From Distance Constraints to Multi-domain Protein Architecture

BPJ_110_10.c1.inddThe architecture of multi-domain proteins is crucial for their function. However, the determination of such structures in native-like environments to high resolution remains difficult. Electron Paramagnetic Resonance (EPR) spectroscopy is a highly sensitive technique that can be used to study samples in near native-like conditions using small sample volumes. Proteins are site-specifically labelled with nitroxides and Pulsed Electron-electron Double Resonance (PELDOR) is used to measure the interactions between pairs of nitroxide spin labels. These interactions can be interpreted as a distribution of distances (in the range 2 to 8 nm) between the two nitroxide labels bound to the protein, which includes information on the structure and dynamics of both the biomolecule and the nitroxide label. It is possible to utilize these distance distributions as distance constraints to assist computational structure refinement in order to deduce the architecture of multi-domain proteins.

The cover image shows the crystal structure of the three POTRA domains of anaOmp85 from the cyanobacterium Anabaena sp. PCC 7120 (PDB code: 3MC8). The nitroxide spin-labels used in our study have been added to the original crystal structure, each site is represented by a different coloured collection of rotamers. These were derived from molecular dynamics simulations performed with YASARA and depict the intrinsic flexibility of the label used. An experimental data set, so-called PELDOR time trace, which oscillates with a frequency dependent on the interaction and distance between the nitroxide labels, is shown in the lower left corner. The long lasting and pronounced modulations of that signal are remarkable for a biological system. Distance distributions are extracted from the time domain signals using a mathematical approach called Tikhonov regularization. The distribution corresponding to the trace shown is displayed in the upper right corner.

In our study (Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy. Biophysical Journal 110/10) we describe the application of these distance distributions as distance constraints in structure refinement unravelling a restricted conformational flexibility of the POTRA domains compared to unrestricted molecular dynamics simulations. The structures we derive also show a change in the architecture of the domains compared to the crystal structure, which may be a result of the conditions under which crystallization was achieved.

The combination of distance restraints from PELDOR with structural refinement has the potential to be applied in many different protein systems and therefore is a valuable tool in the armoury of protein structure refinement techniques.

– Reza Dastvan, Eva-Maria Brouwer, Denise Schuetz, Oliver Mirus, Enrico Schleiff, Thomas Prisner

A Participant’s Perspective: STEM on the Hill

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Daniel Richman, Ellen Weiss, and Catherine Zander in front of the U.S. Capitol.

Every year, the Science-Engineering-Technology Working Group (SETWG), a network of researchers, educators, and business professionals, gather in Washington DC to meet with congressional representatives. The objective is to discuss the impact of federal funding on their work and communities, and to emphasize that the federal funding of research and development is a long-term investment in our country’s future.  Because of my background in political science and long standing interest in policy, I wrote to the Biophysical society asking how I could be more involved in their advocacy efforts. As a fantastic stroke of luck, they were looking for interested society members to visit congress for STEM on the Hill day on April 12 and 13th, 2016.

Despite being involved in university and national groups and organizations, I had no active experience with federal politics. I hoped that by taking part in STEM on the Hill day, that not only would I get to be part of delivering an important message, but I would also gain understanding of the workings of congressional offices and staffers. In the first day at Washington, SETWG held an orientation session in the Capitol Visitor’s Center to prepare us all for meeting with the Senate and Congressional offices. The orientation was run by congressional staffers and advisors, and representatives from many organizations including The American Association for the Advancement of Science (AAAS), The Institute of Electrical and Electronics Engineers (IEEE), The Association of Public and Land-grant Universities (APLU), and The Alliance for Science & Technology Research in America (ASTRA). They presented on topics including the President’s 2017 budget request, the organization of committees, bills, and appropriations, to the best method of communicating with the legislators and staff.  This gave us a working understanding of how the federal government allocates funds for different organizations.

