Using Biophysics to Understand Cataracts

June is Cataract Awareness Month in the US. By age 80, more than half of all Americans either have a cataract or have had cataract surgery, according to the National Eye Institute. To recognize this month, we spoke with BPS member Doug Tobias, University of California, Irvine, about his research on crystallin proteins, the proper functioning of which is key to lens clarity. 


What is the connection between your research and cataracts?

The eye lens, which focuses light on the retina, is composed of bundled fiber cells that lose their nuclei, ribosomes, and organelles during embryonic development. The transparency and refractive properties required for proper lens function results from the liquid-like order of structural proteins called crystallins, which are present in these cells at concentrations exceeding 300 g/L. Because the central nucleus of the lens undergoes very little protein turnover, crystallin proteins must remain stably in solution for a lifetime. A cataract is an opacification of the eye lens caused by the loss of solubility of the crystallin proteins, leading to the formation of aggregates that scatter light.

Tobias image 2

All-atom model of AQP0 tetramer embedded in a hydrated lipid bilayer and complexed with two CaM molecules. Reference: S. L. Reichow, D. Clemens, J. A. Freites, K. L. Németh-Cahalan, M. Heyden, D. J. Tobias, J. E. Hall, and T. Gonen, Allosteric mechanism of water-channel gating by Ca2+–calmodulin, Nat. Struct. Molec. Biol. 20, 1085-1092 (2013).

Our research seeks to understand cataract formation on the molecular level from two different angles, both involving atomically-detailed computer simulations (molecular dynamics, Brownian dynamics, and Monte Carlo), in concert with experiments carried out by our collaborators. On one front, we are attempting to elucidate how minor changes to the chemical structure of the crystallin proteins – such as the single-point mutations that are associated with congenital, early onset cataract, or post-translational modifications (e.g., truncation, disulfide bond formation, UV damage, deamidation, etc.) in the case of the much more common age-related cataract – give rise to altered interprotein interactions, which, in turn, lead to protein aggregation.

On the other front, we are learning how the protein aquaporin 0 (AQP0) is regulated. AQP0 is expressed exclusively in the eye lens, where it comprises roughly half the membrane protein content and functions as a water channel and, hence, is a key player in maintaining lens homeostasis. AQP0’s water permeability is regulated by pH and Ca2+, whose concentrations change dramatically with distance into the lens. Defects in AQP0 can lead to cataract. We are using molecular dynamics simulations, in conjunction with experimental measurements carried out by our collaborators, to determine the mechanism of the alteration of AQP0 permeability by certain mutations, as well as by changes in pH and Ca2+ levels.

Why is your research important to those concerned about cataracts?

There is good reason to be concerned about cataracts. In its latest assessment of priority eye diseases, the World Health Organization estimated that cataract is responsible for more than 50% of world blindness, which amounts to roughly 20 million people. Presently, cataracts are treated by surgical replacement of the opaque lens with an artificial intraocular lens.  Due to lack of access to eye care, millions of people in the developing world remain blind from cataract. The long-term goal of our research is to gain new insights into cataract formation that could ultimately guide the development of new therapies for cataract prevention and treatment.

How did you get into this area of research?

I got into this area of research through interactions with colleagues at UC Irvine, specifically, Jim Hall and Rachel Martin. Jim Hall is a lens physiologist in the Department of Physiology and Biophysics and an expert on AQP0, and Rachel Martin is a biophysical chemist in the Department of Chemistry and an expert on the crystallins. Their enthusiasm for their respective subjects was infectious and irresistible, and I jumped at the opportunity to collaborate with them. Our collaboration with Jim Hall was initiated by a graduate student in his lab who wanted to spend time in my lab learning molecular modeling. Our work with Rachel Martin began as a summer undergraduate research project.

How long have you been working on it?

We started dabbling in the field about five years ago and then increased our effort substantially after receiving funding for the work about three years ago.

Do you receive public funding for this work? If so, from what agency?

Our work on the crystallin proteins is supported by both the National Institutes of Health and the National Science Foundation, and our work on AQP0 is supported by the National Institutes of Health.

Tobias image

Computational model of an aggregate formed by the congenital cataract-related W42R mutant of human gD-crystallin in a solution at 220 g/L concentration. The N-terminal domains are colored red and the C-terminal domains blue. The aggregates formed by the W42R mutant display enhanced interprotein contacts involving the N-terminal domain, where the mutation is located, vs. the wild-type protein, which displays primarily non-specific interactions at the same concentration.

Have you had any surprise findings thus far?

Ca2+ regulation of AQP0 is mediated by the calcium binding protein calmodulin (CaM), which forms a complex with AQP0 of unusual stoichiometry (2 CaM to 1 AQP0 tetramer). We expected that CaM binding would lower AQP0 permeability by simply blocking the water conducting pores. However, we found instead that CaM binding lowers the open probability of a gate deep within the pore through long-range allosteric interactions. We are now learning, also unexpectedly, that pH regulation occurs through a similar (allosteric) mechanism involving a second gate on the other end of the pore.

