Max-Kade Scholarship 2015: Short-Reports

/ November 9, 2015/ News, Students

In 2015 eight students on Bachelor- or Master level got the Max-Kade Scholarship to participate in ongoing research projects between Vanderbilt University and the University of Leipzig.

Hereby their short research and travel reports are listed…


Ryan Xin (Undergraduate Student at Vanderbilt University):
Semisynthetic Preparation of Ga Fusion Proteins to Investigate Receptor Interaction
(Laboratory of Prof. Dr. Annette Beck-Sickinger & Stefan Ernicke / May-July 2015)

In my time at Leipzig University, I learned a great deal from my mentor, Stefan Ernicke, and the rest of the laboratory. Before my internship with the Max Kade Foundation, I had never worked extensively in molecular biology. After my time with the Beck-Sickinger lab, I believe that I have a solid foundation to continue molecular biology research. I worked with Stefan on discerning the function and interaction method of G-protein coupled receptors. GPCR’s are the largest family of membrane receptors, yet not much is known about the mode of interaction between the receptor and the G-protein. They are quite important in many body processes. This makes it an ideal candidate for future pharmaceutical research. The G-protein contains a GTPase domain (5 α-helices and 6 antiparallel β-sheets) that is activated by interacting with the GPCR. This GTPase domain hydrolyzes GTP and “powers” the rest of the process. During the summer, my main goal was to produce and isolate a specific peptide of interest, the C-terminal Gαi peptide. The peptide was synthesized using solid phase peptide synthesis, a very interesting technique that I have never encountered before. The peptide was joined in a fusion protein to an intein protein and a chitin-binding domain, allowing for isolation and elution of protein in a chitin-binding column. Unfortunately, the protein proved very difficult to isolate and elute. It is quite large and difficult to fold correctly. My main focus was to produce the protein in a soluble form. I used protein expression in E. coli to achieve this. I tested several different strains, including T7 Shuffle, Arctic Express, BL21, and ER 2566. These strains all had unique characteristics that allowed for efficient folding, cell vitality, and more. In addition to different strains, several chaperones and protein tags were used to enhance protein folding and elution. Chaperones included GroEL/ES and DnaK; protein tags included NusA, and MBP. I used different combinations of E.coli strains, chaperones, and protein tags to find the ideal combination for protein expression and elution. Both SDS-PAGE and western blots were used to verify the existence of protein in insoluble and soluble portions and the amount of protein in said portions. Eventually, I was able to find a suitable combination that allowed for a fair amount of the protein of interest in the soluble portion. Throughout the summer, I learned and used a variety of laboratory techniques, including DNA transformation, protein expression, protein purification/extraction, gel production, SDS-PAGE, western blots, midi-prep, and cell culturing. Overall, my experience in the Max Kade Foundation internship was overwhelmingly positive and definitely helped me advance my skills in science.

Another important part of my summer experience was living and traveling in Europe. Leipzig was the perfect place to live in the summer. It was an amazing city; it not too crowded or too busy. Leipzig had all the charm of a small European town but had the sights and sounds of a city. I particularly enjoyed the entertainment of the city center and the nearby Cospundener Sea. The most stark difference that I faced was the prevalence of biking in the city. I gained a new appreciation for bikes after using one for the entire summer. My friends and I also loved the food in Leipzig and Europe. Our favorite food by far was the Döner, an incredible-tasting and incredibly cheap delicacy only found in Europe.

I was able to travel all around Europe on the weekends. I visited Berlin, Dresden, Barcelona, Munich, the Alps, Amsterdam, Prague, Salzburg, Venice, Rome, and Florence. Throughout my trip, I gained a great appreciation for each country’s culture and customs. I was able to fulfill my dream of traveling through Europe and especially, Italy. Additionally, I made a lifelong friend while traveling with a fellow Vanderbilt student, Mike Mercier. My time in Germany was incredible and I am glad to have engrossed myself in German culture. I am truly grateful to the Max Kade Foundation for allowing me this amazing opportunity.

