Associate Professor Shohei Koide, Biochemistry and Molecular Biology, The University of Chicago

Engineering protein interaction interfaces with high affinity and specificity has been a major goal in protein engineering and protein-based drug development. Traditionally, protein engineers have aimed to reproduce large and chemically complex interfaces so as to mimic those found in natural proteins. I will present recent studies demonstrating that high-performance interaction interfaces can be produced using synthetic "minimalist interfaces" that are small and chemically and architecturally simple, which have important implications in protein engineering and biology.

October 3, 2007, Distinguished Speakers Series
Engineering Building II, Room 205/206

Distinguished Professor Shu Chien, Bioengineering and Medicine and Whitaker Institute of Biomedical Engineering, University of California, San Diego

Shear stress resulting from blood flow and mechanical stress due to transmural pressure can modulate cellular functions by activating sequentially the mechano-sensors, signaling pathways, and gene and protein expressions. This presentation will summarize work done in our laboratory on mechanotransduction in vascular endothelial cells (ECs).

Laminar or pulsatile shear stress with a significant forward direction causes a transient activation of expression of pro-atherosclerotic genes, but the long-term effects are the down-regulation of these genes and also the activation of growth-arrest genes. The disturbed flow seen at curved regions and branch points of the arterial tree in vivo or oscillatory perfusion flow in vitro, which do not have a significant net forward direction, exert the opposite effects. Thus, flow patterns without a definite direction are pro-atherogenic; in contrast, flows with a net forward direction, which are seen in the straight part of the arterial tree, are protective against atherogensis.

ECs respond differentially to uniaxial and biaxial stretches. Uniaxial stretch causes a Rho-dependent orientation of actin stress fibers perpendicular to the direction of stretch, but this is not seen following biaxial stretch. Uniaxial stretch causes the stretch-induced JNK activation to be transient, as the stress fibers undergo reorganization, and this is followed by the down-regulation of JNK and protection of ECs against apoptosis. In contrast, biaxial stretch, which does not induce stress fiber reorganization, causes a sustained JNK activation that leads to EC apoptosis.

In summary, temporal and spatial variations in shear stress and stretch, especially the presence vs. absence of directionality, result in differential modulations of signal transduction, gene expression, and protein expressions, as well as EC functions. Interdisciplinary studies at the interface of biology, medicine and engineering are needed to elucidate the mechanisms of mechanotransduction in various physiological and pathophysiological conditions in health and disease.

Dr. John Frangos, Founder, Principal Scientist, President & CEO, La Jolla Bioengineering Institute

The ability of cells to respond to hydrodynamic stimuli is ubiquitous amongst all cells and organisms, and has been implicated in a number of physiological and pathological processes. While many of the biochemical transduction pathways have been characterized, the primary mechanoreceptor(s) remain(s) unknown. It is our hypothesis that hydrodynamic shear destabilizes the plasma membrane, leading to a decrease in membrane microviscosity, or more precisely, an increase in membrane free volume. Mechanochemical transduction is proposed to occur when membrane-associated signaling proteins are activated by the increase intramolecular mobility.

A number of studies have implicated a role of heterotrimeric G proteins in the mediation of cellular responses to fluid shear stress and stretch. Studies from our lab demonstrate that heterotrimeric G proteins are rapidly activated by hydrodynamic shear, representing the earliest known biochemical response to mechanical stimulation presented. Furthermore, both fluid shear stress and membrane fluidizing agents activate these G proteins in the absence of classical G protein coupled receptors. Using fluorescent molecular rotors it was recently shown that hydrodynamic shear increases membrane free volume. Taken together, these results demonstrate that hydrodynamic shear stress stimulates cellular responses by increasing membrane fluidity and activating heterotrimeric G proteins.

Assistant Professor Volkmar Heinrich, Biomedical Engineering, University of California, Davis

The development of ultrasensitive force probes (AFM, optical tweezers, biomembrane force probe etc.) continues to fuel discovery in nano-to- microscale bioengineering. Examples like our recent work on the mechanoregulation of leukocyte adhesion demonstrate how it often takes a multiscale, molecular-to-cellular approach to validate the biological relevance of insight gained from single-molecule techniques. This is partly due to the 'weakness' (or susceptibility to thermal activation) of biomolecular interactions. Consequently, their interaction strength is a dynamic quantity that depends on the stress history experienced by the participating molecules. This history, in turn, is affected by the mechanical properties of the supporting subcellular structures like membranes or the cytoskeleton.

