Category Archives: Biochemistry and Biophysics

Exploring a protein’s turf with TIRF

Investigating Otoferlin

Otoferlin is a protein required for hearing. Mutations in its gene sequence have been linked to hereditary deafness, affecting 360 million people globally, including 32 million children. Recently graduated PhD candidate Nicole Hams has spent the last few years working to characterize the activity of Otoferlin using TIRF microscopy. There are approximately 20,000 protein-coding genes in humans, and many of these proteins are integral to processes occurring in cells at all times. Proteins are encoded by genes, which are comprised of DNA; when mutations in the gene sequence occur, diseases can arise. Mutations in DNA that give rise to disease are the focus of critical biomedical research. “If DNA is the frame of the car, proteins are the engine,” explains Nicole. Studying proteins can provide insight into how diseases begin and progress, with the strategic design of therapies to treat disease founded on our understanding of protein structure and function.

Studying proteins

Proteins are difficult to study because they’re so small: at an average size of ~2 nanometers (0.000000002 meters!), specific tools are required for visualization. Enter TIRF. Total Internal Reflection Fluorescence is a form of microscopy enabling scientists like Nicole to observe proteins tagged with a fluorescent marker. One reason TIRF is so useful is that it permits visualization of samples at the single molecule level. Fluorescently-tagged proteins light up as bright dots against a dark background, indicating that you have your protein.

Another reason why proteins are hard to study is that in many cases, parts of the protein are not soluble in water (especially if part of the protein is embedded in the fatty cell membrane). Trying to purify protein out of a membrane is extremely challenging. Often, it’s more feasible for scientists to study smaller, soluble fragments of the larger protein. Targeted studies using truncated, soluble portions of protein offer valuable information about protein function, but they don’t tell the whole story. “Working with a portion of the protein gives great insight into binding or interaction partners, but some information about the function of the whole protein is lost when you study fragments.” By studying the whole protein, Nicole explains, “we can offer insight into mechanisms that lead to deafness as a result of mutations.”

Challenges and rewards of research

Nicole cites being the first person in her lab to pursue single molecule studies as a meaningful achievement in her graduate career. She became immersed in tinkering with the new TIRF instrument, learning from the ground up how to develop new experiments. Working with cells containing Otoferlin, in a process known as tissue culture, required Nicole to be in lab at unusual hours, often for long periods of time, to make sure that the cells wouldn’t die. “The cells do not wait on you,” she explains, adding, “even if they’re ready at 3am.” Sometimes Nicole worked nights in order to get time on the TIRF. “If you love it, it’s not a sacrifice.”

Why grad school?

As an undergraduate student studying Agricultural Biochemistry at the University of Missouri, Nicole worked in a soybean lab investigating nitrogen fixation, and knew she wanted to pursue research further. She had worked in a lab work since high school, but didn’t realize it was a path she could pursue, instead convinced that she wanted to go to medical school. Nicole’s mom encouraged her to pursue research, because she knew that it was something she enjoyed, and her undergraduate advisor (who completed his post-doc at OSU) suggested that she apply to OSU. She feels lucky to have found an advisor like Colin Johnson, and stresses the importance of finding a mentor who is personally vested in their graduate student’s success.

Besides lab work…

In addition to research, Nicole has been actively involved in outreach to the community, serving as Educational Chair of the local NAACP Chapter. Following completion of her PhD, Nicole intends to continue giving back to the community, by establishing a scholarship program for underrepresented students. Nicole remembers a time when she was told and believed that she wasn’t good enough, and while she was able to overcome this discouraging dialogue, she has observed that many students do not find the necessary support to pursue higher education. Her goal is to reach students who don’t realize they have potential, and provide them with resources for success.

Tune in on December 3rd  at 7pm to 88.7 KBVR Corvallis or stream the show live right here to hear more about Nicole’s journey through graduate school!

Thanks for reading!