        


Michelle Chapmann (Bachelor Student at Vanderbilt University):
OVEREXPRESSION OF GPCR FOR STRUCTURAL STUDIES
(Laboratory of Prof. Dr. Norbert Sträter / Marco Stelter, Ulrike Obeck, Björn Kieslich / May-July 2015)

Traveling to Germany this past summer was indeed a life changing experience. Germany was a place I had always wanted to go, ever since I watched them play in the 2006 World Cup. Never had I dreamed that I would go to Germany for research and end up having one of the best experiences of my life.

In the lab, I was able to cultivate new skills as I learned about and practiced cell culture techniques under the guidance of Dr. Sträter and my two mentors, Ulrike Obeck and Mandy Geisler. Everyone in the lab was incredibly welcoming and would help me without hesitation if I was ever unsure of a particular procedure or concept. After a week of acclimation, I began the project that would define my time in the lab. Ultimately, the goal was to express a particular construct of the neuropeptide Y2 receptor (NPY2R) in insect cells with the hope that one day, the structure of that receptor would be elucidated through crystallization. Expression was achieved using various cell culture techniques, such as restriction digest, transformation of E. coli cells, bacmid DNA isolation, PCR gel electrophoresis, transfection of insect cells, viral amplification, protein test expression, SDS-PAGE, and western blotting. In the end, protein expression was achieved but not at high levels as is required for crystallization. Although these results were not the best results possible, I was also able to work with my mentor on the solubilization of a second NPY2R, one that had been successfully expressed before my arrival. While the expression of this second construct was successful, we struggled to solubilize the protein. However, by the end of my time in Leipzig, by changing conditions repeatedly, the protein was successfully solubilized, leaving my mentor with promising next steps to continue toward the ultimate goal of crystallization.

Of course, the entirety of my German experience was not spent in the lab, and I was able to dive into the culture of Leipzig. Since I arrived in Germany 3 weeks before any other Vanderbilt student, I was forced to navigate this foreign, strange world on my own. Fortunately, by a chance encounter as I walked to the lab one morning, I met a group of people that I will never forget. This group consisted of other Leipzig students, most on track for a Master’s degree. Within this group, I met people from all over the world – Costa Rica, Belgium, Norway, Spain, Italy, Ireland, Brazil, Lithuania, Netherlands, South Africa, and, of course, Germany. They showed me the city and took me to all the best restaurants and bars. We played soccer together every Tuesday night, and on weekends, we would share drinks and cook for one another, something from our home countries. They even taught me how to count to ten in German. These friends made Leipzig more than just a city in Germany that I visited for a few months. Whenever I left Germany for the weekend to travel, I always had people to come home to, and I think that’s what I’ve gained most from this experience. I realized that no matter where I went – Berlin, Munich, Prague, Athens, Bergen, or Oslo – it’s the people I met along the way that made a culture real, that made traveling worthwhile. And so, while I could go on and on about my amazing sightseeing experiences – from standing in the shadow of the Parthenon to hiking 12 hours to the top of a fjord – instead I have told you about the people I met, the friends I made.

I will forever be grateful for the experiences and memories.     


Michael Mercier (Bachelor Student at Vanderbilt University):
DYNAMICS OF Y RECEPTORS STUDIED BY SOLID-STATE NMR
(Laboratory of Prof. Dr. Daniel Huster & Dr. Ulrike Krug / May-July 2015)

My twelve-week summer research fellowship sponsored by the Max Kade Foundation was an educational and cultural experience of a lifetime. The opportunity to conduct research at a world-renowned institution with devoted faculty not only helped provide preliminary data for a promising project, but also taught me a valuable set of translatable laboratory skills. The Vanderbilt–Leipzig exchange program’s wonderful infrastructure and inter-institutional coordination made my time spent in Germany an incredibly productive, enriching opportunity that has shaped my perspective as a scientist and American student.