Two examples of biomolecular interactions will be discussed in this context: one intermolecular, the other intramolecular. First, the P-selectin:PSGL-1 catch bond is at the molecular root of an intricate biosystem of switches, fuses, and shock absorbers that mechano-regulate the leukocyte response to inflammation. Second, direct observation of the folding of individual spectrin repeats under force is changing our view of the molecular basis of the red-blood-cell membrane's hyperelasticity, enabling red cells to their amazing feat of 3-4 months circulatory survival.

Dr. Abbas Yaseen, Bioengineering, Rice University

Investigators continue to develop new applications using deep penetrating near infrared (NIR) light for the noninvasive treatment and diagnosis of disease. More recent techniques involve the use of reagents sensitive to NIR light, such as Indocyanine Green (ICG) dye, as a means to improve diagnostic efficacy and treatment selectivity. Although ICG is FDA approved and absorbs and fluoresces in the NIR region, it suffers from several drawbacks, including optical instability and rapid removal from the bloodstream. We have developed a carrier system to encapsulate ICG to overcome its limitations for NIR light-based medical applications. Charge-assembled capsules with controllable size and adjustable coatings efficiently encapsulate ICG and effectively stabilize its optical properties. When encapsulated, ICG retains its pronounced light absorption and fluorescence properties in the near infrared spectral range. I will discuss the optical characterization and in vivo applications of these capsules for laser-mediated diagnosis and therapy. The capsules show promise for a variety of medical applications, including phothothermal treatment of tumors and vascular lesions as well as fluorescence-based pulmonary imaging.

Distinguished Professor Andy McCammon, Chemistry and Biochemistry, University of California, San Diego

J. Andrew McCammon is the Joseph E. Mayer Chair Professor of Theoretical Chemistry and Distinguished Professor of Pharmacology at UCSD, and is an Investigator of the Howard Hughes Medical Institute. He received his B.A. from Pomona College, and his Ph.D. in chemical physics from Harvard University, where he worked with John Deutch. In 1976-78, he developed the computer simulation approach to protein dynamics in Martin Karplus’s lab at Harvard. He joined the University of Houston as Assistant Professor of Chemistry in 1978, and became the M.D. Anderson Chair Professor of Chemistry in 1981. He moved to UCSD in 1995. Professor McCammon has invented theoretical methods for accurately predicting and interpreting molecular recognition, the rates of reactions, and other properties of chemical systems. In addition to their fundamental interest, these methods play a growing role in the design of new drugs and other materials. Professor McCammon is the author with Stephen Harvey of “Dynamics of Proteins and Nucleic Acids” (Cambridge University Press), and is the author or co-author of more than 500 publications on a variety of subjects in theoretical chemistry and biochemistry. About 50 of his former students have tenured or tenure-track positions at leading universities or research institutes. In the 1980’s, Professor McCammon guided the establishment of the computer-aided drug discovery program of Agouron Pharmaceuticals (now Pfizer Global Research and Development, La Jolla Laboratories), and contributed to the development of the widely prescribed HIV-1 protease inhibitor, Viracept. The McCammon group’s studies of HIV-1 integrase flexibility contributed to the discovery of the first in a new class of antiviral drugs by Merck & Co., named Isentress (raltegravir) in 2007. Professor McCammon received the first George Herbert Hitchings Award for Innovative Methods in Drug Design from the Burroughs Wellcome Fund in 1987. In 1995, he received the Smithsonian Institution’s Information Technology Leadership Award for Breakthrough Computational Science, sponsored by Cray Research. He is the recipient of the American Chemical Society’s 2008 national award for computational chemistry. He is a Fellow of the American Academy of Arts and Sciences, the American Association for the Advancement of Science, the American Physical Society, and the Biophysical Society.

Assistant Professor Jacob J. Schmidt, Bioengineering, University of California, Los Angeles

Membrane channel proteins are major targets of drug discovery and screening and recent work has also shown their potential as single molecule sensors. Conventional freestanding membranes housing these proteins can be problematic to form in a device and are extremely fragile, limiting channel protein-based sensing technology. Our goal is to develop membrane platforms that require little to no expertise to operate, result in robust long-lived membranes, and can be created in an automated high throughput manner. Toward this goal, we developed two new membrane technologies: in situ hydrogel encapsulation of lipid membranes and automated microfluidic membrane formation. The hydrogel encapsulated membranes are created by forming a conventional freestanding lipid bilayer membrane in the presence of a hydrogel precursor solution. A hydrogel is then formed by polymerizing the precursor solution using chemical or photo-initiation. The resulting polymer network molds to the self-assembled membrane, stabilizing its structure.