The next day, prepared with the advice and practice pitches from the orientation, Ellen Weiss, the Director of Policy and Outreach for the Biophysical Society, guided Dr. Richman from the Georgia Institute of Technology, and me to meet with our legislators. Ellen had arranged for us to meet with six legislators, three from Georgia and three from Alabama. In 2015, the University of Alabama at Birmingham, where I am a postdoctoral researcher, received $243,263,382 from NIH grants. As Birmingham’s largest employer (23,000 jobs reported in 2013), federal funding is vital for the city’s economy. Unsurprisingly, because of the huge impact that federal funding has on Alabama’s economy, Senator Shelby, our first office visit, is a champion of the sustained support of the NIH.  I, coincidentally, work in the Richard C. and Annette N. Shelby Interdisciplinary Biomedical Research Building at UAB, for which Senator Shelby was instrumental in securing the funds. In most of the offices we visited, we found the same supportive attitude. In the offices that do not have the long history of NIH or NSF support the Legislative Correspondents and Assistants were curious to hear about the applications of our research, and our stories about work that was lost or delayed because of breaks in or lack of funding. In each office we visited, we asked for the support of, at minimum 34.5 billion for the NIH, 8 billion for the NSF, and 5.672 billion for the DOE Office of Science; we left behind materials that broke down the economic impact upon the state. After our meetings, we wrote thank you letters to the offices encouraging them to contact us whenever we can be of assistance.

As Tip O’Neill stated, “All politics is local.” What I understand more fully after taking part in STEM on the Hill day is that regardless of political affiliations, the men and women who run for public office have a desire to help their constituents, but as their constituents, we also have an active role to play. We met a wide array of people waiting to meet with their representatives. From Georgia based poultry farmers and food distributors, to bus companies and airport architects, they are all meeting with their legislators to have their needs and problems heard and addressed.  The scientific community as a whole must adopt this hands on approach. With that, I encourage you all to invite your local and state representatives to your labs and facilities. We must start sharing the importance of our work, not just at academic conferences, but also with our representatives. This is the best way to show that the federal funds given to scientific and technological disciplines are a beneficial investment in our professional communities and in the future of our nation.

–Catherine Zander, University of Alabama at Birmingham

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.

 

Interview with Dr. Raz Jelinek

BPJ_110_9.c1.inddBJ: How did you compose the cover image for Biophysical Journal 110/9?

RJ: The image is designed to highlight the thrust of our paper; the C-dot-phospholipid conjugates are seamlessly embedded within the lipid bilayer “carpet” representing the cell membrane.  The fluorescence of the protruding C-dots (green spheres) , constituting the core property of the C-dots and the primary analytical tool, is represented by the “hallows” around the spheres.

BJ: How does this image reflect your scientific research?

RJ: We are deeply interested in cellular membranes, and have been active over the years trying to decipher fundamental structural and functional properties of membranes, primarily as related to their lipid bilayer scaffolding.   The image reflects the research presented in the paper—development of a new analytical platform for studying membranes and membrane processes.

BJ: Can you please provide a few real-world examples of your research?

RJ: An important research program in my lab focuses on the relationship between membranes and amyloid diseases, particularly Alzheimer’s disease.  While amyloid diseases are incurable, there is a growing evidence for the intimate roles of cellular membranes in the onset and pathogenicity of the diseases.  We aim to elucidate these putative relationships and assess the relevance of membrane interactions toward development of therapeutic treatments.

BJ: How does your research apply to those who are not working in your specific field?

RJ: We believe that introduction of new tools for imaging membranes and analysis of membrane dynamics could aid research efforts of scientists in other disciplines, including biologists, biochemists, and bioengineers.

BJ: Do you have a website where our readers can view your recent research?