You can download Nicole’s iTunes Podcast Episode!

Earlier in the show we discussed current events, specifically how the tax bill moving through the House and Senate impact students. Please see our references and sources for more information.

Motor proteins—and people—can change directionality

It took three years of adventures after college—including stints as a ski instructor, barista and a commercial chemist—before Andrew Popchock knew that he wanted to return to the lab to pursue a PhD at OSU’s Department of Biochemistry and Biophysics.

Two microtubules slide across each other by the walking of motor proteins sandwiched between them

Andrew’s research takes place at Dr. Weihong Qiu’s Single-Molecule Biophysics Laboratory and focuses on kinesin-14s—motor proteins found in eukaryotic cells. These motor proteins in cells travel along microtubules to create and maintain the mitotic spindle, which are macromolecular structures that are responsible for chromosome segregation during cell division.

By using an imaging technique called TIRF microscopy, a team of researchers from Dr. Qiu’s lab discovered that a kinesin-14 found in fungus cells called KlpA can change direction along its cytoskeleton tracks. KlpA is the first motor protein of its kind that researchers have discovered that demonstrates this type of bidirectional movement. The results of their study were recently published in Nature Communications.

Total Internal Reflection Fluorscence (TIRF) microscopy image of two microtubules sliding across each other

The motor protein that Andrew studies could be important in helping researchers understand cancer growth. This could have implications for drug treatment therapy, potentially guiding the creation of motor protein-based molecular devices for more controlled drug delivery in cancer treatments.

 

Andrew on the Oregon Coast

Growing up, Andrew was interested in physics and biology, but it wasn’t until he worked in a lab under the direction of a graduate student at Washington State University that he began to consider graduate studies. While working as a chemist in Idaho, he realized that he quickly reached the limit of his creative capacity and that returning to a laboratory as a graduate student at OSU would help him continue to develop his skills as a researcher.

To learn more about Andrew’s research and his path to graduate school, tune in to hear our conversation on Sunday, May 14th at 7:00 pm on 88.7 FM KBVR Corvallis or listen live online.

Elucidating protein structure with crystals

Kelsey in the lab pipetting one of her many buffers!

Proteins are the workhorse molecules of the cell, contributing to diverse processes such as eyesight, food breakdown, and disabling of pathogens. Although cells cannot function without helper proteins, they’re so small that it’s impossible to view them without the aid of special tools. Proteins are encoded by RNA, and RNA is encoded by DNA; when DNA is mutated, the downstream structure of the protein can be impacted. When proteins become dysfunctional as part of disease, understanding how and why they behave differently can lead to the development of a therapy. In Andy Karplus’ lab in the Department of Biochemistry & Biophysics, PhD candidate Kelsey Kean uses a technique known as protein x-ray crystallography to study the relationship between protein structure and function.

Protein crystals. On the left, each blade making up this cluster is an individual crystal that needs to be separated before we can use them.

Protein diffraction. An individual crystal is placed in front of an x-ray beam and we collect the diffraction resulting from the x-ray hitting each atom in the protein crystal . Using the position and darkness of each spot (along with some other information), we can figure out where each atom in the crystal was originally positioned.

An electron density map. After collecting and processing our diffraction images, we get an electron density map (blue)- this shows us where all the electrons for each atom in the protein are- and this guides us in building in the atomic coordinates (yellow) for each part of the protein. It’s like a puzzle!

Crystallization of protein involves many steps, each of which presents its own unique challenges. A very pure protein sample is required to form an ordered crystal lattice, and hundreds of different buffer solutions are tested to find the ideal crystallization conditions. Sometimes crystals can take weeks, months, or a year to grow: it all depends on the protein. Once a crystal is obtained, Kelsey ships it to the synchrotron at Lawrence Berkeley National Laboratory, which provides a source of ultra powerful x-ray light beams. Exposure of the protein crystal to x-ray light results in a diffraction pattern, which is caused by the x-ray light diffracting off of all the atoms in the crystal. A map of electron density is generated from the diffraction pattern, and then the electron density map is used to determine where the atoms are located in the protein, like a complex puzzle. X-ray protein crystallography is really amazing because it allows you to visualize proteins at the atomic level!

