biological interest, including proteins, peptides, lipids, nucleic acids, carbohydrates and small molecule ligands, as they occur in solution, crystals, and membrane environments. For the study of such systems, the program provides a large suite of computational tools that include numerous conformational and path sampling methods, free energy estimators, molecular minimization, dynamics, and analysis techniques, and model-building capabilities. In addition, the CHARMM program is applicable to problems involving a much broader class of many-particle systems. Calculations with CHARMM can be performed using a number of different energy functions and models, from mixed quantum mechanical-molecular mechanical force fields, to all-atom classical potential energy functions with explicit solvent and various boundary conditions, to implicit solvent and membrane models. The program has been ported to numerous platforms in both serial and parallel architectures. This paper provides an overview of the program as it exists today with an emphasis on developments since the publication of the original CHARMM paper in 1983.

Dengue virus is responsible for Ϸ50 -100 million infections, resulting in nearly 24,000 deaths annually. The capsid (C) protein of dengue virus is essential for specific encapsidation of the RNA genome, but little structural information on the C protein is available. We report the solution structure of the 200-residue homodimer of dengue 2 C protein. The structure provides, to our knowledge, the first 3D picture of a flavivirus C protein and identifies a fold that includes a large dimerization surface contributed by two pairs of helices, one of which has characteristics of a coiled-coil. NMR structure determination involved a secondary structure sorting approach to facilitate assignment of the intersubunit nuclear Overhauser effect interactions. The dimer of dengue C protein has an unusually high net charge, and the structure reveals an asymmetric distribution of basic residues over the surface of the protein. Nearly half of the basic residues lie along one face of the dimer. In contrast, the conserved hydrophobic region forms an extensive apolar surface at a dimer interface on the opposite side of the molecule. We propose a model for the interaction of dengue C protein with RNA and the viral membrane that is based on the asymmetric charge distribution of the protein and is consistent with previously reported results.D engue virus, a member of the flavivirus genus of enveloped RNA viruses, is one of the most significant mosquito-borne viral pathogens, given the impact of the recent resurgence of dengue fever and dengue hemorrhagic fever (1). Other members of the flavivirus genus are also important human pathogens and include such viruses as yellow fever, West Nile virus, and Japanese encephalitis (2). The structure of dengue virus was recently described by using cryo-electron microscopy, which permitted visualization of certain viral protein components (3). The organization of the major envelope glycoprotein (E) in the virion was obtained by fitting the atomic structure of the ectodomain from the related tick-borne encephalitis (TBE) E protein into the outermost layer of density (4). Density internal to the E ectodomain demonstrated a host-derived lipid bilayer with transmembrane helices from E and the small structural protein M (5). Interior to the bilayer is the nucleocapsid core that comprises multiple copies of the capsid, or core, protein (C) and a single 10.7-kb genome RNA. Surprisingly, the organization of the C protein within the nucleocapsid core layer is not discernable. The lack of strong density for C may suggest a rather unique architecture of the flavivirus nucleocapsid core that is distinct from the structural organization of morphologically related viruses such as alphaviruses, a genus in the Togaviridae family of mosquito-borne viruses.The dengue C protein is essential in virus assembly to ensure specific encapsidation of the viral genome. The critical role of C is evident from the existence of subviral particles that are released from infected cells but lack C protein and genome RNA (6). The me...

The high mortality and financial burden associated with prostate cancer can be partly attributed to a lack of sensitive screening methods for detection and staging of the disease. Guided by in silico docking studies using the crystal structure of PSMA, we designed and synthesized a series of PSMA-targeted (99m)Tc-chelate complexes for imaging PSMA-expressing human prostate cancer cells (LNCaP cell line). Of the six targeted radioimaging agents synthesized, three were found to bind LNCaP cells with low nanomolar affinity. Moreover, the same three PSMA-targeted imaging agents were shown to localize primarily to LNCaP tumor xenografts in nu/nu mice, with an average of 9.8 +/- 2.4% injected dose/g tissue accumulating in the tumor and only 0.11% injected dose/g tissue retained in the muscle at 4 h postinjection. Collectively, these high affinity, PSMA-specific radioimaging agents demonstrate significant potential for use in localizing prostate cancer masses, monitoring response to therapy, detecting prostate cancer recurrence following surgery, and selecting patients for subsequent PSMA-targeted chemotherapy.