My work on series of experiments taught me a variety of invaluable lab techniques. First, the DNA that coded for our receptor of interest was inserted into E. coli cells, and then grown to a high density via fermentation. The receptor was then isolated from inclusion bodies, which are cytoplasmic aggregates of the Y2R sequestered by E. coli. The isolated inclusion bodies were solubilized using a detergent, and then purified using ligand affinity chromatography. The purified receptor was then reconstituted into a native lipid environment using a novel bicelle incorporation protocol. Modifying the phospholipid to detergent ratio allowed us to optimize the size and shape of the bicelles for solid-state NMR. Perhaps the most fascinating aspect of my summer research project was learning how to run the NMR machine and its software alongside expert faculty. Preliminary solid-state NMR experiments on our Y2R mutant effectively quenched signals from a variety of forms of isotopically labeled NPY, which will provide insight into future experiments aiming to characterize structural features of the receptor.

In addition to the immersive academic experience that the program provided, spending time traveling throughout Europe was the opportunity of a lifetime. My mentor, Ulrike Krug, was extremely welcoming and was a great resource for any questions I had during my stay. Annett Albrecht did a phenomenal job integrating the Vanderbilt students into life in Leipzig, and even arranged a picnic between the American and German exchange students. Leipzig is a beautiful and livable city that had many of the amenities of a large European city without the typical tourism. Outside of my time spent in Leipzig, I traveled to many different European cities including Berlin, Munich, Prague, Paris, Rome, Venice, Florence, Barcelona, and Salzburg. Leipzig is located in a central region of Europe that made travel often quick, affordable, and logistically convenient. The amount of great friends I made through these travel experiences makes my time in Europe even more special. This unique opportunity provided by the Max Kade Foundation has not only made me a more confident scientist–it has taught me how to be a more effective communicator, how to value other people’s perspectives, and given me new memories and international friends that will last a lifetime. I cannot thank the administrative members at Vanderbilt University, Universität Leipzig, and the Max Kade Foundation for affording me this opportunity–it has truly been a blessing!    


Han Noo Ri Lee (Undergraduate Student at Vanderbilt University):
DYNAMICS OF Y RECEPTORS STUDIED BY SOLID-STATE NMR
(Laboratory of Prof. Dr. Ulf Wagner & Elisabeth Jäger / May-July 2015)

My research project at Leipzig University involved investigating the role of G-Protein coupled receptor family C group 6 member A (GPRC6A) and its functional similarity to calcium sensing receptors (CaSR). Not much is known about the recently discovered GPRC6A, but it is suspected that it plays a similar role to CaSR, as it was hypothesized from in vivo experiment in rats. Various techniques were incorporated in order to further investigate and identify its roles as part of a structure of the inflammasome; techniques that I have learned include cultivating cells and maintaining the cell culture, staining the receptors, transfection, microscopy, and protein assays including ELISA and Western Blot. I cultivated THP1 cell line, which is a monocytic cell line derived from a leukemia patient, in order to study the receptors. Isolated monocytes from subjects were sometimes incorporated into the study as well, as they tend to yield better results than cultivated cells. In the first half of the project, I mainly focused on staining the receptors and looking them under the electron microscope. Staining technique involved using fluorescent dye coupled antibodies to localize the receptors on cells. Staining was repeated multiple times, each time incorporating different cells or under different conditions such as by controlling the level of calcium in the medium the cells were cultivated in. Despite multiple tries, the results were largely unproductive due to various errors including lack of cell growth.

In order to carry out a better-controlled experiment, human embryonic kidney (HEK) cells, which naturally lack CaSR, were transfected with the CaSR gene and was then incubated in calcium for different lengths of time. For a positive control, a control vector was transfected into HEK cells. The specific transfection technique that was incorporated was lipofection, in which the newly inserted genes are surrounded by lipid molecules and inserted into cells by endocytosis and incorporated into the DNA.

