Our research lies at the intersection of surface and colloid science, polymer materials engineering, and biointerface science. These broad inquiries deal with fundamental behaviors of soft-wet materials on surfaces and interfaces.
Current projects include:
(1) fabrication, manipulation, and characterization of stimulus-responsive biomolecular and bio-inspired polymeric nanostructures on surfaces;
(2) nanomechanics of soft-wet materials and hybrid biological/non-biological microdevices;
(3) interaction of proteins with lipid membranes in the context of HIV-1;
(4) the role of glycoproteins in boundary lubrication of cartilage.
This research is motivated by the observation that rare, broadly neutralizing antibodies (NAbs), 4E10 and 2F5, associate with HIV-1 lipids as part of a required first step in neutralization before binding to membrane-proximal antigens. Subsequently, induction of these types of NAbs may be limited by immunologic tolerance due to autoreactivity with host cell membranes. Despite the significance of this lipid reactivity there is little experimental evidence detailing NAb-membrane interactions.
Simple and efficient screening assays are needed to further dene and understand NAb neutralization and autoreactivity, specically in the context of how exposed chemical groups from lipid membranes help drive antibody interactions. We recently have developed a surface plasmon resonance (SPR) spectroscopy based assay that monitors antibody binding to thiol monolayers, which mimic salient surface chemical properties of lipid membranes. Specically, we probed the relative importance of charge and hydrophobicity on antibody-surface interactions. We found that NAb binding to hydrophobic thiol surfaces was signicantly greater than that of control mAbs. Furthermore, we conrmed the importance of charge mediated antibody surface interactions, originally suggested by results from mAb interactions with conventional lipid vesicle/bilayer surfaces. Our approach thus provides an efficient and useful tool to screen interactions of mAbs and lipid-reactive NAbs with a broad range of biologically relevant surface chemistries.
We now have begun to extend this research and use supported lipid bilayers that mimic the viral membrane. In-vitro studies by my collaborator Dr. Munir Alam (Duke Human Vaccine Institute) have shown that NAbs 2F5 and 4E10 successfully inhibit the fusion process of HIV-1 by binding the membrane-proximal external region (MPER) of gp41 during a two-stage mechanism: the NAbs first interact nonspecically with the viral lipid membrane and then with the target MPER antigen. One explanation for this two-stage interaction is that NAb-membrane interactions likely direct high NAb concentrations toward the viral surface, where the NAb’s ability to diffuse within the viral membrane could better position it to encounter its sparse MPER antigen. Besides binding kinetics, there is very little, if any, experimental evidence detailing this lipid reactivity in HIV-1 neutralization. This unexpected interaction between lipids and 2F5/4E10 is potentially mediated by their long complementary determining region (CDR) H3 loop. CDR H3 contains unusually large numbers of hydrophobic and membrane-reactive residues that can embed in the viral membrane. CDR H3 regions mediate a reversible attachment to the viral membrane; which is a required first step for neutralization. This phenomenon may explain why simple peptide immunogens that mimic neutralizing epitopes on gp41 do not elicit NAbs in vivo, and it is clear that peptide sequence is not the sole determinant of neutralizing ability. The native viral Env is heterogeneous, representing a mosaic of lipid rafts, protein and antigen clustering, and possibly various gradients of diffusivity. In addition, the HIV-1 Env contains a lipid composition that differs from that of host cell membranes. Major differences include elevated levels of cholesterol, sphingomyelin, and anionic lipids, all of which have been shown to contribute to heterogeneous lipid domain formation. Lipid domains that have high lipid diffusivity or an increased presence of anionic lipids may drive NAb-membrane interactions and control conformations of membrane embedded antigens. Yet, the size, physical properties, and dynamics of such lipid domains are poorly characterized for the HIV-1 lipid Env, and it is unknown how lipid domains contribute to NAb-membrane interactions and antigen presentation.
Our research, in collaboration with Dr. Munir Alam (Duke Human Vaccine Institute), Dr. Frank Heinrich (NIST’s Center for Neutron Research), and Prof. Joseph Shapter (Flinders University, Adelaide, Australia), thus focuses on understanding how membrane properties, such as composition, lipid domain organization, and lipid diffusivity contribute to 2F5/4E10-membrane interactions and antigen localization at the membrane interface. To this end, my lab has developed supported lipid bilayers (SLBs) whose compositions model the HIV-1 lipid Env. These SLBs have planar surfaces that facilitate the use of quantitative surface-characterization techniques such as high resolution scanning-probe imaging, detection of fluorescence recovery after photobleaching, and neutron reflection measurements. We have begun to use these techniques to i) visualize domains of lateral membrane organization; ii) determine lipid diusivity within domains; iii) determine dierences in adhesion force (surface energy) of domains; and iv) correlate these differences with details of NAb-membrane binding, NAb/antigen localization, and, conformational details of NAbantigen interactions at the membrane interface. Because current 2F5/4E10 immunogens have not yet elicited antibodies with this required membrane reactivity, our research is important in that it will reveal molecular details of the role of lipids underlying 2F5/4E10 antigen binding. This information will elucidate how membrane properties could enhance antigen recognition and thus enable the design of next generation HIV-1 immunogens.