The tertiary structure of CA is conserved across the major retroviral genera, yet sequence variations are sufficient to cause change in associative behavior. CA forms the exterior shell of the viral core in all mature retroviruses. However, the core morphology differs between viruses. Consistent with this observation, we find that the capsid proteins of RSV and human immunodeficiency virus type 1 exhibit different associative behavior in dilute solution and assemble in vitro into different structures.

The post-mortem brains of individuals with Parkinson’s disease (PD) and other synucleinopathy disorders are characterized by the presence of aggregated forms of the presynaptic protein α-synuclein (aSyn). Understanding the molecular mechanism of aSyn aggregation is essential for the development of neuroprotective strategies to treat these diseases. In this study, we examined how interactions between aSyn and phospholipid vesicles influence the protein’s aggregation and toxicity to dopaminergic neurons. Two-dimensional NMR data revealed that two familial aSyn mutants, A30P and G51D, populated an exposed, membrane-bound conformer in which the central hydrophobic region was dissociated from the bilayer to a greater extent than in the case of wild-type aSyn. A30P and G51D had a greater propensity to undergo membrane-induced aggregation and elicited greater toxicity to primary dopaminergic neurons compared to the wild-type protein. In contrast, the non-familial aSyn mutant A29E exhibited a weak propensity to aggregate in the presence of phospholipid vesicles or to elicit neurotoxicity, despite adopting a relatively exposed membrane-bound conformation. Our findings suggest that the aggregation of exposed, membrane-bound aSyn conformers plays a key role in the protein’s neurotoxicity in PD and other synucleinopathy disorders.

The capsid proteins of two flaviviruses, yellow fever virus and dengue virus, were expressed in Escherichia coli and purified to near homogeneity suitable for biochemical characterization and structure determination by nuclear magnetic resonance. The oligomeric properties of the capsid protein in solution were investigated. In the absence of nucleic acid, both proteins were predominately dimeric in solution. Further analysis of both proteins with far-UV circular dichroism spectroscopy indicated that they were largely alpha-helical. The secondary structure elements of the dengue virus capsid were determined by chemical shift indexing of the sequence-specific backbone resonance assignments. The dengue virus capsid protein devoid of its C-terminal signal sequence was found to be composed of four alpha helices. The longest alpha helix, 20 residues, is located at the C terminus and has an amphipathic character. In contrast, the N terminus was found to be unstructured and could be removed without disrupting the structural integrity of the protein.The Flaviviridae family of enveloped RNA viruses causes significant disease in both humans and agriculturally important animals. Flavivirus, the largest of the three genera of Flaviviridae, comprises over 70 viruses, mostly arthropod transmitted, including yellow fever virus (YF), dengue virus (DEN), West Nile virus, and tick-borne encephalitis virus (TBE) (15). The mature flavivirus particle is spherical with a diameter of 50 nm and contains multiple copies of three different structural proteins (C, M, and E), a host-derived membrane bilayer, and a single copy of a positive-sense RNA genome of approximately 11,000 nucleotides. The RNA genome is translated from a single open reading frame generating a polyprotein that is processed by viral and host proteases to yield the three structural proteins located at the N terminus followed by at least seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (23). The nonstructural proteins associate to form the viral replicase complex, for which several enzymatic functions have been identified, including protease, helicase, methyltransferase, and RNA-dependent RNA polymerase (15). The replicase complex is associated with intracellular membranes of the infected host and induces discrete membrane structures (16). Recent evidence suggests that the process of particle assembly may be coupled to genome replication (6,12). This coupling of replication and virus assembly has made analysis of flavivirus assembly difficult, and thus, little is known about this important aspect of the virus life cycle.The structure of DEN, recently determined by cryoelectron microscopy (cryo-EM) and three-dimensional image reconstruction, has elucidated the molecular organization of the end product of the flavivirus assembly pathway (11). The organization of the E protein within the DEN particle was determined by modeling the atomic resolution structure of TBE E protein (22) into the outer density of the cryo-EM reconstruction. The model consist...