These encapsulated membranes can withstand severe mechanical perturbation and are significantly longer-lived than their unencapsulated counterparts as a result of the intimate hydrogel/membrane contact. We have also synthesized a cross-linkable lipid which can participate in the polymerization reaction with the hydrogel. Membranes created from cross-linkable lipids bond directly to the hydrogel matrix, further strengthening the membrane and resulting in lifetimes over ten days. These membranes retain the ability to support the incorporation and measurement of single channels. The automated microfluidic formation device enables the creation and manipulation of lipid membranes through a novel membrane formation process in a microfluidic channel. The microfluidic channels are molded in PDMS, and the solvent absorptive properties of the PDMS are used to mediate solvent extraction from a droplet of lipid-containing organic solvent. The lipid is left behind, eventually forming a lipid bilayer membrane, into which single channel proteins can be incorporated and measured. This new method of membrane formation lends itself very readily to further miniaturization and in an array format. We also have preliminary results with two new methods:

• one in which freezing membrane precursors which may allow for an indefinite lifetime and permits shipping,
• and another which uses lipid monolayers resulting from inverted phases to form lipid bilayers able to be created in an automated high throughput process with robotics.

These technologies have potential applications for drug discovery and screening as well as small molecule sensing.

Professor Enrico Gratton, Bioengineering and Physics, University of California, Irvine

The 3D spatial position of a particle can be determined by scanning the excitation volume of a 2-photon microscope in a three-dimensional orbit around the particle and by subsequently analyzing the fluorescence intensity profile along the orbit. If the analysis is computed very rapidly in comparison to the orbit time, it is possible to use it in a feedback loop to constantly monitor the particle's position and re-center the orbit at the particle, in the case the particle moves. The individual feedback steps reconstitute the particle's 3D spatial trajectory. Trajectories of organelles in vivo, for example, have been obtained in this way with a spatial and temporal resolution in the order of 10 nm and 32 ms respectively.

Enrico Gratton was born in Italy and received his doctorate in physics from the University of Rome. He came to the University of Illinois at Urbana-Champaign (UIUC) in 1976 and began as a research associate in the Department of Biochemistry under the direction of Professor Gregorio Weber. At Illinois Professor Gratton holds a joint appointment in the Departments of Physics and Biophysics. In 1986, Dr. Gratton was awarded a grant from the National Institutes of Health to establish the first national facility dedicated to fluorescence spectroscopy: the Laboratory for Fluorescence Dynamics (LFD). The LFD is a state-of-the-art fluorescence laboratory for use by local, national, and international scientists. It is committed to service in a user-oriented facility, as well as to research and development of fluorescence instrumentation and theory.

The LFD has reached international recognition for the development of instrumentation for time-resolved fluorescence spectroscopy using frequency domain methods. In 2006 the LFD moved to its current location at the new Natural Sciences II building at the University of California, Irvine. Dr. Gratton remains Principal Investigator of the LFD and holds joint appointments as Professor in the departments of Biomedical Engineering and Physics, and also in the College of Medicine. His research interests include design of new fluorescence instruments, protein dynamics, hydration of proteins, and I.R. spectroscopy of biological substances. He has over 400 publications in refereed scientific journals.

Assistant Professor Mohammad Mofrad, Bioengineering, University of California, Berkeley

Force-induced biological activities in the cell play a central role in development and in various disease processes that integrate mechanics and biology. How these mechanical and biochemical pathways interact remains largely unknown. To study the biomechanics of cell function requires a multi-scale and multi-physics approach. Stresses transmitted through adhesion receptors are distributed throughout the cell, leading to conformational changes that occur in individual proteins which in turn lead to increased enzymatic activity or altered binding affinities. The challenge is to couple the macro-scale stresses to micro-scale (individual protein) deformation. We are developing robust computational tools, drawing from molecular dynamics, Brownian dynamics, and large-scale continuum models of mechanics needed to numerically simulate the response of the cytoskeleton to mechanical stimuli. These quantitative models, predicated upon comprehensive and systematic experimental measurements made across multiple length scales, will enable the coupling of continuum with meso- and molecular-level simulations for studying large cellular deformation wherein the whole cell is divided into numerous elements and the response of each element is derived from a concurrent mesoscale simulation consisting of a small number of actin links and actin-binding proteins.

Professor Robert Phillips, Applied Physics and Mechanical Engineering, California Institute of Technology

Cells respond to their environments as a result of a number of different external cues. One intriguing way in which cells respond to environmental perturbations result from forces on the cell membrane. In this talk, I will describe various types of mechanosensation with special emphasis on the way that bacterial cells respond to membrane tension.