In addition to her lab work, Kelsey is extensively involved in teaching and STEM outreach. For the past 3 summers, she has organized a week-long summer biochemistry camp through STEM Academy, with the help of a group of biochemistry graduate students. Kelsey has also been involved in Discovering the Scientist Within, a program providing 150 middle school girls with the opportunity to perform science experiments, including isolation of strawberry DNA and working with mutant zebrafish.

Kelsey completed her undergraduate degree in biochemistry with a minor in math at the University of Tulsa, where she was also a Division I athlete in rowing. She attributes her work ethic and time management skills to her involvement in Division I athletics, which required a significant commitment of time and focus outside of lab and coursework. During one summer when she wasn’t busy with competitive rowing, she performed experiments related to protein crystallography at the Hauptman-Woodward Medical Research Institute associated with the University at Buffalo.

Kelsey knew she wanted to pursue science from an early age. She grew up surrounded by scientists: her mom is a biochemist and her dad is a software engineer! She recalls playing with Nalgene squirt bottles as a kid, and participated in the Science Olympiad in middle school, where she engineered a Rube Goldberg machine. She cites early exposure to science from her family as one reason why she feels strongly about STEM outreach to students who might not otherwise receive encouragement or support. In the future, Kelsey would like to teach at a primarily undergraduate institution.

Please join us this Sunday, April 23rd on KBVR Corvallis 88.7FM at 7 pm PST  to hear much more about x-ray protein crystallography, STEM outreach, and to hear an awesome song of Kelsey’s choosing! You can also stream this episode live at www.kbvr.com/listen.

Kean on Science!

This evening on our special pre-Inspiration Dissemination interview, we had a wonderful conversation with Kelsey Kean, a PhD candidate in the department of Biochemistry & Biophysics. While discussing the Tsoo King Lecture series, we stumbled into a myriad of tangential topics including CRISPR/Cas9 and Peter Walter’s discovery machine. As promised, we’re including some links to more information here. Click away for some awesome reading, watching, and listening!
TsooKingFlyer

Tsoo King Lectures with Peter Walter; Vilcek Award winner on the unfolded protein response

CRISPR-Cas9, revolutionary tool for genome editing.

 

Looking For the Link Between Centromeres and Cancer

DNA, the “building blocks of life”, can be bent and broken. While it is the source code for every creature on the earth, DNA is also the source of some of the most difficult diseases that plague humanity. Tonight at 7PM PST, Steve Friedman joins us from the department of Biochemistry and Biophysics to discuss characterizing centromeres of a filamentous fungi called Neurospora crassa. Centromeres, the part of the chromosome that is targeted by proteins that aid in cell division, are studied to understand how genetic mutations and resulting abnormalities in cells can lead to genetic disease and cancer.

Flasks containing strains of Neurospora crassa

Flasks containing strains of Neurospora crassa

Fungi serve as a model organism for the study of centromeres in Steve’s work because their genetic code is more complex than the yeast (Saccharomyces cerevisiae) that have been used in older studies, but still easier to study and understand than the complicated human genome.

Understanding how the human genetic code controls the production of proteins that are implicated in diseases like cancer, and how these proteins relate to centromeres that are crucial parts of a natural and healthy process of cell division, is the long term goal of such research.

To learn more about Steve and his work, tune in at 88.7 KBVR FM, or stream the show live!

microscopy images of GFP/RFP tagged centromere proteins (taken in Galya Orr's Lab at PNNL)

Microscopy images of GFP/RFP tagged centromere proteins (taken in Galya Orr’s Lab at PNNL).

Steve enjoys some time away from the lab

Steve enjoys some time away from the lab