RJ: http://www.bgu.ac.il/~razj/

– Sukhendu Nandi, Ravit Malishev, Susanta Bhunia, Sofiya Kolusheva, Jurgen Jopp, Raz Jelinek

BPS Summer Program Alumni Spotlight: Manuel Castro

Mac_Castro_PhotoWe recently had a chance to catch up with Manuel “Mac” Castro from BPS’s Summer Program in Biophysics Class of 2015. Mac is currently finishing up his BS in Biochemistry, with a focus in Medicinal Chemistry, at Arizona State University (ASU) where he works as a Research Assistant in the Van Horn Lab. Starting in the fall of 2016, Mac has accepted an offer to attend Vanderbilt University through the Interdisciplinary Graduate Program (IGP), a PhD program intended to help students transition from undergraduate to graduate style biomedical research. Additionally, Mac has also recently been awarded a prestigious NSF Graduate Research Fellowship, which recognizes and supports outstanding graduate students in NSF-supported science, technology, engineering, and mathematics disciplines.

What is your research focus?

My current research focus in Dr. Van Horn’s lab can be generalized as structural and functional studies of Transient Receptor of Potential (TRP) ion channels, transmembrane proteins that are involved in a plethora of signaling pathways in larger organisms (metazoans). Namely, I work on TRPM8, 1 of 27 TRP channels expressed in humans, which has great implications in pain and cancer therapy and offers an overall better understanding of neurology and physiology. To study these ion channels, I produce the proteins in bacteria, purify them, and probe them using solution –state nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy techniques.

When and how did you first become interested in this type of research?

To be honest, I was thrown into this research as a college sophomore and had no idea what the bigger picture was. However, throughout my time in this lab, I have come to fall in love with both the problems that our research is trying to address and the various techniques we use to address them.  Now that my time in this lab is nearing its end, I can say that I enjoy this research because neurology has always been one of my favorite topics in science. The Van Horn lab twists the traditional approaches that neuroscience generally employs by taking a more biochemical and biophysical approach towards understanding how these proteins work both inside  and outside of cells.

What was the most important thing you learned or took away from the summer program that helped you get where you are at now?

The summer program was a fantastic review of my physical chemistry, biology, and biochemistry classes. It really solidified what topics from my classes were going to be central to my future as a biomedical scientist. However, I think the most immediately beneficial part of the summer program was working with Dr. Matt Redinbo in his crystallography lab. I learned a series of new techniques while I was there and also solidified my last necessary letter of recommendation for graduate school.  Without the experience and connections I attained that summer, I may not have made it where I wanted to be.

What was your favorite thing about the summer program?

My favorite thing about the summer program was how much the people who sponsored the program tried to make the interns feel like they were being taken care of. The hospitality that was offered was unlike anything I had experienced and the excursions that they planned for us made for some of the best memories of my life. I also made some good friends who will likely be so for the rest of my life, and that type of experience is hard to replace.

Have mentors played a role in your success? If so, how?

I have had two impactful mentors during my undergraduate years, and each of them offered different insights in my life. Wade Van Horn helped me turn my education around and would consistently challenge me to do better than I was already doing. He would never accept mediocrity from me and his guidance kept my eyes on the prize (being a successful scientist). David Capco encouraged me to channel my desire to help other students into becoming a legitimate mentor for freshman undergraduates and middle/high-school students alike.  My mentoring experience helped me understand the importance of being a mentor to others in a similar way that Drs. Van Horn and Capco were to me. It is safe to say that the person I was before my interactions with them was a very different person than I am today.

What have been some of your toughest challenges so far in advancing your career?

The toughest challenge in my career had to be my grades. In my early college career, I was a terrible student and had no motivation to excel in my classes. This left me at a huge disadvantage after my sophomore year because my GPA hit a trough of cumulative 2.8. Since then, I have had to get basically all As in every class in every semester in order to prove to graduate admissions committees that the person I was in the beginning of college was not who I became during the middle and at the end. I ended up applying to graduate schools with a cumulative GPA of 3.25, which was the best I could get to. However, I plan to make up for my poor decisions by hitting the ground running in graduate school and not letting my prior habits from early undergraduate translate into the future of my career.

