QM/MM methods for crystalline defects. Part 1: Locality of the tight binding model

The tight binding model is a minimal electronic structure model for molecular modelling and simulation. We show that the total energy in this model can be decomposed into site energies, that is, into contributions from each atomic site whose influence on their environment decays exponentially. This result lays the foundation for a rigorous analysis of QM/MM coupling schemes.Comment: 35 pages, 3 figure

Active site voltage clamp fluorometry of the sodium glucose cotransporter hSGLT1.

In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescence indicated that the environment of the chromophores became more polar with opening of the external gates as the protein transitioned from the inward to outward facing state. Structural analyses showed an increase in polar side chains and a decrease in hydrophobic side chains lining the vestibule, and this was reflected in solvation of the chromophore. The results demonstrate the opening and closing of external gates in real time, with the accompanying changes of polarity of the sugar vestibule

Fractional-order susceptible-infected model: definition and applications to the study of COVID-19 main protease

We propose a model for the transmission of perturbations across the amino acids of a protein represented as an interaction network. The dynamics consists of a Susceptible-Infected (SI) model based on the Caputo fractional-order derivative. We find an upper bound to the analytical solution of this model which represents the worse-case scenario on the propagation of perturbations across a protein residue network. This upper bound is expressed in terms of Mittag-Leffler functions of the adjacency matrix of the network of inter-amino acids interactions. We then apply this model to the analysis of the propagation of perturbations produced by inhibitors of the main protease of SARS CoV-2. We find that the perturbations produced by strong inhibitors of the protease are propagated far away from the binding site, confirming the long-range nature of intra-protein communication. On the contrary, the weakest inhibitors only transmit their perturbations across a close environment around the binding site. These findings may help to the design of drug candidates against this new coronavirus.Comment: 21 pages, 2 figure

A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the beta-19' position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (Delta-F/F) approximate to 10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for Delta-F agreed well with those for epibatidine-induced currents, but were shifted approximate to 100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the alpha-gamma-binding site than to the alpha-delta site and (ii) ACh binds with reverse-site selectivity, these data suggest that Delta-F monitors an event linked to binding specifically at the alpha-delta-subunit interface. In experiments with flash-applied agonists, the earliest detectable Delta-F occurs within milliseconds, i.e., during activation. At low [ACh] (less than or equal to 10 muM), a phase of Delta-F occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from Delta-F is complete before the slowest phase of recovery from desensitization (time constant approximate to 250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the alpha-delta-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes

Applications of a tight-binding total energy method for transition and noble metals: Elastic Constants, Vacancies, and Surfaces of Monatomic Metals

A recent tight-binding scheme provides a method for extending the results of first principles calculations to regimes involving $10^2 - 10^3$ atoms in a unit cell. The method uses an analytic set of two-center, non-orthogonal tight-binding parameters, on-site terms which change with the local environment, and no pair potential. The free parameters in this method are chosen to simultaneously fit band structures and total energies from a set of first-principles calculations for monatomic fcc and bcc crystals. To check the accuracy of this method we evaluate structural energy differences, elastic constants, vacancy formation energies, and surface energies, comparing to first-principles calculations and experiment. In most cases there is good agreement between this theory and experiment. We present a detailed account of the method, a complete set of tight-binding parameters, and results for twenty-nine of the alkaline earth, transition and noble metals.Comment: 24 pages (REVTEX), 6 figures (epsf.tex required). Several new results have been added. Re-submitted to Physical Review

Conformational Dynamics of the Plug Domain of the SecYEG Protein-conducting Channel

The central pore of the SecYEG preprotein-conducting channel is closed at the periplasmic face of the membrane by a plug domain. To study its conformational dynamics, the plug was labeled site-specifically with an environment-sensitive fluorophore. In the presence of a stable preprotein translocation intermediate, the SecY plug showed an enhanced solvent exposure consistent with a displacement from the hydrophobic central pore region. In contrast, binding and insertion of a ribosome-bound nascent membrane protein did not alter the plug conformation. These data indicate different plug dynamics depending on the ligand bound state of the SecYEG channel.

A study of CIS-acting elements required for dosage compensation in Drosophila Melanogaster : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand

Dosage compensation (the equalisation of X-linked gene products) occurs in Drosophila melanogaster by a two fold transcriptional up-regulation of X-linked gene expression in males. This involves the binding of five proteins, MSL-1, MSL-2, MSL-3, MLE, MOF, and potentially an RNA (roXl or roX2), to hundreds of sites along the male X chromosome. The cis-acting X-linked DNA sequences required for dosage compensation (called dosage compensation regulatory elements or DCREs) remain elusive, despite numerous attempts of identify them. An insulated reporter gene assay system has been developed to minimise problems previously encountered with identification of these elements. The reporter system consists of the constitutive armadillo promoter fused to the lacZ reporter gene (called arm-lacZ). This reporter construct is flanked by SCS/SCS' insulator elements to block potential repressive effects of an autosomal chromatin environment. The role of the roX genes during dosage compensation was investigated. Initially both the roXl and roX2 RNAs were expressed from within the arm-lacZ insulated system. Expression of either RNA lead to a significant increase in lacZ expression in males, although consistently less than two-fold. These results suggested that either the MSL complex was binding to the roX genes or the expression of the roX RNAs in cis lead to male-specific hypertranscription of lacZ. To test these possibilities roX1 and roX2 cDNAs were inserted into the arm-lacZ reporter. Insertion of either cDNA lead to a significant increase in lacZ expression in males, suggesting that the transcribed regions of the roX genes contain binding site(s) for the MSL complex. Interestingly the level of lacZ hypertranscription in males was significantly higher in homozygous roX1 cDNA lines than homozygous roX1 gene lines. This may indicate that too high a local concentration of roX1 RNA has a dampening effect on the level of hypertranscription meditated by the MSL complex. In a set of experiments designed to identify the MSL binding site(s) in roX1, two regions of the cDNA sequence were amplified and inserted into the arm-lacZ system. One of these fragments, containing a proposed DNAseI hypersensitivity site and possible GAGA binding sites, increased lacZ expression in males, but to levels lower than the entire cDNA. This suggests there may be more than one MSL biding site in roX1. A second method of dosage compensation is thought to occur in Drosophila, independently of the MSL proteins. The arm-lacZ insulated reporter system was used to investigate the hypothesis that some genes may be dosage compensated due to repression by Sex-lethal (Sxl) in females. Several genes have been found to contain three or more Sxl binding sites in their 3' UTRs. with some also carrying Sxl binding sites in the 5' UTR. Fragments from the Sxl, Cut and Small Forked genes, containing numerous Sxl binding sites from the 3' UTR, were inserted into the 3' UTR region of arm-lacZ. Males carrying autosomal insertions of the construct had on average 1.07 - 1.50 times the level of β-galactosidase in females. This suggests that some genes could be partially compensated through Sxl repression in females. In addition to inserting 3' UTR fragments into arm-lacZ, a synthetic oligonucleotide containing a long Sxl binding site was inserted into the 5' region of an arm-lacZ construct already carrying the Runt 3' UTR fragment. Males carrying autosomal insertions of the construct had levels of β-galactosidase activity similar to those lines carrying autosomal insertions of the 3' UTR fragments alone. This suggests that other factors such as RNA binding proteins or RNA secondary structure may be required in order to obtain efficient translation repression by Sxl. Finally three X-linked DNA fragments, from the 1C region, were inserted individually between the SCS' element and the armadillo promoter. If the X-linked fragment contained a DCRE then males carrying autosomal insertions of the construct would produce twice the β-galactosidase activity of females. However, males and females expressed the same levels of lacZ