The Structure and Function of the Biological Membrane and its Associated Macromolecules
The biological membrane and its associated macromolecules are essential to the survival of all living organisms. My main research theme is to elucidate biological membranes' biophysical and biochemical dynamics and their related proteins because of their crucial role in living organisms, using both in vitro and in vivo techniques. The diverse functions of the biological membrane can easily be understood by elucidating its biophysical and biochemical dynamics.
My research interest is in the role of HIV envelope glycosylated proteins and dendritic cell receptors in HIV transmission, membrane transport proteins, signal transduction membrane proteins, synthetic biology, and antimicrobial peptides. My research is grouped into five main broad areas:
1. HIV-1 Envelope Structure-Function and Viral Fitness
The research in this area is divided into two parts;
the first part involves analysis of the differences in glycosylation profile of HIV-1 subtype C transmitter/founder (T/F) and chronic viruses using mass spectroscopy as detailed below:
This study aims to investigate the mechanism whereby HIV crosses the genital epithelium and infects CD4 T lymphocytes. The project involves determining the composition of the carbohydrate structures of gp120, the presence and absence of N-glycan sites during transmission, and specific gp120 glycosylation patterns essential for HIV transmission. An HIV-1 genetic and phenotypic bottleneck occurs at transmission because, in eight out of 10 cases, only a single HIV infectious unit results in productive clinical infection. The presence of sexually transmitted infections (STIs) is associated with an increased risk of HIV infection and the transmission of multiple viruses. This suggests that an intact genital epithelium is highly protective against HIV infection and that variants that can overcome this barrier are selectively transmitted. Transmitted variants are enriched for env sequences with shorter variable loops and fewer N-glycan sites than variants from chronically infected individuals. This suggests that transmission might depend on the presence or absence of specific N-glycan sites, and those lectins (carbohydrate-binding proteins) might be involved in HIV infection. Dendritic cells are located at the rectal and genital mucosa and express a lectin (DC-SIGN) that has been shown to bind to gp120 and mediate HIV trans-infection of CD4+ T cells. Therefore, DC-SIGN might selectively bind to variants depending on the presence or absence of specific N-glycan sites on gp120. This study tests the hypothesis that HIV variants are successfully transmitted because they carry an optimal arrangement of high mannose-type N-glycans that facilitate their uptake by dendritic cells through binding to DC-SIGN (or an as yet unidentified lectin) and enable their infection of CD4+ T cells.
the second part involves biochemical and biophysical characterization, antigenicity assays of native-like gp140 trimers, and pseudovirus neutralization assays, which may pave the way for HIV preclinical and clinical trials.
2. Membrane Proteins Folding and Stability
Protein folding into functional three-dimensional structures is one of the most important events in living organisms. Its aberration has led to many fatal diseases, such as cystic fibrosis, Parkinson’s, Alzheimer’s, and atherosclerosis. It is still a Levinthal paradox to predict conditions that will fold a sequence of amino acids into a functional protein. This project aims to devise new methods to unfold and refold membrane proteins under specific requirements. It will provide insights into membrane protein folding and the role of the biological membrane-associated macromolecules in the folding process. The functions of membrane lipids in bacterial strains or mammalian cell lines that have their lipid composition properly regulated or determined will be elucidated through the protein's different conformations and functional states. Similarly, the mutants will provide the structural and functional details of specific regions of the proteins. Furthermore, obtaining a properly folded and stable functional protein is a significant prerequisite in determining its three-dimensional molecular structure using structural biology techniques.
3. Membrane Transport Proteins
Membrane Transport proteins are very important in transporting metabolites and toxins across the cell membrane, and they have been implicated in several antibiotics and cancer resistance to medications. Although the mechanisms of some transport proteins have been elucidated in great detail, a large number of them still need to be. This is mainly due to requiring more protein qualities for biophysical and biochemical studies. This project will provide thermodynamics and kinetics of transport membrane proteins, which in turn provide insights into structural and functional changes induced when certain amino acids are removed or substituted. This information is critical in rational drug design and understanding pathogenesis. Membrane transport proteins that have shown some stability and are produced in large quantities will be set up for 2-D Electron crystallization and 3-D X-ray crystallization trials to obtain crystals for atomic resolution structure determination.
4. Synthetic Biology (Antimicrobial properties of natural peptides and engineered
genetically augmented polymers)
“Synthetic Biology can revolutionize major industries in bio-energy and biotechnology in the UK. If we develop this exciting area to its full potential, there are fantastic opportunities in sectors such as biofuel and medical care that are largely untapped. This roadmap positions the UK as a leader in global synthetic biology, which presents significant growth and employment opportunities.” Minister for Universities and Science, David Willetts. Please download more details about the roadmap below.
This multidisciplinary research involves gene design and construction and applied protein design that will help to control and create protein interaction networks. I am mainly interested in genetically augmented sequence-controlled polymers to develop and program intracellular interaction networks. Sequence-controlled macromolecules have significant applications in nanomedicine and can provide vital information on the relationship among primary, secondary, and tertiary structures of proteins or polymers. The research project involves introducing coding information into polymers and incorporating unnatural amino acid sequences into proteins.
5. Molecular Mechanisms of Antimicrobial Peptides
Antimicrobial peptides are part of innate immunity and are found in all living organisms. They target bacteria, viruses, fungi, and cancer cells. The molecular mechanisms of most of these peptides need to be better understood. Antimicrobial peptides contact the target organisms through the biological membrane. With an increase in antibiotic resistance of major pathogens since the 1980s, antimicrobial peptides are in the pipeline to become the next-generation biopharmaceuticals. Indeed some people have resorted to using viruses to fight bacteria infections since the resistance worsens worldwide. Some antimicrobial peptides disrupt the membrane, while others have intracellular targets. Those with intracellular targets must be transported across the membrane by ATP-binding cassette transporters. This project will try to elucidate the molecular mechanisms of antimicrobial peptides and their transporters. It will also provide details of interactions between the antimicrobial peptides and the lipid bilayer. Some antimicrobial peptides have already reached the market, and many more are in the pipeline in major pharmaceutical companies. Enfuvirtide, currently marketed as Fuzeon (Roche), is a biomimetic peptide that inhibits HIV.
The above studies will be approached using molecular biology, biochemical and biophysical methods in collaboration with other research groups. Some of these methods are recombinant DNA technology, gel electrophoresis, SDS-PAGE, gene design and construction, western blotting, DNA sequencing, affinity, ion exchange and size exclusion chromatography, circular dichroism, radiolabeling, site-direct mutagenesis, absorption spectroscopy, mass spectroscopy, ITC, transmission electron microscopy, peptide mass fingerprinting, X-ray crystallography, fluorescence spectroscopy, solid-state NMR, bioinformatics, cDNA synthesis, transport assays, membrane protein reconstitution in liposomes, single particle analysis, etc.