What advice would you give for current undergraduates interested in pursuing a higher degree?

Do it. That is my advice. Don’t think too hard about it and just do it. Although, since I have not started graduate school yet, I would argue that my opinion isn’t the most important. Another piece of advice I could give would be that if you are serious about pursuing a higher degree, look into PhD programs and not masters programs. They offer you more training, a more qualified degree, and they will give you great financial support. Don’t sell yourself short and shoot for the stars!

Neurons, Brains, and Biophysics at the U.S.’s largest science fair

15,500 pipe cleaners.

160 showings of The Human Brain:  Images to Atoms

6000 individuals at the BPS exhibit booth.

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BPS Council member Bob Nakamoto, University of Virginia, helps elementary school students with their neuron models.

In a nutshell, these numbers wrap of the Biophysical Society’s participation in the 4th Annual USA Science and Engineering Festival held in Washington, DC, April 15-17, 2016.

In three short days, Biophysical staff and member volunteers gave over 6000 individuals a glimpse of the power and beauty of biophysics research through a short planetarium style movie showcasing images of neurons and proteins in the brain, as well as a hands on activity– making neuron models out of pipe cleaners.  Pretty amazing numbers considering the Society’s booth was one of over 1000 exhibitors at the Festival.

With a booth at the entrance of one of the exhibit floors (Yes, there was more than one at the festival!), the Biophysical Society’s exhibit was bumping throughout the entire event.  On Friday, school groups made up the majority of attendees, while on the weekend, the attendees were primarily families.  An estimated 345,000 people attended the free event, and it was very heartening to see the interest in science from the diverse crowd.

The Society would like to thank its member volunteers for showing up, being amazing educators, and sharing their passion for science with the next generation.  The Society would  also like to thank its partners in bringing the Dome to the event:  Wah Chiu and Matt Doherty, from Baylor College of Medicine, and the Houston Museum of Science for the use of their equipment.

Want to make a neuron model out of pipe cleaners at home or at a local outreach event?  Here are the instructions on How To Make a Neuron Model.

 

 

The Secrets of Fertile Gamete

April 24-30, 2016 has been designated National Infertility Awareness Week by RESOLVE, the National Infertility Association.  Basic research plays an important role in our understanding of infertility.  Here, BPS member Polina Lishko, Department of Molecular and Cell Biology
University of California, Berkeley,  shares information about her research on male infertility, what makes human sperm fertile, and the path that brought her to use her biophysics background in the field of reproductive biology.  

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Infertility constitutes a global problem, with male infertility contributing to half of all cases. About 80% of male infertility cases are considered idiopathic, which means we don’t know the cause. The only available treatment in such cases is limited to assisted reproductive technologies. This huge gap in our understanding of etiology of male infertility is partially attributed to our insufficient knowledge of physiology of human sperm cells. Frankly, we still do not fully understand sophisticated machinery that regulates human sperm motility and their fertilizing potential. Sperm cells or spermatozoa as we call them, are diverse and species specific not only in their morphology or overall appearance, but also in their choice of molecular mechanisms that drive fertility. Essentially, what works for mouse or sea urchin sperm, may not necessarily work for human spermatozoa. This is why our lab and other reproductive biology laboratories embark on the comprehensive study to define what makes human sperm fertile. Our ultimate goal is either decrease sperm fertility by developing novel contraceptives or increase sperm fertilizing potential to help infertile couples to conceive. But the first step in this task- is to define what makes human sperm fertile and what impacts this ability. Once one knows all major regulatory units of human sperm cell, one can develop a comprehensive diagnostic test that could help men test their fertility potential. This knowledge also will be helpful to develop novel contraceptives for both men and women, and ultimately, this knowledge will be vital to help infertile couples.

