Introduction to our lab

We are interested in the use and development of novel physical techniques to study biomolecular interactions including proteins, DNA and RNA at the level of individual molecules. The advantages of single-molecule detection are many, apart from the fascination of looking at individual biomolecules at work, single-molecule techniques can measure intermediates and follow time-dependent pathways of chemical reactions and folding mechanisms that are difficult or impossible to synchronize at the ensemble level. Thus, using single-molecule fluorescence in combination with advanced microscopes and manipulation techniques, we can make "molecular movies" of biological processes that help to propose and understand the underlaying molecular mechanism.

Molecular basis of RNA-mediated gene regulation-riboswitches

Since the discovery that RNA can catalyze biochemical reactions and more recently that RNA elements, so-called riboswitches, built into mRNA can sense the concentration of a particular metabolite and turn gene expression on or off in response, RNA does not any longer play a passive role in cellular processes and RNA research has entered a phase in which its central importance as a bridge between genomics and proteomics has been emphasised. The main goal of this project is to dissect the molecular mechanisms involved in ligand- and ion-assisted folding of these RNA switches and understand how their highly dynamic nature is used to trigger gene expression modulation. We will focus on the glycine riboswitch, the only known natural metabolite-binding riboswitch that makes use of a tandem aptamer configuration to achieve activation and repression of genes encoding the glycine cleavage system. We will use a powerful combination of ensemble-FRET, single-molecule fluorescence spectroscopy, 2-aminopurine fluorescence and standard biochemical techniques (gel electrophoresis mobility assays, in-line probing) to dissect the mechanism of glycine-induced folding and characterize the structural key features linked to the recognition and cooperative binding of glycine to both aptamers.
Nowadays, that it is clear that bacteria and fungi are becoming more resistant to existing antibiotics, the emerging field of riboswitches is becoming a very promising ground to develop anti-bacterial drugs that target essential metabolic pathways in these organisms. Therefore, the results obtained from these studies not only will greatly expand the existing knowledge on RNA folding and riboswitch gene regulation mechanisms but also could be very valuable in the development of a new generation of novel antimicrobial drugs targeting riboswitches for use as antibiotics.

Single-molecule studies on protein-nucleic interactions involved in DNA processing pathways:

From the very beginning of life, there has been a challenge to maintain and replicate the genetic material DNA. The integrity of DNA is constantly challenged by the damaging effects of numerous chemical and physical agents, compromising the informational content. Every living organism devotes considerable resources to these vital tasks. Often this involves enzymes that detect aberrant or intermediate DNA structures, manipulate them, and process them, before passing the products on to other enzymes or proteins in a pathway. We are interested in the study of DNA processing pathways at single-molecule level. In particular, biochemical analysis has produced a common general outline of the nucleotide repair mechanism, which includes recognition of the DNA lesion, unwinding of the DNA double helix around the damage and, finally, excision of an oligonucleotide containing the damage bases. This process involves the ordered assembly of several proteins and its interaction with the double helix, and undoubtedly requires complex intermediate structures. The exact composition and lifetime of the different intermediates, and thus, the precise reaction mechanism, is not known. Single-molecule fluorescence methods provide a way to target and identify distinct intermediate species dynamically exchanging (protein-protein and protein-DNA complexes), quantify their prevalence and determine their lifetime. Our aim is to answer fundamental questions such as: a) how do structure-specific DNA-binding proteins find their targets? b) Define the conformational changes that occur in both proteins and DNA during binding, and c) Determine the sequence of events involved in the formation of active complex and d) by making use of single-molecule enzymatic techniques determine the degree of static and dynamic disorder in the cleavage rate constants and its origin. This project is a collaboration between the School of Physics and Astronomy and the Biomolecular Sciences Centre at St Andrews.
 
Protein misfolding and aggregation

During their life cycle proteins undergo a sequence of conformational changes in order to achieve their functional three dimensional structures in a process known as protein folding. This process is so important that organism have developed systems such as Chaperonins that consume energy to assist in the folding process. However, sometimes the folding process follows an anomalous route where the proteins no longer adopt their natural active structure but aggregate into insoluble fibrils or plaques. These structures, so-called amyloids have been linked to over 20 diseases including Alzheimer, Parkinson, type II diabetes and spongiform encephalopathies. Recent evidence has shifted some of the focus from amyloid fibrils to prefibrillar aggregates as the main cause of Alzheimer´s disease symptoms and there is growing evidence that amyloid can bind mitochondrial proteins such as ABAD (Amyloid Binding Alcohol Dehydrogenase) opening and additional toxic channel inside the cell. In this project our aim is to provide a mechanistic understanding of the amyloid-assembly process, the physical interactions and structural changes involved and the factors that influence the aggregation/disaggregation rates at all stages of the process.  The advantage of a single-molecule approach lies on its ability to differentiate and classify the different species and intermediates involved in amyloid formation by shape, size, kinetic behaviour and stability not easy to obtain by any other technique. Of particular interest will be to asses the participation of difficult-to-detect-and-quantify small aggregates responsible for the early stages of amyloid formation. We will study the effect of environmental variables such as temperature, pH, helix promoting solvents and denaturant and reducing agents on the amyloid assembly mechanism. A mechanistic understanding at the level of single-molecules will certainly provide new handles and probes for the physiological interactions that cause amyloidosis and will allow better approaches to the prevention at early stages of amyloid-related diseases. This project is collaboration between Dr. Frank Gunn-Moore at the Bute Medical School and the School of Physics and Astronomy at St Andrews.

Force-induced conformational dynamics in biomolecules reported by single-molecule FRET: a combined single-molecule fluorescence and force manipulation microscope.

Traditionally, the single-molecule field has been classified in those techniques based on fluorescence detection and those methods that analyse mechanical effects on single-molecules as a response to an applied force. It is clear that the combination of single-molecule force and fluorescence in a single experiment and in a single-molecule will offer a much powerful and versatile approach to monitoring spatial or conformational changes in a temporal manner. Thus, single-molecule FRET could be used to report in which region of a particular biomolecule the force applied is having the biggest impact and thus, allowing the direct correlation between mechanical changes and specific transitions. However, because of the intrinsic characteristics of both techniques, the marriage between both methods was not successfully achieved until that Block et al reported in 2003 an instrument setup capable of performing simultaneous and spatially coincident optical trapping and single-molecule fluorescence. Although, this achievement is a breakthrough in the field of single-molecule detection, its wide application has been very rare, mainly because of the strong expertise and expensive technical requirements that limit its use to very few labs around the world. In this project we are aim to follow an alternative path to develop a combined single-molecule force and fluorescence microscope based on the replacement of the optical trapping beam by a magnetic trap. This will avoid a number of problems arising from the high photon flux of the optical trap and therefore its ability to quench the fluorescence dyes, reduces the number of expensive optical components such as knotch filters to remove unwanted trapping light in the vicinity of the fluorophores and allows the use of much shorter molecular tethers between the magnetic bead and the biomolecule under study, with the subsequent increase in the possible biomolecular geometries that can be targeted. We expect this technique to become a standard gold tool widely used in the biophysics community.

This multidisciplinary program lies in the interface between physics, chemistry and biology providing not only new insights into very important biological and health problems and bringing innovations to biotechnology, but also creates an ideal multidisciplinary platform in the physics-life science interface for preparing young scientists. In our lab, students with physical science background have opportunities to apply their expertise in instrumentation and quantitative analysis to interesting biological problems; and students with biological background are exposed to state-of-the-art or emerging physical technologies that can substantially expand their biological research capabilities.