DNA hybridization assays are fundamental to many biological measurements and particularly in applications of microarray technology. A major objective of these measurements is to identify single nucleotide polymorphisms (SNPs) – mutations of a single nucleic acid base which may be used to map genes involved in disease, to predict drug metabolism and to develop models for biochemical processes. Current recognition mechanisms are limited by energy differences in Watson-Crick base pairing (hydrogen bonding) which makes SNPs difficult to detect. Many recognition modes also require target DNA strands to be fluorescently labelled. This project is aimed at developing label-free SNP detection using a molecular bioswitch based on a variant of the DNA Holliday Junction – a branched recombination intermediate. Together, the probe and target form an ion-controlled switch where the states are the open and folded conformations of the junction. The switch characteristics and folded structure are found to be highly sensitive to single base changes located at the branch point of the junction. Sensitivity to mismatches is found to be higher than the limit imposed by H-bond enthalpies. Only the probe (not the target) strand is fluorescently labeled and structural characterization is made using steady-state and time-resolved fluorescence resonance energy transfer. The project aims to use advanced ultrafast laser spectroscopies and fluorescence resonance energy transfer to determine structure and to reveal the nature of the energy landscape of these very unusual molecules. We are also interested in driving down the detection sensitivity to its ultimate limit- single molecule resolution. The project is highly cross-disciplinary with collaboration between physics, chemistry, engineering and medicine.
Amino acids are the building blocks of all protein structures. There are 20 commonly occurring amino acids in nature, characterized by the chemistry of their side chains. In a protein they are joined together by carbon-nitrogen peptide bonds to form a polymer of possibly many hundreds of amino acids. The shape and function of the protein in its native state is determined ultimately by the amino acid sequence: for example some proteins undertake complex chemical reactions within a cell, others provide recognition, while others have a structural role. However the process by which a protein folds to its native state is strongly coupled to the environment in which the protein exists. Protein structure formation takes place in a complex aqueous solution, the precise conditions of which are closely controlled by the surrounding cell medium. Alter that environment and the protein may become “denatured” or fold incorrectly. Mechanisms often exist to correct for natural errors in folding, but such misfoldings can also result in diseases, for example those related to amyloid forms, and loss of cell function. The proposed research will investigate at the atomic level the way these amino acids, and also the macromolecules to which they belong, respond to different aqueous environments. Our strategy is to combine state-of-the-art neutron diffraction and optical spectroscopy. Neutron diffraction is the premier technique by which the structure of hydrogen bonded liquids such as water, alcohols and simple acids have been determined. We will be using a unique instrument on ISIS target Station II which relies on the longer wavelengths to increase the upper limit of the accessible correlation lengths, while also extracting atomic resolution from the shorter wavelengths. Building upon the detector technology of its forerunner instrument, SANDALS, the instrument bridges the traditional gap between SANS and wide-angle neutron scattering, by using a common calibration procedure for all Q-scales. The data obtained from will therefore enable the development of detailed and well-constrained models of complex scattering systems.
The most urgent need in HIV therapy is for new treatments that are effective against strains of the virus that are resistant to currently approved drugs including reverse transcriptase and protease inhibitors. From a therapeutic perspective inhibition of viral entry/fusion is one of the most attractive intervention points. The search for effective fusion inhibitors of HIV remains a major goal of AIDS research and understanding the structure of potentially theraputic peptides is an important element of this activity. In collaboration with the National Physical Laboratory, this project is aimed at using sophisticated optical spectroscopic tools to determine the structure of peptides relevant to HIV fusion inhibition. Specific techniques include Raman spectroscopy, Fourier Transform Infrared spectroscopy, and circular dichroism, The results are compared to those obtained from molecular dynamics simulations to infer structure property relationships.
Blue Gene grew out of an IBM Research project dedicated to exploring the frontiers in supercomputing, computer architecture, software required to program and control massively parallel systems, and in the use of computation to advance understanding of important biological processes such as protein folding. Blue Gene is now the world’s most powerful supercomputer. As part of a major collaboration with IBM’s TJ Watson research Center in New York, we have developed fine-grained parallel implementations of classical and ab-initio molecular dynamics simulation for complex systems with a particular emphasis on liquid state biophysics simulations. In particular, a novel scalable parallelization strategy for Car-Parrinello molecular dynamics has been developed using the concept of processor virtualization. This, coupled with low latency has given unprecedented scaling on thousands of processors – performance that is impossible to achieve using standard approaches. We are seeking postgraduate students to support the work in two areas of the collaboration:
Polarisable Model Development: In the effort to develop atomistic models capable of accurately describing nanoscale systems with complex interfaces, it has become clear that simple treatments with rigid charge distributions and dispersion coefficients selected to generate bulk properties are insufficient to predict important physical properties. The quantum Drude oscillator model, a system of one-electron pseudoatoms whose "pseudoelectrons" are harmonically bound to their respective "pseudonuclei," is capable of treating many-body polarization and dispersion interactions in molecular systems on an equal footing due to the ability of the pseudoatoms to mimic the long-range interactions that characterize real materials. We are actively developing this methodology using quantum Monte Carlo and path integral techniques.
Parallel Tempering Molecular Dynamics: At room temperature, complex molecules such as polypeptides can become trapped in local energy minima. To fold into their native structure, they have to transverse rugged energy landscapes and escape the energy traps. These barrier crossing processes are believed to be the most time consuming steps in protein folding. They are also prohibitively costly for conventional molecular dynamics techniques in which configurations become trapped for times that are long compared to the simulation time. Parallel tempering MD (PTMD) is a powerful approach that can sample the conformational space more effectively than constant temperature MD. In this technique, simulations at a series of temperatures are made. Swaps of configurations between these parallel simulations allow the room temperature system of interest to escape from local free energy minima where it might otherwise have been trapped. We have now implemented parallel tempering molecular dynamics efficiently on Blue Gene and are completing tests on saline solutions and solutions of amino acids. Graduate students are sought to refine the implementation and apply it to biophysical problems in peptide folding and molecular structure determination.
Despite the chemical specificity, high resolution and optical sectioning capability of fluorescence-based imaging techniques enabled by laser-scanning confocal detection, multi-photon excitation, resonance energy transfer and time-resolved lifetime measurements, all share one fundamental limitation. Namely, that the molecular constituents of interest must be stained by incorporating "foreign" labels to create image contrast. In the most benign cases, these labels act as robust but passive dyes which have no effect other than to render visible the molecules to which they are attached. In some circumstances, however, the dyes are neither robust nor passive. Specifically, prolonged exposure to the excitation beam often leads to permanent damage to the dye molecules. This photo-bleaching degrades image contrast and forces compromises between between illumination intensity, image quality and acquisition speed. Image quality may also suffer from fluorescence quenching whereby non-radiative electronic relaxation processes become significant and suppress fluorescence yield. Beyond these practical issues connected to obvious loss of signal, other problems are more subtle. Namely, free radical reaction products generated during photo-bleaching may be toxic for biological specimens. Moreover, fluorescent labeling itself can cause non-negligible perturbations to chemical or biophysical pathways and to cellular functions the severity of which cannot be assessed a priori. CARS is a nonlinear optical process in which molecular vibrations are driven by optical fields and used to create highly selective molecular maps of complex objects such as living cells and artificially structured materials. CARS microscopy thus has the potential of becoming a major intrinsically selective optical imaging technology with the following advantages:
The project involves optical instrument development and refinement as well as applications to cellular structure and signalling.