Crystallin proteins are constantly bumping into one another due to their high concentration in the lens. “Healthy” (wild type) crystallins stay in solution because they don’t stick to each other, i.e., their interactions are non-specific. It is fascinating to me that a single-point mutation in a single class of crystallin proteins can lead to early-onset cataract. The aggregation mechanisms of these mutant proteins are only beginning to be worked out, and it is likely that different mechanisms will be at play in different cataract-related variants. In one case that we are currently studying, we found that the W42R mutation “cracks open” the interface between the two domains in gD-crystallin, exposing a sticky patch that promotes the formation of large-scale protein clusters.

What is particularly interesting about the work from the perspective of other researchers?

Our work produces atomically-detailed descriptions of molecular mechanisms that are often critical to the interpretation of experimental observations. We take great pains to validate our simulations so that their predictions can be trusted.

What is particularly interesting about the work from the perspective of the public?

Cataract is the leading cause of blindness worldwide.  Even in developed countries like the US, where cataract surgery is widely available, and safe and effective in the vast majority of cases, delaying it for just a few years would greatly reduce health care costs. We hope that our research will yield new fundamental insights that will inform non-surgical approaches to cataract treatment.

Biophysics and Bleeding Disorders

March is Bleeding Disorders Awareness Month in the US. More than three million Americans who have hemophilia, von Willebrand disease, and other rare bleeding disorders. These conditions prevent blood from clotting the way it should, which can lead to prolonged bleeding after injury, surgery, or physical trauma. We spoke with Biophysical Society member Valerie Tutwiler, an American Heart Association graduate research fellow in the lab of John Weisel at the University of Pennsylvania, about her hemostasis and thrombosis research.

What is the connection between your research and bleeding disorders?

BPJ_112_4.c1.indd

This recent cover of Biophysical Journal shows Tutwiler, Wang, Litvinov, Weisel, and Shenoy’s image of a colorized scanning electron microscope image of a coronary artery thrombus extracted from a heart attack patient.

Blood clotting or hemostasis is the process that stems bleeding. On one hand if you have insufficient clotting this can result in prolonged bleeding, on the other hand a hypercoagulable state can result in thrombosis. Thrombi can result in the obstruction of blood flow, which can cause heart attacks and strokes. My thesis research pertains largely to studying one portion of the coagulation process blood clot contraction, or the volume shrinkage of the clot, which has been implicated to play a role in hemostasis and the restoration of blood flow past otherwise obstructive thrombi.

Why is your research important to those concerned about bleeding disorders?

While there is much known about the various aspects of blood clotting relatively little is known about the process of clot contraction despite the clinical implications of its importance in the formation of a strong hemostatic seal and the restoration of blood flow past otherwise obstructive thrombi. The study of clot contraction is a highly interdisciplinary problem and as a result can be of interest to researchers from many different fields. Platelets are active contractile cells, which interact with an extracellular matrix of fibrin, a naturally occurring polymer with unique mechanical properties. The fibrin matrix can be imbedded with other blood cells, such as red blood cells, as well. From a biophysical standpoint the mechanisms of clot contraction have not been well understood. To better elucidate this process, we performed a systematic study on how the molecular and cellular composition of the blood influences the rate and extent of clot contraction along with the mechanical properties of the contracting clot using a novel application of an optical tracking system.

Additionally, to further explore the mechanical nature of the clot contraction process we developed a mathematical model that couples active platelets with a passive viscoelastic matrix made up of fibrin and red blood cells. The model predicts the process of clot contraction and explains some of the experimental observations of clot size, structure and mechanical forces. Interestingly, we found that clot contraction is altered in thrombotic states such as ischemic stroke patients. Collectively, these findings show that the study of clot contraction has the potential to inform the development of diagnostics and therapeutics.

How did you get into this area of research?

Since beginning research I have been interested in applying engineering techniques to answer biological questions. I became interested in hemostasis and thrombosis research while completing my first co-op experience in undergrad.

How long have you been working on it?

I began doing hemotology research during my undergraduate career. However, I started studying clot contraction specifically when I started my PhD research.

Do you receive public funding for this work? If so, from what agency?

I am currently funded by the American Heart Association as a pre-doctoral fellow, although we also receive funding from the National Institute of Health and National Science Foundation.

Have you had any surprise findings thus far?

We were surprised to find such a striking decrease in the extent of clot contraction in ischemic stroke patients compared to healthy subjects. Correlations with stroke severity suggest that clot contraction may be a potential pathogenic factor in ischemic stroke. These findings have led us to expand our study to other pathological conditions as well.

What is particularly interesting about the work from the perspective of other researchers?

Due to the conservation of the basic principles of contractile proteins and motility, the information learned from the development of a mathematical model of active contractile cells interacting with a viscoelastic matrix can be applied to a variety of different processes.

What is particularly interesting about the work from the perspective of the public?

