Guy H. Grant

Distance measurements in the borderline region of applicability of CW EPR and DEER: A model study on a homologous series of spin-labelled peptides

Inter-spin distances between 1 nm and 4.5 nm are measured by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) methods for a series of nitroxide-spin-labelled peptides. The upper distance limit for measuring dipolar coupling by the broadening of the CW spectrum and the lower distance limit for the present optimally-adjusted double electron electron resonance (DEER) set-up are determined and found to be both around 1.6-1.9 nm. The methods for determining distances and corresponding distributions from CW spectral line broadening are reviewed and further developed. Also, the work shows that a correction factor is required for the analysis of inter-spin distances below approximately 2 nm for DEER measurements and this is calculated using the density matrix formalism.

The Distinct Conformational Dynamics of K-Ras and H-Ras A59G

Ras proteins regulate signaling cascades crucial for cell proliferation and differentiation by switching between GTP- and GDP-bound conformations. Distinct Ras isoforms have unique physiological functions with individual isoforms associated with different cancers and developmental diseases. Given the small structural differences among isoforms and mutants, it is currently unclear how these functional differences and aberrant properties arise. Here we investigate whether the subtle differences among isoforms and mutants are associated with detectable dynamical differences. Extensive molecular dynamics simulations reveal that wild-type K-Ras and mutant H-Ras A59G are intrinsically more dynamic than wild-type H-Ras. The crucial switch 1 and switch 2 regions along with loop 3, helix 3, and loop 7 contribute to this enhanced flexibility. Removing the gamma-phosphate of the bound GTP from the structure of A59G led to a spontaneous GTP-to-GDP conformational transition in a 20-ns unbiased simulation. The switch 1 and 2 regions exhibit enhanced flexibility and correlated motion when compared to non-transitioning wild-type H-Ras over a similar timeframe. Correlated motions between loop 3 and helix 5 of wild-type H-Ras are absent in the mutant A59G reflecting the enhanced dynamics of the loop 3 region. Taken together with earlier findings, these results suggest the existence of a lower energetic barrier between GTP and GDP states of the mutant. Molecular dynamics simulations combined with principal component analysis of available Ras crystallographic structures can be used to discriminate ligand- and sequence-based dynamic perturbations with potential functional implications. Furthermore, the identification of specific conformations associated with distinct Ras isoforms and mutants provides useful information for efforts that attempt to selectively interfere with the aberrant functions of these species.

Characterization and tissue‐specific expression of two lepidopteran farnesyl diphosphate synthase homologs: Implications for the biosynthesis of ethyl‐substituted juvenile hormones

The sesquiterpenoid juvenile hormone (JH) regulates insect development and reproduction. Most insects produce only one chemical form of JH, but the Lepidoptera produce four derivatives featuring ethyl branches. The biogenesis of these JHs requires the synthesis of ethyl‐substituted farnesyl diphosphate (FPP) by FPP synthase (FPPS). To determine if there exist more than one lepidopteran FPPS, and whether one FPPS homolog is better adapted for binding the bulkier ethyl‐branched substrates/products, we cloned three lepidopteran FPPS cDNAs, two from Choristoneura fumiferana and one from Pseudaletia unipuncta. Amino acid sequence comparisons among these and other eukaryotic FPPSs led to the recognition of two lepidopteran FPPS types. Type‐I FPPSs display unique active site substitutions, including several in and near the first aspartate‐rich motif, whereas type‐II proteins have a more “conventional” catalytic cavity. In a yeast assay, a Drosophila FPPS clone provided full complementation of an FPPS mutation, but lepidopteran FPPS clones of either type yielded only partial complementation, suggesting unusual catalytic features and/or requirements of these enzymes. Although a structural analysis of lepidopteran FPPS active sites suggested that type‐I enzymes are better suited than type‐II for generating ethyl‐substituted products, a quantitative real‐time PCR assessment of their relative abundance in insect tissues indicated that type‐I expression is ubiquitous whereas that of type‐II is essentially confined to the JH‐producing glands, where its transcripts are ∼20 times more abundant than those of type‐I. These results suggest that type‐II FPPS plays a leading role in lepidopteran JH biosynthesis in spite of its apparently more conventional catalytic cavity. Proteins 2006.