The second part of the project involved protein assays such as ELISA and Western Blot. ELISA was performed to identify the levels of IL1-beta secretion in cells incubated in calcium for different lengths of time, and Western Blot was performed in order to identify the protein levels in cells incubated in calcium for different lengths of time. The results from protein assays were largely coherent, as it was possible to identify the presence and different levels of GPRC6A and CaSR in cells incubated in calcium for different lengths of time.

Over the span of 10 weeks, I visited eight different cities in six different countries, including Germany. Travelling around Europe was one of the items on my bucket list, and being able to achieve a little bit of my wish felt incredible. I was very much grateful for the opportunity. Travelling around so many countries by myself was an adventurous journey. It was truly an eye-opening and inspirational experience that made me realize the wonders of the world and the need to broaden my perspective. Looking back, I really believe that my experience over the summer – travelling across countries and encountering difficult situations by myself – has allowed me to mature as an individual and adopt a different attitude towards life, as I am now able to view life in a much more relaxed and broader sense.        


Lisa Pankewitz (Bachelor Student at Leipzig University):
STRUCTURE AND DYNAMICS OF THE Y2 RECEPTOR
(Prof. Dr. Jens Meiler, Brain Bender / July-September 2015)

In January 2015 I was very excited when I received the invitation for a research internship at the Vanderbilt University. In the ten weeks of my internship I got to be part of Prof. Dr. Jens Meiler’s lab and had the great opportunity to work with Dr. Soumya Ganguly as my supervisor on determining the structure of the membrane protein DsbB by using NMR (Nuclear magnetic resonance spectroscopy). He introduced me to expression, purification and labeling of the protein DsbB with paramagnetic lanthanoid ion tags to measure special restraints of the protein with NMR. The resulting restraints can be used to refine the three dimensional structure of DsbB with the modeling software ROSETTA. These techniques could be transferred to figure out detailed three dimensional structures of more complex membrane proteins. All things considered, I not only had the opportunity to gain a lot of knowledge about the whole process of expressing a protein to the determination of its structural features but also improve my spoken English. Moreover to the scientific aspects of the work in the wetlab and computational lab I enjoyed talking to the members of Jens Meilers lab to learn about their lifestyle, cultural interests, and personal history.

Beside the scientific part of the internship I utilized the opportunity to get to know Nashville and to visit a few places around Nashville. As a group of exchange students we went e.g., to the Nashville Zoo, Atlanta and New York City. By watching a baseball game or going to the concerts live on the Greens in Nashville we got to know the American way of Life and could get in touch with the American culture.

All in all, the trip to Nashville was not only an excellent possibility to improve my scientific skills but also to connect to different people from different places in the world and to make some new friends. I would highly recommend this amazing experience to everybody!        


Minh Ganther (Master Student at Leipzig University):
MECHANISM OF NPY RECEPTOR MEDIATED G PROTEIN ACTIVATION
(Laboratory of Prof. Dr. Heidi Hamm, Ali I. Kaya / July-September 2015)

One major research topic of Prof Heidi Hamm’s group is the investigation of G protein conformational changes, mediated by the activation of G protein coupled receptors (GPCR). GPCRs represent a major fraction of pharmacological drug targets and therefore their interactions with G proteins are of great significance and interest.

My lab work included the construction and expression of a G protein mutant suitable for electron paramagnetic spin resonance (EPR) experiments. The protein mutant was constructed using mutagenesis PCR and was expressed in E. coli bacteria. The cultures were harvested and the protein was purified using several chromatography techniques. After purification, protein functionality was verified to ensure that the mutations do not affect protein function. The protein was labelled with MTSSL, introducing paramagnetism, at two amino acid residues. The labelled protein was joined with the human Y2 receptor to investigate the conformational changes when bound to the receptor using EPR and double-electron-electron resonance experiments. The human Y2 receptor was obtained by harvesting membranes from human embryonal kidney cells which stably express the receptor.