Bleeding and thrombotic conditions remain leading causes of death and disability worldwide. Gaining a more thorough understanding of the processes involved in hemostasis and thrombosis will lead to the development of more effective diagnostic tools and more targeted therapeutics.

Highlighting Biophysics Research During American Heart Month

February is American Heart Month. Heart disease is the leading cause of death for men and women in the United States, causing 1 in 4 deaths each year. In recognition of this awareness month, we spoke with BPS member Anna Grosberg, University of California, Irvine, about her research on cardiomyocytes, which play an essential role in contracting the heart and pumping blood.

ImageJ=1.46r unit=um

What is the connection between your research and heart disease?

My laboratory studies the structure dynamics, and function of cells. We are mostly focused on cells that make-up the heart muscle – cardiomyocytes, which play the essential role in contracting the heart and pumping the blood. Recently, we have made discoveries elucidating how the organization on multiple length-scales affects the ability of engineered heart tissues to generate force. This is especially important for the future progress in developing therapies following heart attacks and for understanding heart diseases, such as dilated and hypertrophic cardiomyopathies.

Why is your research important to those concerned about heart disease?

The label “heart disease” encompasses a wide variety of patient symptoms and pathologies. Yet in many presentations of the disease, the structure of the heart is changed over a wide variety of length-scales. Indeed, the normal heart consists of sheets of elongated cardiomyocytes arranged to generate maximal force in parallel with each other. Conversely after an infarct, a scar forms that disturbs this organization and prompts the muscle to remodel. Often such remodeling causes the cells to become less organized within the tissue, and it can also occur without an initiating scar. Understanding how these progressive architectural changes affect function is important to identifying the dominant mechanism to target in modulating heart disease. Through the type of research my lab does, it was discovered that changes to the organization also cause downstream variations to expression levels of a variety of genes some of which are linked to both electrophysiological and contractile function. This makes it problematic to determine how much of the force reduction of diseased muscle is caused by disorganization, and to what extent it is influence by the downstream biological effects. We have recently found that providing muscle cells with local organizational cues, while maintaining a global organization results in engineered cardiac muscle with predictable organization-force generation relationship based on simple physical understanding.

In general, remodeling of the heart muscle and, even, the enlargement of the heart are part of normal physiology. For example, it is known that for both competitive athletes and pregnant women, the heart can enlarge for the duration of the higher load, but it will remodel to normal size once the excessive exercises are over or the baby is born, respectively. Thus, there is hope that by understanding the functional aspects of remodeled heart tissue, it will be possible to reverse some of the pathological remodeling observed in heart disease. Additionally, we also work with cells from patients with genetic mutations that cause heart disease. A deeper understanding of the mechanisms that make these cells abnormal can provide more clues for how genetically normal patients can still develop heart disease.

How did you get into this area of research?

In the past decade, advances in tissue engineering have allowed researchers to guide the architecture of engineered heart tissues. It has also been possible to image the detailed structures within the tissues – thus visualizing the organization of the sarcomeres (the force producing units within muscle cells). However, it was also found that the reduction in force generated by badly organized tissues was two times higher than predicted from basic physics principles. Unfortunately, it was not possible to change the organization of the tissue without also triggering biological downstream effects that affected force generation in complex, poorly understood ways. When I started my lab, one of our big goals was to create a system to both quantify organization of cardiac muscle over multiple length-scales and to build an experimental system where the organization could be changed without triggering any downstream effects. These tools now allow us to greatly deepen our understanding of the relationship between structure and function in both normal and diseased heart tissues.

How long have you been working on it?

I have been working on the relationship between structure and function in the heart for the past 15 years. However, only in the past few years we have built-up the tools to study multi-scale organization and its connection to force generation.

Do you receive public funding for this work? If so, from what agency?

Yes, our work has been funded by both the National Science Foundation and the National Institutes of Health.

Have you had any surprise findings thus far?

We have found that in the absence of guidance cardiac tissue forms spontaneous patches of organization. Yet, the force generated by such a tissue is almost two times weaker than if the patches of organization are introduced with guided parquet patterns. The first surprising finding was that it is possible through very basic physical principles (i.e. force vector addition) to predict the amount of force generated tissues if their organization was guided in parquet tiles. Second, we found that the spontaneously organized patches are only 3 times smaller than the engineered parquet tiles. This implies that either the cells are very sensitive to a narrow range of local signals, or the temporal change in self-assembly provided by guidance cues plays an important role in resultant force generation. Our current aim is to solve this mystery.

What is particularly interesting about the work from the perspective of other researchers?

Our results have shown that in the case of eliminating biological downstream effects, simple physical principles are predictive for cardiac tissue force generation. This provides a way to interpret the reduction in force observed in stem-cell derived cardiomyocytes that are incapable of achieving sarcomeric structures that fully match the adult or even neonatal hearts.

What is particularly interesting about the work from the perspective of the public?