Modeling Aromatic Liquids: Toluene, Phenol, and Pyridine.

Aromatic groups are now acknowledged to play an important role in many systems of interest. However, existing molecular mechanics methods provide a poor representation of these groups. In a previous paper, we have shown that the molecular mechanics treatment of benzene can be improved by the incorporation of an explicit representation of the aromatic π electrons. Here, we develop this concept further, developing charge-separation models for toluene, phenol, and pyridine. Monte Carlo simulations are used to parametrize the models, via the reproduction of experimental thermodynamic data, and our models are shown to outperform an existing atom-centered model. The models are then used to make predictions about the structures of the liquids at the molecular level and are tested further through their application to the modeling of gas-phase dimers and cation-π interactions.

Binding modes of diketo-acid inhibitors of HIV-1 integrase: A comparative molecular dynamics simulation study

Graphical abstract Highlights ► Comparative molecular dynamics simulations on HIV-1 IN bound with L-731,988, L-708,906 and S-1360. ► The acidic end of all the DKA inhibitors studied formed favourable ionic interactions with Lys159. ► The keto–enol parts of these compounds were consistently coordinated to Mg. ► The catalytic residue Glu152 formed a favourable ion–pair interaction with the negatively charged Arg199 on α6 in the most potent DKA inhibitors. ► The complexation with Merck inhibitors and S-1360 significantly constrained the flexible surface loop into an extended or open conformation.

The Structure of Liquid Benzene.

The interactions of aromatic groups have been identified as playing a crucial role in many systems of interest. Unfortunately, conventional atom-centered force fields provide only an approximate representation of these molecules owing to their failure to consider the quadrupole moment arising from the π electrons. In this paper the structure of liquid benzene, the prototypical aromatic system, is investigated using a novel approach to Monte Carlo simulation, parametrized against experimental thermodynamic data, which incorporates an explicit representation of the aromatic π electrons. In contrast to previous simulations of liquid benzene it is found that a perpendicular arrangement of benzene molecules is preferred to a parallel arrangement. This result is in good agreement with experimental data.

Computational Analysis of Phosphopeptide Binding to the Polo-Box Domain of the Mitotic Kinase PLK1 Using Molecular Dynamics Simulation

The Polo-Like Kinase 1 (PLK1) acts as a central regulator of mitosis and is over-expressed in a wide range of human tumours where high levels of expression correlate with a poor prognosis. PLK1 comprises two structural elements, a kinase domain and a polo-box domain (PBD). The PBD binds phosphorylated substrates to control substrate phosphorylation by the kinase domain. Although the PBD preferentially binds to phosphopeptides, it has a relatively broad sequence specificity in comparison with other phosphopeptide binding domains. We analysed the molecular determinants of recognition by performing molecular dynamics simulations of the PBD with one of its natural substrates, CDC25c. Predicted binding free energies were calculated using a molecular mechanics, Poisson-Boltzmann surface area approach. We calculated the per-residue contributions to the binding free energy change, showing that the phosphothreonine residue and the mainchain account for the vast majority of the interaction energy. This explains the very broad sequence specificity with respect to other sidechain residues. Finally, we considered the key role of bridging water molecules at the binding interface. We employed inhomogeneous fluid solvation theory to consider the free energy of water molecules on the protein surface with respect to bulk water molecules. Such an analysis highlights binding hotspots created by elimination of water molecules from hydrophobic surfaces. It also predicts that a number of water molecules are stabilized by the presence of the charged phosphate group, and that this will have a significant effect on the binding affinity. Our findings suggest a molecular rationale for the promiscuous binding of the PBD and highlight a role for bridging water molecules at the interface. We expect that this method of analysis will be very useful for probing other protein surfaces to identify binding hotspots for natural binding partners and small molecule inhibitors.