My scientific journey into reproductive biology was not straightforward. I was trained as biophysicist and neuroscientist and has spent significant portion of the graduate and postdoctoral research studying how ion channels regulate excitability of neurons, as well as studying molecular mechanisms of vision and pain. However, ion channels are important regulatory switches in many different cell types, and they have long been suspected to play huge role in physiology of gametes. The area of sperm ion channel physiology was relatively terra incognita in comparison to neuroscience and muscle physiology, and I was very excited to go there and explore. Dr. Stanley Meizel, well-known reproductive biologist, once called sperm cell a neuron with a tail, and this is indeed quite smart comparison. While sperm cell are not known to generate action potential, they resemble sensory neurons in their ability to react to various physiological cues provided by female reproductive tract and use these cues to successfully navigate in their search for the egg.

So, in late 2007, while being a postdoctoral fellow at UCSF, I decided to begin my unforgettable and fun journey into reproductive biology. This path was not without a certain risks. While reproductive biology is very important and exciting field of research, paradoxically, it is one of the least funded one. For example, NICHD funding rate is way below 10%, and very few extramural funding opportunities currently exist for students and postdoctoral researchers who decide to devote their research to reproductive biology. Myself, I have been struggling for several years to secure NIH funding, and while my lab is currently fortunate for being supported by two NIH grants: NIGMS (R01) and NICHD (R21), as well as by private funding from Pew Charitable Trust (thanks to the neuroscience portion of my research) and Alfred P. Sloan Foundation, we need to secure more research support, as the work we do requires significant investments.

various mammalian sperm

Fig. 1. Examples of sperm morphological diversity. Spermatozoa of different species are shown with cytoplasmic droplets indicated by yellow arrows. Shown are: human (Hs; Homo sapiens), mouse (Mm; Mus musculus), rat (Rn; Rattus norvegicus), rhesus macaque (Mmu; Macaca mulatta), boar (Sd; sus scrofadomesticus), and bull (Bt; Bos taurus) sperm cells.

Why should we study reproductive biology and what is there for me, may you ask? Reproductive biology field holds many surprises for everyone, and has unlimited translational potential. For example, while working on identification of non-genomic progesterone receptor of human sperm, we have uncovered a novel signaling pathway that links steroid hormones with endogenous cannabinoids. And this bioactive lipid signaling is not sperm specific, but likely plays role in various tissues. Why is this important? Steroid hormones, such as progesterone, estrogen, testosterone or other steroids control fundamental organism function by regulating gene expression via their cognate nuclear receptors. However, fast and potent non-nuclear membrane signaling can also be initiated by steroids. Such phenomena as sperm activation, egg maturation and progesterone–induced analgesia are operated via a non-nuclear pathway, the key molecular regulators of which remained unknown. After five years of search for sperm membrane progesterone receptor we have finally revealed its molecular identity- monoacylglycerol lipase ABHD2. This protein is highly expressed in sperm, possesses progesterone-stimulated hydrolase activity and directly regulate sperm principal calcium channel that is crucial for male fertility. But what we actually found, is an unconventional pathway that links steroid hormones with the levels of bioactive lipids, such as endocannabinoid monoacylglycerol and arachidonic acid. ABHD2 is a member of large ABHD family of lipid enzymes, and it is possible that other members of the same family could be influenced by other steroids in similar manner. Of course, this hypothesis requires rigorous testing and we hope that it will be done in the near future. ABHD2 is not sperm specific: it is highly expressed in testis, microglia and lungs, and all these tissues are known to be regulated by endocannabinoid monoacylglycerol- the bioactive lipid that is eliminated by ABHD2 in progesterone-dependent manner.  Therefore, targeting ABHD2 in neurons or lungs may provide a new target for novel pharmacological approaches to improve pain management, as well as treat respiratory diseases. ABHD2 can also serve as a biomarker for male fertility and may help clinicians understand why some couples are unable to conceive naturally.

The link between steroids and endocannabinoids is just one of the many surprises that gametes hold in their treasure box. These cells are more sophisticated than what we think of them and will reward greatly those researchers who dare to wonder in the unexplored fields of reproductive biology.

–Polina Lishko