Heart disease is still the number one cause of death in the developed world. Yet, there are no cures – only therapies that help manage the disease. By exploring the basic physical principles behind heart muscle structure and function, it might be possible to provide better targets for therapies or surgical intervention. Additionally, it might become realistic to use the principles we discover to build better engineered cardiac tissues, which would provide better predictability for testing pharmaceuticals for cardiac toxicity.

Using Biophysics to Understand Diabetes

November is National Diabetes Month in the United States. Twenty-nine million people in the US live with diabetes. To recognize this awareness month, we spoke with BPS member Roger Cooke, University of California, San Francisco, about his biophysics research related to the disease.

figure-cooke

This cartoon shows the 3 states of myosin. In the active state the myosin head is attached to the actin filament producing force and motility. In the super relaxed state, shown above, myosin heads are bound to the core of the thick filament, where they have a very low ATPase activity. In the disordered relaxed state myosin heads extend away from the core of the thick filament where they have a much higher ATPase activity and are available for binding to actin.

What is the connection between your research and diabetes?

Our laboratory has studied the physiology and biophysics of skeletal muscle for many decades. Recently we have concentrated on the metabolic rate of resting skeletal muscle. Skeletal muscle plays a major role in diabetes as it is the organ response for metabolizing a large fraction of the carbohydrate that we consume. Recently we have discovered a mechanism, which we believe can be manipulated to up regulate the metabolic rate of resting muscle, thus metabolizing more carbohydrate. This would be particularly helpful in Type 2 diabetes.

Why is your research important to those concerned about diabetes?

Type 2 diabetes is thought to be caused by or an overconsumption of carbohydrate coupled with a sedentary lifestyle that does not need the carbohydrate as fuel.  The excess carbohydrate leads to high levels of serum glucose. Our laboratory has focused on the motor protein myosin, which is responsible for producing the force of active muscle and also responsible for using much of the energy ingested in the form of lipids and carbohydrates. We have shown that myosin in resting muscle has 2 states with vastly different functions and metabolic rates. In one of these, the super relaxed state, the myosin is bound to the core of the thick filament where its metabolic rate is inhibited, See Figure.  In the other, the disordered relaxed state, the myosin is free to move about and its metabolic rate is more than10 fold higher.  By analogy with another motor, myosin in active muscles is akin to a car racing down the road. Myosin in the disordered relaxed state is similar to a car stopped at a traffic light with the motor idling, and the counterpart of the super relaxed state is a car parked beside the road with the motor off.

For energy economy in resting muscle most of our myosins are in the super relaxed state. If all of these myosins were transferred out of the super relaxed state into the disordered relaxed state they would consume an additional 1000 kilo calories a day. This is a large fraction of the standard daily consumption, which is approximately 2000 kilo calories a day.  Thus a pharmaceutical that destabilized the super relaxed state would lead to the metabolism of a greater amount of carbohydrate providing an effective therapy for Type 2 diabetes. Such a pharmaceutical would address one of the fundamental problems in Type 2 diabetes the consumption of more carbohydrates than are required as fuel.

How did you get into this area of research?

In 1978 a group in England showed that purified myosin in a test tube had a much greater activity than it has in living fibers. This observation showed that myosin in vivo spent much of its time in a state that had a very low metabolic rate. I felt that this inhibited state of myosin could have important consequences for resting muscle and whole body metabolic rates. Although we studied this problem for a number of years, we were not able to find an in vitro system that replicates the in vivo activity. In 2009 we started using quantitative epi-fluorescence spectroscopy to measure single nucleotide turnovers in relaxed skinned muscle fibers, and finally we were able to observe the elusive inhibited state of myosin, the super relaxed state.  This ability allowed us to now study the properties of this state.

How long have you been working on it?

I have been interested in this problem since the original observation in 1978, described above. However it was not until 2009, and the discovery of the in vitro assays, which allowed us to observe the super relaxed state, that this project became the central focus of our laboratory.

Do you receive public funding for this work? If so, from what agency?

Our work has been funded by the National Institutes of Health.

Have you had any surprise findings thus far?

The Holy Grail in this area of research is to find pharmaceuticals that will destabilize the super relaxed state. Recently we were able to devise new methods of measuring the population of the super relaxed state, methods that were amenable to use in high throughput screens. We screened over 2000 compounds looking for ones that destabilized the super relaxed state.   We found only one compound that did so, a compound named piperine, which provides the pungent taste in black pepper. After working for over a year developing assays and running the screen, to our surprise the one molecule we discovered was already known to mitigate Type 2 diabetes in rodents. Although piperine lowered serum glucose, no one knew how it did this. We propose that piperine acts by destabilizing the super relaxed state, thus up-regulating the metabolic rate of resting skeletal muscles. We showed that piperine had no effect on active muscle and no affect in cardiac muscle, both desirable qualities to have in a pharmaceutical targeting resting skeletal muscle to treat Type 2 diabetes. Although piperine is effective in lowering blood glucose in rodents, it only does so at very high doses, too high to be useful as a therapeutic in humans.  We now need to find molecules whose action is similar to piperine, but which bind with greater affinity.