Electronic Circular Dichroism Spectroscopy of 1-(R)-Phenylethanol: The “Sector Rule” Revisited and an Exploration of Solvent Effects

The sensitivity of the electronic circular dichroism (ECD) of a chiral molecule to structural and environmental changes has been investigated using 1-(R)-phenylethanol (1-PE) as the benchmark solute and cyclohexane and water as the trial solvents. Rotatory strengths associated with the π π* (1Lb) electronic transition have been calculated ab initio for: (a) isolated 1-PE, as a function of the dihedral angles within its chiral side chain and between the side chain and the aromatic ring: these confirm the validity of the empirical “sector rule” but with the signs reversed; (b) the singly and doubly hydrated clusters of 1-PE, isolated in the gas phase; (c) 1-PE and its singly and doubly hydrated clusters embedded in a polarizable dielectric continuum; and (d) 1-PE in an aqueous solution interacting with the local hydration shell and with the polarizable dielectric continuum, using averaged solute structures computed from the fluctuating solute and solvent configurations generated via molecular dynamics simulations. The ab initio calculations (conducted with GAUSSIAN 98) employed configuration interaction singles (CIS) or time-dependent density functional theory (TD-DFT) procedures using basis sets of double- and triple-zeta quality. The results have been related to experimental observations and discussed in terms of changes in the molecular geometry and solvent-induced perturbations.

Pinpointing anthrax-toxin inhibitors

118 findings often generate such hyperbole, and so biotechnology companies would benefit by keeping their own appraisals modest and emphasizing the often long-term nature of biomedical research findings. 3. Scientists working in areas that involve difficult or controversial ethical issues should seek ethical advice from external sources. Acting on this principle, ACT invited a distinguished group of bioethicists, health-care lawyers, clinical medical specialists, and community members to form an independent Ethics Advisory Board to furnish advice and guidelines for our research. Chaired by Ronald M. Green of Dartmouth College, this Board has met on a quarterly basis since the inception of our therapeutic cloning research and has laid down strict guidelines for the conduct of that research and the associated egg donor program. Members of that EAB have no financial stake in ACT’s research and remain free to publicly criticize our conduct. We are by no means trying to suggest that everything we have done is flawless. Like others working in a new and controversial area of research, we are trying to steer our way through a thicket of challenging new issues and are learning as we proceed. The opportunities afforded by cellular reprogramming through nuclear transfer are broad and could substantially affect future health-care practice. The ability to manufacture nearly any cell type through a nuclear transfer–derived embryonic stem-cell line could make it possible to produce many cells not currently available to treat a wide array of disorders including Parkinson’s disease, diabetes, heart failure, and renal failure, as well as many others. Therefore, we at ACT regard our role in this debate with the highest degree of gravity. The debate is greater than any of us. The accusation that ACT is, by its actions, inviting an outright legislative ban on therapeutic cloning research is therefore a serious charge. We would point out that the US House of Representatives voted to ban all uses of cloning in the human species (including therapeutic cloning) months before ACT’s report and that similar prohibitions were actively being discussed in the US Senate. To advise that researchers working in this controversial area must halt their work or withdraw publication of their results until legislators make a final law is, in our opinion, to display a lack of confidence in the strength of US democracy. The United States plays an important role as one of the leaders in biotechnology. It is not only our opportunity, but our duty, to inform the public of our research objectives, keep the public appraised of our progress, and display confidence that a free and informed public is capable of appreciating the motives of the biotechnology industry. Robert P. Lanza, Jose B. Cibelli, and Michael D. West, Advanced Cell Technology, Worcester, MA 01605 (rlanza@advancedcell.com)

Unraveling evolutionary constraints: a heterogeneous conservation in dynamics of the titin Ig domains.

The giant protein titin, which comprises immunoglobulin (Ig) domains, acts as a bidirectional spring in muscle. The unfolding of Ig domains has been extensively studied, but their dynamics under native states have not been well‐characterized. We performed molecular dynamics simulation on a single titin Ig domain and multi‐domains. Mobile regions displaying concerted motions were identified. The dynamics of Ig domains are constrained by evolutionary pressures, in such a way that global dominant motion is conserved, yet different flexibilities within Ig domains and in linkers connecting neighbouring domains were observed. We explain these heterogeneous conserved dynamics in relation to sequence conservation across species and the sequence diversity among neighbouring Ig domains.