Human glutaredoxin (GRx), also known as thioltransferase, is a 12 kDa thiol-disulfide oxidoreductase that is highly selective for reduction of glutathione-containing mixed disulfides. The apparent pK(a) for the active site Cys22 residue is approximately 3.5. Previously we observed that the catalytic enhancement by glutaredoxin could be ascribed fully to the difference between the pK(a) of its Cys22 thiol moiety and the pK(a) of the product thiol, each acting as a leaving group in the enzymatic and nonenzymatic reactions, respectively [Srinivasan et al. (1997), Biochemistry 36, 3199-3206]. Continuum electrostatic calculations suggest that the low pK(a) of Cys22 results primarily from stabilization of the thiolate anion by a specific ion-pairing with the positively charged Lys19 residue, although hydrogen bonding interactions with Thr21 also appear to contribute. Variants of Lys19 were considered to further assess the predicted role of Lys19 on the pK(a) of Cys22. The variants K19Q and K19L were generated by molecular modeling, and the pK(a) value for Cys22 was calculated for each variant. For K19Q, the predicted Cys22 pK(a) is 7.3, while the predicted value is 8.3 for K19L. The effects of the mutations on the interaction energy between the adducted glutathionyl moiety and GRx were roughly estimated from the van der Waals and electrostatic energies between the glutathionyl moiety and proximal protein residues in a mixed disulfide adduct of GRx and glutathione, i.e., the GRx-SSG intermediate. The values for the K19 mutants differed by only a small amount compared to those for the wild type enzyme intermediate. Together, the computational analysis predicted that the mutant enzymes would have markedly reduced catalytic rates while retaining the glutathionyl specificity displayed by the wild type enzyme. Accordingly, we constructed and characterized the K19L and K19Q mutants of two forms of the GRx enzyme. Each of the mutants retained glutathionyl specificity as predicted and displayed diminution in activity, but the decreases in activity were not to the extent predicted by the theoretical calculations. Changes in the respective Cys22-thiol pK(a) values of the mutant enzymes, as shown by pH profiles for iodoacetamide inactivation of the respective enzymes, clearly revealed that the K19-C22 ion pair cannot fully account for the low pK(a) of the Cys22 thiol. Additional contributions to stabilization of the Cys22 thiolate are likely donated by Thr21 and the N-terminal partial positive charge of the neighboring alpha-helix.

SynopsisIn an unfavorable solvent environment, DNA (and other polymers) undergo a conformational transition to a collapsed form, accompanied by a dramatic reduction in the effective volume of the molecule. Solvent conditions leading to the collapse are the same as those that cause aggregation. We give here a thermodynamic description of the collapse and its relations t o aggregation (or precipitation). This is formulated in terms of the Flory-Huggins theory of the thermodynamics of polymer solutions. The results show that it is possible for three different states of DNA to be stable under different conditions: (1) the extended random coil, (2) the collapsed coil, and (3) a concentrated phase of aggregated random coils. The collapsed coil is predicted to he stable against aggregation only at high dilutions, of the order of parts per million. For DNA the transition between the extended coil and the collapsed coil is predicted to he discontinuous, in the sense that intermediate states are not present, because of the relatively high stiffness of the chain. The transition should appear diffuse because of the small size of the single molecule in comparison to macroscopic systems.