Professor Vladislav Yakovlev, Physics, University of Wisconsin, Milwaukee

Ever since the discovery of a microscope, the imaging of a single molecule was always presented as one of the greatest challenges for scientists. Fifty years ago, Schrodinger wrote that he does not believe that it will be ever conceivable. Nowadays, due to numerous technical improvements, it becomes possible to watch the motion of individual single molecules by monitoring in real-time their position with nanometer precision and measuring forces generated by molecules with piconewton accuracy. This allows Physics to step into a world of Biology by introducing a quantitative analytical approach to long-standing problems.

In my talk I will introduce some basic biology and physics necessary for understanding the physics of biological molecular motors, and show how single-molecule spectroscopy can minimize some of the mysteries in this field, while leading to a better understanding of how the motion of those molecules can be initiated by variation of physical, rather than biochemical, parameters.

Professor Michael K. Gilson, Center for Advanced Research in Biotechnology, University of Maryland

BindingDB (www.bindingdb.org) is a free database of protein-ligand binding affinities which is designed to support computational methods development and computer-aided drug discovery. The data in BindingDB raise interesting questions about the limits of achievable affinity and the significance of entropy-enthalpy compensation, and motivate a discussion of the nature and origins of entropy changes, and their role in defining the affinities of small molecules for protein targets. In particular, our calculations of changes in configurational entropy also show strong entropy-energy compensation in most cases, but a series of unusual host-guest complexes overcome this compensation to bind with affinities rivaling that of streptavidin and biotin. Applications of these ideas to protein-ligand binding and molecular design will be presented.

Professor Manbir Singh, Radiology and Biomedical Engineering, University of Southern California

Magnetic resonance Imaging (MRI) based methodology to probe brain function, called functional MRI or fMRI, has now reached the stage where it is well established and used widely in diverse fields such as neurosurgery, cognitive neuroscience and economics. Similar developments are now occurring in another MRI based modality called Diffusion Tensor Imaging or DTI, which enables one to map axonal connections through white-matter in the brain.

This talk will focus on novel methodology that has been developed for both fMRI and DTI in Dr. Singh's laboratory at USC. Commonly used fMRI approaches present a snapshot view of brain function after a task has been completed.

This talk will review new methodology developed in Dr. Singh's laboratory to achieve dynamic fMRI where the fMRI signal is tracked through the brain at different time points during the performance of a task. Examples will be presented showing the sequential activation of different brain regions during a simple motor task and also during a more complex task where the subject is involved in making decisions in high risk versus low risk situations. likewise, novel methodology to perform whole-brain tractography with DTI data, and potential applications including a search for biomarkers of Alzheimer Disease and quantification of damage to brain connectivity in Traumatic Brain Injury will be discussed.

Distinguished Professor Harry Gray, Chemistry and Chemical Engineering, California Institute of Technology

We have shown that laser-induced electron transfer can be used to trigger and monitor protein folding on nanosecond timescales; we have estimated that the folding speed limit for heme proteins is about 100 ns. Experimentally validated energy landscapes for native and metal-substituted cytochromes confirm that the ligand substitution step is greatly inhibited when the central metal is cobalt(III), allowing early events in the search for topologically productive conformations to be examined thoroughly. We are investigating compact and extended structures of both denatured and natively unfolded proteins through analysis of fluorescence energy transfer kinetics. Much of our work has centered on labeled cytochrome c and on alphasynuclein, a protein implicated in Parkinson’s disease. Partially folded polypeptide structures are key intermediates in both the proper assembly of proteins, and in the formation of harmful misfolded structures. Knowing the structures, energetics, and dynamics of these transient species will lay a foundation for elucidation of their benign and malignant pathways.

Dr. Bongsu Jung, Department of Biomedical Engineering & Center for Nano & Molecular Science & Technology The University of Texas at Austin

Recently, label-free detection techniques in array type sensors have been extensively studied to solve the current limitations of fluorescent labeling technique such as toxicity, false positive results, photo-bleaching, concentration dependence of the fluorescence, interference, non-specific interactions, and complicated and labor intensive processing of labeling. High-density array formats also require a less technically involved and more fast parallel detection technique than existing labeling methods. Localized Surface Plasmon Resonance (LSPR) is a label-free technique that is fast, highly sensitive to the dielectric environmental changes, suitable for high-throughput screening, nanoscale detection up to femto-molar, and can be detected by simple absorption and scattering measurement in a UV-Vis spectrometer or a dark-field microscopy. We developed new fabrication technique which is a carbon-based template stripping technique to create nano-textured ultraflat surface. This is called carbon-based ultraflat nanosphere lithography (c-UNSL). With the technique, it is possible to create partially embedded periodic truncated tetrahedron MNPs and to control the size and the shape of MNPs in a glass. The resulting UNSL-MNPs are capable of detecting thiol surface modification, and biotin-streptavidin protein binding events. Since each gold or silver particle principally acts as an independent sensor, on the order of a few thousand molecules can be detected, and the sensor can be miniaturized without loss of sensitivity. Our Finite-difference-time-domain (FDTD) and finite-element-method (FEM) numerical calculations show the influence of the sharp features on the resonance peak position. The maximum near-field intensity is dependent on the polarization direction, the sharpness of the feature, and the near-field confinement from the substrate. It is also found that the near-field electric field intensity also directly affects the far-field extinction. 3D FDTD simulation shows the local refractive index sensitivity of the gold truncated tetrahedron, which is in agreement with our experimental result. Both experimental and numerical calculations show that each particle can act as its own sensor for non-labeling array type assays..