What is particularly interesting about the work from the perspective of other researchers?

Our results provide the proof of concept that pharmaceuticals targeting resting muscle metabolic rate, can be found, using the high throughput screens we developed.  These new pharmaceuticals have the potential of more effectively treating Type 2 diabetes.

What is particularly interesting about the work from the perspective of the public?

Type 2 diabetes is a growing problem worldwide. Almost 10% of the US public has Type 2 diabetes.  Our research holds out the hope that new pharmaceuticals will be found to treat this disorder more effectively than those available today.

Our studies have also shown that the super relaxed state is destabilized when muscles are activated, and that it will remain destabilized for a number of minutes afterwards, due to phosphorylation of myosin. This extended period of destabilization adds to the metabolic cost of activity, particularly during light and intermittent activities. In fact a number of studies have shown that even modest and intermittent activity will improve serum glucose, help prevent weight gain and lead to better health. The worst thing that people can do is to sit for extended periods of in front of a computer or TV screen. For example when working at a computer I get up and walk around the room every 10 minutes or so, and I avoid elevators, taking the stairs to my laboratory on the 4th floor.

Biophysics on World Hepatitis Day 2016

July 28 is World Hepatitis Day. Viral hepatitis is inflammation of the liver caused by a virus. There are five different hepatitis viruses, hepatitis A, B, C, D and E. Hepatitis C affects approximately 250 million people worldwide. We spoke with Jiawen Li, University of Texas at Austin, Institute of Cellular and Molecular Biology, about her research related to hepatitis C, for which there is currently no vaccination.  

What is the connection between your research and hepatitis C?

Here in the Johnson lab we use transient-state kinetic approaches to characterize viral polymerases, specifically to measure nucleotide specificity, polymerase fidelity and dynamics. More importantly, we apply these methods to understand the mechanisms of action of nucleoside analogs and non-nucleoside inhibitors that are developed to target viral polymerases. For example, to combat HIV, reverse transcriptase is primarily targeted for anti-AIDS therapy. As the RNA-dependent RNA polymerase for Hepatitis C virus, NS5B is considered an important target for effective antivirals as well. Thus the focus of our research is to develop assays to determine kinetic parameters governing RNA dependent RNA replication by NS5B and establish the mechanisms of action and efficiency of various clinically relevant anti-HCV drugs.

Why is your research important to those concerned about hepatitis C?

Hepatitis C affects approximately 250 million people worldwide and chronic infection can lead to hepatitis, liver cirrhosis, and cancer. There is no vaccine available, but combination therapies with direct-acting antivirals including nucleoside analogs and non-nucleoside inhibitors targeting NS5B have been recently advanced and have dramatically improved the potency of HCV treatment. Surprisingly, besides the identification of binding site on NS5B, very little is known about the inhibition mechanisms of drugs that are currently on the market. Two pharmaceutical companies, Gilead Science and Alios Biopharma, have generously provided us with some of their inhibitors to study. Our primary goal is to analyze a handful of these inhibitors in depth to establish their mechanisms of inhibition and to set evaluation guidelines for the effectiveness of each class of inhibitor. Ultimately, we want to apply our methods to each FDA-approved inhibitor for HCV treatment to aid information for the development of even better therapeutics.

How did you get into this area of research?

With a bachelor’s degree in Biochemistry, I was accepted into the Biochemistry graduate program at UT Austin in 2011. During my rotation in the Kenneth Johnson lab, I was fascinated by transient-state kinetic methods such as combining rapid quench-flow and stopped-flow techniques to accurately measure and analyze nucleotide incorporation by HIV RT. Of course I immediately joined the lab and I was very enthusiastic to work on other viral polymerases. The hepatitis C viral RNA-dependent RNA polymerase, NS5B, is known to catalyze de novo RNA synthesis, which means RNA replication is divided into two distinct mechanistic phases: initiation and elongation. Previous studies in our lab along with other groups in the field have made tremendous efforts to develop assays for efficient NS5B replication, but were always hindered by the slow and inefficient initiation phase. Therefore, although the crystal structure of NS5B was solved a decade ago, kinetic characterization of enzyme mechanism, specificity and fidelity are limited, and little is known about the mechanistic basis for inhibition. Finally in 2012, Zhinan Jin, who graduated from our lab and worked for Roche at the time, succeeded in developing conditions for formation of highly active HCV elongation complex. I then continued the work he has accomplished and further optimized the kinetic assays for NS5B inhibition analysis.

How long have you been working on it?

It has been four years since I started working on HCV NS5B in 2012 as a second year graduate student here at UT Austin. I know several lab members had tried to establish conditions for efficient NS5B replication over a decade ago. I am glad this project is brought back to life again!

Do you receive public funding for this work? If so, from what agency?

Yes, we received funding from the Welch Foundation and the National Institutes of Health.

Have you had any surprise findings thus far?