In my opinion, one of the greatest thing about America is the general air of friendliness, helpfulness and hospitality emanating from most people. Whereas in Germany people tend to keep to themselves and mind their own business, American people are extraordinarily helpful and kind. They are also quite straight-forward and hard-working people. We did a lot of trips in and around Tennessee and even went to New York City for a weekend. I highly recommend visiting the numerous national parks America has to offer, the nature there is beautiful and stunning and there are a lot of outdoor activities like hiking and swimming. But also visiting the great metropolises was quite a new experience comparing the smaller cities in Germany.


Tobias Haensch (Master Student at Leipzig University):
ELUCIDATION OF THE STRUCTURAL BASIS OF ARRESTIN-3-MEDIATE SCAFFOLDING OF ASK1-MKK4/7-JNK1/2/3 SIGNALING CASCADES
(Laboratory of Prof. Dr. Vsevolod V. Gurevich, Qiuyan Chen / July – September 2015)

In the summer holidays 2015 I had the great possibility to work in the lab of Prof. Dr. Vsevolod V. Gurevich. I got the chance to participate in a quiet new and interesting topic about arrestins. Arrestins interact with one of the most important receptor class in the human body, the G-protein coupled receptors. After their interaction with a ligand like hormones or neurotransmitters these receptors are phosphorylated by a G-protein receptor kinase allowing high affinity binding of arrestins to the G-protein coupled receptors. Besides different functions like desensitization and internalization, arrestins play an important role in signaling, too, by working as a scaffold for the protein kinases of the MAP kinase cascade. In my project I investigated the binding of one known binding partner of arrestin-3, the JNK3, whose activation is implicated with neurodegenerative diseases like Alzheimer’s, Parkinson’s and Huntington’s disease. I examined the binding of a JNK3 mutant to an arrestin-3 fragment for crystallization trays and therefore the elucidation of the interaction site of arrestin-3 with JNK3. I expressed and purified my own proteins, used different binding assays and methods like western blot in this practical course.

In the evenings we explored Nashville with a trolley tour, visited different restaurants, ate typical American food and enjoyed free concerts and lots of live music with Tennessee whiskey. On the weekends we did lots of trips to get familiar with the American culture and the surroundings. We were hiking in the Great Smoky Mountains, stayed in different cities like Memphis, Knoxville and Chattanooga in Tennessee, Atlanta in Georgia, Birmingham in Alabama and lots more. One very special trip was the flight to New York exploring the metropolis, drinking cocktails in a roof-top bar and watching a Broadway musical. We also watched typical American games like baseball and football together. I savored every moment in the US.        


Isabel Kratochvil (Master Student at Leipzig University):
ION MOBILITY-MASS SPECTROMETRY FOR THE STRUCTURAL RESOLUTION OF BIOMOLECULES
(Laboratory of Prof. Dr. John A McLean, Ewa Jurneczko / July – September 2015)

In the summer 2015 I had the great opportunity to visit the lab of Prof. Dr. John McLean at the Vanderbilt University for 10 weeks. Prof. Dr. John McLean and his group work with ion mobility-mass spectrometry were the species are separated based on their charge, mass and sizes/shape what makes it possible to differentiate between molecules with the same molecular weight and the same charge state but different shape. That is not possible with single mass spectrometry.

During my stay I investigated different conformations of proteins that occur due to the charge state of this protein. This proteins were investigated under native and nonnative conditions. So I learned during my visit a lot about the use of ion mobility-mass spectrometry for the structural resolution of biomolecules and the different ways to analyze the received data especially on basis of the structural resolution of biomolecules. Furthermore I enjoyed the nice atmosphere in the lab and the interesting talks and discussions with my colleagues.

Beside the scientific work I had the chance to visit a few places in and around Nashville. For example we did trips to the Great Smoky Mountains, Knoxville and Chattanooga, as well as to Atlanta and Memphis. Also we watched a Baseball game: Nashville sounds vs. Omaha Storm Chasers. Though nobody of us knew the concrete rules it was a great time due to the amazing atmosphere in the stadium.

All in all I learned during my stay in Nashville not just about the ion mobility-mass spectrometry and the way of working in US labs but also about the American way of life that includes both long working hours and kindliness.