Assistant Professor Michelle Khine, Engineering, University of California, Merced

In this post-genomic era, we are well positioned to investigate dynamic processes at the cellular systems level and to leverage this to engineer therapeutically useful cells. Understanding how individual cells make decisions will help realize the potential of systems biology to completely transform biomedical research. Previous limitations in sensitivity of analytical methods meant that most cellular measurements were traditionally taken across large pools of cells. Such bulk population experiments output the mean value of a parameter of interest, whereas single-cell experiments allow for investigating the parameter's distribution. This is an important distinction because even cells that are identical genetically exhibit marked variations in genetic expression and cellular response. By leveraging the advantages of microfluidics in terms of improved spatial and temporal resolution, we can address individual cells using multiplexed electrical and chemical cues in our microfabricated systems. Specifically, we are developing tools to: 1) understand cancer metastasis behavior 2) control stem cell differentiation 3) provide low-cost point-of-care diagnostics for infectious disease and 4) monitor air quality. The goal of our lab is to provide advanced yet low- cost solutions for diagnostics, sensing, and analytical instrumentation. To this end, we are also developing novel microfabrication approaches.Most recently, we have developed Shrinky-Dink Microfluidics. Leveraging the inherent shrinkage properties of the pre-stressed thermoplastic polystyrene sheets of the children's toy Shrinky-Dinks, we can rapidly develop deep and rounded channels, and even complex 3D chips.

Professor Shankar Subramaniam, Bioengineering, Chemistry and Biochemistry and Biology , University of California, San Diego

CANCELLED

Assistant Professor Jennifer Cochran, Bioengineering, Stanford

Nature has provided stable peptides that we can exploit as scaffolding materials to create novel biomolecules. Using molecular cloning, we grafted biologically-active sequences from large cell adhesion proteins into several of these constrained peptide scaffolds and showed that they were able to bind to integrin receptors with modest affinity. Since three dimensional conformation is critical for a peptide to bind its target with high affinity and specificity, prototype molecules were affinity-matured using yeast surface display. We showed that these engineered high affinity integrin binding peptides specifically modulated cell adhesion, and are promising in-vivo tumor targeting agents for molecular imaging and therapeutic applications.

Dean and Professor Howard Stone, Engineering and Applied Science, Harvard

We describe several microfluidic approaches for cellular-scale hydrodynamics. In particular, (i) we demonstrate a microfluidic technique for measuring Michaelis-Menten rate constants, which should be broadly applicable to enzymatic reactions, and investigate the idea using experiments, numerics, and scaling arguments, and (ii) we consider the influence of shear stress on the kinetics of ATP release from red blood cells that flow through constrictions, which may be helpful for understanding extracellular ATP transport and signalling. These microfluidic projects have in common the use of hydrodynamic principles to explore quantitatively new questions in nonlinear chemical kinetics and cellular mechanotransduction.

Professor Tonya Kuhl, Chemical Engineering and Materials Science, University of California, Davis

Polymers – both synthetic and natural – are increasingly used for a variety of biomaterial applications, such as protein separation, initiating cell fusion, creating biocompatible surfaces and controlling the binding rate and circulation of drug-delivery vehicles. In order to optimize their performance, there has been a large effort to understand the way polymers modulate biomolecular and cell surface interactions at the molecular level. In many cases, existing theories have been quite successful in explaining polymer-mediated interactions of cells and biomembranes. In this talk, I will emphasize the impact of using flexible polymer tethers and dynamics to modulate the formation of specific ligand-receptor bonds and complementary adhesion between membranes. Both experimental and theoretical results as a function of tether molecular weight, ligand-receptor affinity, and polymer architecture will be discussed. The optimization of targeting architectures for drug delivery and biosensing applications will be highlighted.