Yes, we have had several surprise findings along the way. Firstly, we now have successfully developed robust kinetic assays to monitor RNA replication by NS5B from initiation to elongation. To our surprise, once the elongation complex is formed, it is extremely stable with half-life of more than a week, which makes the crystal structure of NS5B ternary complex highly promising to obtain in the near future.

Secondly, we have been able to establish modes of action for four classes of non-nucleoside inhibitors. One class of NNIs, the thumb site II inhibitors (NNI2) were shown to be most interesting. NNI2 do not significantly block HCV initiation or elongation; rather they act as allosteric inhibitors to block NS5B transition from initiation to elongation, which is thought to occur with a significant change in enzyme structure. To further examine this allosteric inhibition, we collaborated with Dr. Patrick Wintrode from the University of Maryland and his postdoc Daniel Degrede who mapped the effect of NNI2 inhibitors on the conformational dynamics of NS5B using hydrogen-deuterium exchange kinetics. HDX shows that NNI2 rigidifies an allosteric network extending up to 40 Å from the inhibitor binding site to enzyme active site, providing the rational for blocking NS5B transition at the molecular dynamics level.

NNI2-NS5B HDX (Jiawen Li)

Peptic fragments resulted in significant decrease in HDX upon NNI2 (magenta sticks) binding are shown in dark blue. Rigidification of a large network of enzyme dynamics was observed starting from inhibitor binding site throughout the protein, especially surrounding the enzyme active site, suggesting a long range allosteric effect from inhibitor binding on NS5B conformational change.

Meanwhile, we also explored the mechanisms of NS5B inhibition by nucleotide analogs. We found that both pyrophosphate and NTP mediated excision of incorporated nucleoside analogs were relatively fast reactions, suggesting the important role of pyrophosphorolysis in evaluating the effectiveness of chain-terminating inhibitors. In fact, wild-type NS5B polymerase catalyzes the nucleotide-dependent excision reaction faster than mutants of HIV reverse transcriptase that have evolved to overcome inhibition of nucleoside analogs. This is a significant problem for design of nucleoside analogs to treat HCV infections. We are in the process of publishing this work soon.

What is particularly interesting about the work from the perspective of other researchers?

Our detailed mechanistic studies have provided a fundamental understanding of RNA-dependent RNA replication by HCV NS5B and established the mechanisms of action of different anti-HCV drugs. We hope our experimental and analytical methods will benefit other researchers for studying HCV polymerase or similar viral polymerases and eventually assist screening and design of more effective inhibitors to combat HCV and other viral diseases.

What is particularly interesting about the work from the perspective of the public?

It is great news knowing that more and more anti-HCV drugs are being developed and approved by FDA. With the platform we built for inhibitor analysis, we would like to incorporate more inhibitors into our study and determine their biochemical role of inhibition. We think our work will help providing insights for the development of drugs that are safer and effective against broader range of HCV genotypes.

 

 

Biophysics on World Sickle Cell Day

June 19 is World Sickle Cell Awareness Day. Sickle cell disease refers to a group of inherited red blood cell disorders. The Center for Disease Control and Prevention estimates that sickle cell disease affects 90,000 to 100,000 people in the United States, a majority of whom are African-American. Worldwide, it is estimated that 300,000 children are born each year with sickle cell diseases, though many go undiagnosed in developing countries. We recently spoke with Biophysical Society member Frank Ferrone, Drexel University, about the research his lab conducts related to sickle cell disease. 

What is the connection between your research and sickle cell disease?

We have several projects underway that relate to sickle cell disease.   Let me give two.   In one, we are working to complete a device that can detect the presence of sickle cell disease, or sickle cell trait (and distinguish the two), using a drop of blood, in under a minute, at a cost of a few cents per test.   And we’re almost there!   In a more basic vein (sorry about that ) we are also exploring a radical hypothesis of ours that the structure of the sickle polymers that cause all the trouble in the disease is not understood properly—and if we are correct, our revision of this paradigm has implications for all pathological assembly diseases.

Why is your research important to those concerned about sickle cell disease?

In the US, children get tested, information gets shared (mostly), and emergency medicine is very good…and even here, sickle patients sometimes get misdiagnosed or fall through the cracks.  In certain nations, sickle cell disease afflicts as many as 1 in 7 newborn children, and the resources simply don’t exist to conduct expensive tests, or to track the families whose children are affected.   Thus an inexpensive and rapid test could be a godsend there.   At the same time in the US, the NCAA dictates that student athletes should have their status known, since sickle trait  poses a covert risk—kids can have no symptoms, and then under exertion experience tragic, even fatal, sickle-events.     Now, we supposedly have medical records of everyone’s test—but that’s if you did get tested, and if you have the records.   Imagine that instead of a kid having to wait for results of a test–an expensive one to boot–because his family can’t find his test results, that the school nurse could prick his finger and in a minute pronounce him good to go (or not).

As to the other project I mentioned, well there are currently no drugs that work by interfering with the polymeric structures that generate the pathology.   Maybe if we understood the polymers better, that could change.

How did you get into this area of research?