A central theme of our work in fabrication of surface-confined biomolecular and polymeric micro- and nanostructures has been the development of a comprehensive “nano-toolbox” that consists of stimulus-responsive polymers and biomolecules, and the necessary methodologies to manipulate these molecules at the nanoscale by processes that are compatible with an aqueous environment. Our research has spearheaded the area of polymer brush nanopatterning by combining scanning probe lithography and maskless electron-beam lithography with surface initiated polymerizations. We have also developed novel methods and tools that lead to the functional, oriented immobilization of proteins and biomolecules on surfaces. This research is motivated by the possibility of fabricating protein nanoarrays with well-defined feature size, shape, and spacing. Such structures are important for the fundamental study of the interactions between cells and surfaces: they can function as nanoscale protein purification systems—thus opening the door to single cell lysate screening—and they provide high-throughput, massively-parallel experimentation platforms that enable the interrogation of complex biology at genomic and proteomic levels.
A major new thrust in our research focuses on biophysical approaches to understanding the binding behavior of neutralizing antibodies (NAb) in the context of HIV-1. Vaccine development is a crucial weapon in combating the spread of viral infections such as HIV-1, SARS, and Ebola. Such viral agents infect the body through various means, but share several common characteristics, including an outer membrane decorated with envelope proteins (Env). Here our research is motivated by the observation that rare, broadly neutralizing antibodies, 4E10 and 2F5, associate with HIV-1 lipids as part of an essential first step in neutralization before binding to membrane-proximal antigens. This unique lipid reactivity may explain the rarity of 4E10 and 2F5, because induction of these types of NAbs may be limited by immunologic tolerance due to reactivity with host cell membranes. Overcoming immune tolerance issues is thus one of the major challenges to induce these types of membrane-proximal NAbs and surprisingly, little is known about the NAb-membrane binding mechanism underlying autoreactivity and neutralization. Specifically, we seek to determine whether there are distinct properties, such as lipid heterogeneity, composition, and diffusivity in HIV-1 and host cell membranes that help drive interactions with 2F5 and 4E10. To this end we collaborate with Dr. Munir Alam (Duke University, CHAVI) and Dr. Frank Heinrich (NIST, Center for Neutron Research) to determine interactions of 2F5 and 4E10 on the host cell membrane and the unique lipid environment of the HIV-1 envelope. Neutron reflection measurements of NAb binding to host and viral lipid membranes are currently underway at NIST, and a grant proposal to NIH (R21) is in preparation.
In the subfield of cartilage tribology, we have recently embarked on a new, major research direction. In this research, we use nanotribomechanical measurements on model surfaces and cartilage from genetically engineered mice, combined with several surface-specific physicochemical measurements, to determine the mechanisms by which glycoproteins (such as PRG4 and its product, lubricin) provide lubrication and wear-protection in diarthrodial joints. Our research contributes significantly to the understanding of the mechanisms by which PRG4 mediates normal and friction forces in articular cartilage, in absence of fluid pressurization. Identification of these mechanisms is essential for the development of new treatments, such as tribosupplementation, that exploit mechanical and biochemical modalities for the prevention of disease. In a broader perspective, this research is expected to add significantly to the growing knowledge of biotribology, and the interaction of biomacromolecular polyelectrolytes with natural and man-made surfaces.
SA1: Understand the effect of lubricin on steric, friction and adhesion forces on: (i) the molecular level, using model surfaces that mimic the cartilage surface chemically, and (ii) the tissue level, using a PRG4(-/-) mouse model.
SA2: Assess the implications of lubricin deficiency on the local biomechanical properties of cartilage.
Effect of Lubricin Absence on Cartilage of 2 Week Old Mice
Our research focuses on the use of ferroelectric thin films (FETFs) for the manipulation of matter within aqueous environments. For the past few decades, FETFs have been developed for next generation semiconductor memory devices. FETFs are attractive for memory applications because their polarization states are highly-localized, stable, and switchable. These unique properties, however, are also attractive for applications in micro and nano scale material separation and sensing. FETF integration with lab-on-a-chip (LOC) technology could revolutionize the field of diagnostics. Currently, LOC development has been stifled by complex electrode design, slow response times, and potential for electrochemical degradation. FETFs’ expression of non-Faradaic surface charges may be able to induce long-range, switchable, electrostatic forces in solution sans the risk of electrochemical reaction. With this research we hope to shift the paradigm of FETFs towards applications in biological interfaces and sensing.
Key questions will be addressed, including; Is stability a function of FETF material or composition? Do FETF interfacial properties changes with water exposure? Does FETF atomic organization change as a function of water exposure duration?