For my PhD I did a project on normal hemoglobin, using laser photolysis to trigger a structural change, and following it with kinetic CD (it was a first).   When looking around for a post-doc, I got invited to join Bill Eaton’s group at the NIH, using the photolysis trick to induce sickle cell hemoglobin polymerization.    I knew nothing about sickle cell at the time, but the project sounded interesting, the group was exciting, and, as Bill explained it, “eventually everybody comes to visit the NIH” so the environment was immensely stimulating.

How long have you been working on it?

It’s now on 40 years!   I carried the project with me to Drexel University’s Physics Department, where I’ve continued to work on this.

Do you receive public funding for this work? If so, from what agency?

While we did have many years of generous NIH support, at the moment we don’t have funding.

Have you had any surprise findings thus far?

Plenty!   When I was transitioning to Drexel from the NIH, we came up with the idea that there were two kinds of nucleation that generated the sickle fibers, not just one like everybody thought, mainly because we had these experiments that couldn’t be explained any other way.   That was a big surprise.   And now it’s turned out to be fairly common in pathological assemblies.

We found that the second type of nucleation—onto the surface of polymers—came about because intermolecular contacts that stabilize the interior of polymers also appear on the polymer exterior!   Anybody could have found this from the existing structures, but nobody thought to look.   Even we bumped into it by accident.

When we employed our models to understand the assembly process, we got another big surprise.   The ability of the hemoglobin molecules to oscillate about their equilibrium position in the polymer exercises a HUGE influence on the rate of the assembly, thanks to an effect known as vibrational entropy.   Weak, “sloppy” bonds generate much faster nucleation—which we demonstrated experimentally.

And one of our most recent surprises was that the polymer formation gets hung up in a metastable state because of the extreme crowding.   And in a red cell, that in turn leads to Brownian ratchet forces that can hold cells in narrow channels, like capillaries.

What is particularly interesting about the work from the perspective of other researchers?

Sickle cell is perhaps the “Granddaddy” of all the protein assembly diseases, which now include Alzheimer’s, prion diseases, Huntington’s, Parkinson’s… There is often a lot of hard biochemistry that goes into simply characterizing the assembly that is the root of these.   Sickle hemoglobin can be prepared in large quantities, purified simply, and even reconstructed by site-directed mutagenesis, with the result that much more sophisticated physical questions can be posed and tested without a ridiculous overhead of construction and purification.     Thus, as I mentioned, the double nucleation model, constructed for sickle cell polymerization, has been shown to operate on Aβ assembly.   In addition, to do the analysis, we needed to invent a new mathematical approach, adopting perturbation methods to deal with intractable differential equations.  This has been of great interest to others, too.   Finally, for years it was our “burden” that all the standard kinetic and equilibrium equations we used had to be modified to account for the high concentration of hemoglobin in the red cells.  I hated it!   Fortunately, we succeeded in working it out, but now with the burgeoning interest in molecular crowding, it turns out that our work has a lot of  applicability for another class of problems as well.

What is particularly interesting about the work from the perspective of the public?

Sickle cell disease has been known for over 100 years.   It’s known as the first molecular disease.   When people discuss gene therapy, sickle cell is invariably found in the list of targets.   And yet there is but one drug.   A solution to this long-standing problem generates interest.     Moreover, there are about 240,000 new cases (i.e. sickle cell births) yearly in Africa.   But the other part of the story, which maybe should be told to the public even more prominently, is that these assembly diseases are all connected in a fundamental way.    Our analytical methods led to a novel result in poly-Q assembly.   Our model was adopted for Aβ assembly.    Answering basic questions well generates a tide that indeed floats all boats.

Do you have a cool image you want to share related to this research?

We have a movie!   This was taken in our lab by Dr. Alexey Aprelev.   We have a narrow micro-fluidic channel, 4 µm wide and 1.5 µm deep, and a red cell.  In the movie the cell squeezes in and out just fine in response to externally oscillating the hydrostatic pressure.  The hemoglobin inside the cell has CO bound, which can be removed by light very efficiently.  Just as the cell is entering we turn on a laser, which you can see from the leaked light in the background.   Our oscillatory pressure now does nothing!  And most dramatically, note that the cell cannot exit either!   This trapping is due to Brownian ratchet forces, as we published in a letter in Biophysical Journal in 2012.   And once the laser is turned off, CO rebinds, and the cell once again can move in and out, recovering its deformability.  Click here to view the video.

Biophysics Research and Lyme Disease

May is Lyme Disease Awareness Month in the US. Lyme disease is a bacterial infection primarily transmitted by Ixodes ticks (known as deer ticks) and black-legged ticks that can cause a wide variety of both temporary and chronic symptoms. The CDC estimates that 300,000 people are diagnosed with Lyme disease in the US every year, but Lyme disease is easily misdiagnosed, so the actual number with the disease could be significantly higher. We recently spoke with Biophysical Society member Charles Wolgemuth, University of Arizona, about his research on the bacterium that causes Lyme disease.

What is the connection between your research and Lyme disease?

Many cells are able to actively move themselves through their surroundings.  In order to do this, the cells must exert forces on their environment.  One of the main questions that my research asks is how do cells produce these forces and how do these forces drive the movements of the cells through various environments.  The bacterium that causes Lyme disease, Borrelia burgdorferi, is a fascinating organism.  It is very long (for a bacterium) and is quite thin (being only 300 nm in diameter).  It is also one of the most invasive mammalian pathogens, being able to invade many tissues in the mammal that other bacteria cannot access.  It “swims” through different tissues by undulating its entire body.  We are currently working to understand what about this bacterium’s motility makes it so adept at invading mammalian tissue, a critical aspect of the disease process in Lyme disease.

MATLAB Handle Graphics

This cartoon schematic shows the basic structure of these bacteria. The cell body is green and the helical filaments (flagella) are shown in purple. There are 7-11 flagella per cell end and they are anchored to tiny rotary motors. If you were to peel the flagella away from the cell body, the cell would straighten (as shown on the left side of the schematic).

Why is your research important to those concerned about Lyme disease?

Lyme disease occurs when a person is bitten by an Ixodes scapularis tick, a species of hard tick, infected with Borrelia burgdorferi.  These ticks feed for approximately 4-7 days.  The bacteria reside in the midgut of the tick.  During feeding, the bacteria start replicating and eventually (after about 40 hours) some of the bacteria break through the lining of the tick midgut and swim to the salivary glands.  The bacteria then break into the salivary glands and are deposited in the skin of the mammal through the tick saliva.  Once in the skin, the bacteria are able to move through the mammalian body, infecting many tissues such as the skin, joints, heart, and nervous system.  In order to do all this, these bacteria must be able to maneuver through a large range of different environments.  The symptoms of Lyme disease are caused by our bodies trying to fight off the bacterial infection.  It has been shown that the motility of B. burgdorferi is imperative for the bacterium to set up infection.  Therefore, understanding how this bacterium is so invasive and how its movement allows it to set up infection and evade our immune system is crucial for understanding this disease.

How did you get into this area of research?

Since graduate school, I have been fascinated by figuring out how different cells create the shapes of their bodies and how they move from place to place.  I got into working on Lyme disease when I heard about the shape of B. burgdorferi.  It is shaped like a wave! and achieves this by wrapping helical filaments around a cylindrical body.  The physics for how this works out was perplexing to me and captivated my interest.

How long have you been working on it?

I have been working on this for nearly 15 years.  I started thinking about the problem while I was a postdoc at UC Berkeley and then wrote a grant to work on the shape of B. burgdorferi during my first academic appointment at the University of Connecticut Health Center.

Do you receive public funding for this work? If so, from what agency?

We receive funding for this research from the National Institutes of Health.

Have you had any surprise findings thus far?

One of the first really exciting findings that we had was that we were able to show that the movements of these bacteria through gelatin (such as unsweetened Jello) is very similar to the movements through our skin.  Gelatin is basically a meshwork of protein, which is also true about the dermis of our skin.  Interestingly, the pores in the gelatin are substantially smaller than the diameter of these bacteria.  Therefore, B. burgdorferi has to push apart the gelatin in order to penetrate into it.  This finding has enabled us to develop an in vitro assay for studying how these bacteria invade into different tissues.  We have a couple really new results realted to invasion and the movement through gelatin that we are very excited about.  We haven’t published them yet, so I can’t say too much more than that at this time.

What is particularly interesting about the work from the perspective of other researchers?

I can’t speak for other researchers, but I think that one of the most interesting aspects of our work is that we have been able to link the physics of how these bacteria move to aspects of the disease process.  We recently developed a mathematical model for the early stages of Lyme disease that is based on the physics that we have determined from our gelatin assays.   We were able to show using this model why the rash that accompanies Lyme disease sometimes appear as a bull’s eye pattern.  The model also explains why these rashes grow so fast (around 1 cm in diameter per day).  The ability to go from the basic physics of the movement of these bacteria to an understanding of the disease itself I think is especially exciting.

What is particularly interesting about the work from the perspective of the public?

I would have to say the same thing that I just said:  We have shown that understanding the basic science of these organisms is informative about the disease process. Fifteen years ago when I started working on this, people would ask me what I was working on, and I would tell them that I was trying to figure out how the bacterium that causes Lyme disease creates its shape.  I would often get asked then about the practical application of figuring that out: how would understanding the shape of the bacterium help fight the disease?  How should I respond to this?  At that point of time, I didn’t know what we would figure out.  But it didn’t matter to me; it was an interesting question.  The way I see it, basic knowledge is worth an infinite amount more than any specific practical application.  Knowledge can be built upon and used in ways that no one can predict ahead of time.

With that, I will conclude with one thought for the general public: We must keep funding basic scientific questions, because we never know where a specific line of inquiry may lead us.  Science is not about foreseeable practical ends; it is about discovering things we never thought we would find.