Alana Gudinas: Single-Molecule Mechanics of Ion-Driven Protein Folding

Image

Over the summer of 2024, I had the pleasure of conducting research at the University of Freiburg for three months through Stanford’s Graduate Research and Internship program (GRIP). During my visit I worked with Dr. Bizan Balzer and Prof. Thorsten Hugel in the Single Molecule Lab, housed within the Institute of Physical Chemistry. Single molecule spectroscopy (SMS) uses precise, physics-based techniques to probe the behavior of individual molecules like proteins. Whereas typical biological experiments measure the average behavior of all proteins in a sample, SMS resolves the complex processes that a single protein undergoes at a time. Single molecule experiments enable discovery of the rich molecular-scale physics that gives rise to critical biological processes. 

At Stanford, I investigate how metal ions induce structure in a calcium-binding protein, the repeats-in-toxins (RTX) domain. Ion-binding proteins like RTX perform a wide range of biological processes, but the fundamental principles underlying how specific metal ions trigger protein folding remain elusive. I use a range of experimental techniques to investigate how different metals alter RTX structures, with the aim of eventually designing proteins to capture specific metals from the environment. GRIP provided a unique opportunity to continue this work in a research lab that leads the field in state-of-the-art single molecule studies. 

Research project 

Prior to Freiburg, I established that the calcium-binding RTX domain promiscuously binds to other group II metal ions (magnesium, strontium, and barium). However, it remained unclear whether RTX adopted its fully folded structure or remained partially unfolded in the presence of other metals. I hypothesized that metals of different sizes stabilize partially folded RTX structures and sought to resolve these structural differences. Powerful single molecule techniques like atomic force microscopy (AFM) and single molecule Förster Resonance Energy Transfer (sm-FRET) give insight into the mechanics and dynamics of conformational changes in proteins. 

The AFM Lab, led by Dr. Balzer, recently established a single molecule force spectroscopy (SMFS) technique to apply tension to individual molecules at arbitrary angles. Typically, SMFS experiments apply tension only in the vertical direction; pulling on proteins at other angles enables measuring different chemical bonds along the protein chain. During my time in Freiburg I learned novel protocols for conducting angle-dependent SMFS experiments and established that RTX is compatible with this experimental setup. I have the opportunity continue this collaborative project using a new bio-AFM at Stanford with angle-dependent capabilities.

The Hugel Lab specializes in single molecule FRET, a technique that resolves both protein dynamics and structural heterogeneity. In the Hugel Lab, I worked with students on two custom optical setups: a Total Internal Reflection Fluorescence (TIRF) microscope, where proteins are tethered to surface, and a confocal-based single molecule spectroscopy microscope, where proteins freely diffuse through a volume. With these experiments, we observed that RTX adopts a partially compacted conformation upon incubation with magnesium and barium, with more structural heterogeneity than with calcium. These exciting data are the first demonstration on the single molecule scale that RTX can partially fold with metals beyond calcium. Our results both establish RTX as an excellent sample for sm-FRET and yield new insights into how ion size constrains protein folding. 

The first round of sm-FRET experiments has sparked a longer-term collaborative project. I will return to Freiburg during this academic year to bring new samples for sm-FRET experiments, while a colleague from the Hugel Lab continues measurements with other metal ions. We expect to publish our initial findings this year, with future publications focusing on new ion-binding proteins. Working with the Hugel Lab was an enormous pleasure; I felt extremely lucky to have joined such a welcoming, enthusiastic, and curious group of researchers. I am overwhelmingly grateful to learn from a lab with such specific experimental expertise and look forward to our continued collaboration. 

Outside the lab 

Researching in Germany offered unique opportunities to meet with the larger European scientific community. In the week prior to my arrival in Freiburg, I attended the Bioinspired Materials Gordon Research Conference (GRC) in Les Diablerets, Switzerland, a fantastically picturesque conference site situated amidst the Alps east of Lake Geneva. The Bioinspired Materials GRC was my first chance to spend almost an entire week with a small group of people in my immediate scientific field: designing artificial materials with natural building blocks. Because all researchers attended the same talks and poster sessions, we had lively discussions during every meal and social hour (including awe-inspiring hikes through the Alps!). I was additionally fortunate to attend the IUPAC MACRO World Polymer Congress in Warwick, UK in July. During both meetings, I was exposed to cutting-edge research from some of the top scientists in my field and connected with researchers throughout Europe. 

Outside of research, I enjoyed the lush, beautiful forests surrounding Freiburg and the sprawling city center around the Freiburg Münster, the main cathedral (which began construction in 1200)! Almost every morning I stopped at one the (multiple) bakeries near my apartment to buy a pastry for breakfast and loved to end my day with a walk through the forest. Freiburg has a special culture: residents are exceptionally environmentally conscious and friendly, and the atypically sunny weather means you can find people sitting outside of cafes chatting at every hour of the day. On the weekends I frequented the large daily farmer’s market at the cathedral (Münster Markt) and took the Deutsche Bahn to hike in the Black Forest, visit surrounding lakes, or spend a few days in Munich. Some of the food I’ll miss the most is Swabian-style potato salad, Kaiserschmarrn (a Bavarian dessert whose name means “emperor’s mess”), and a simple fresh-baked pretzel. Prevailing wisdom about Germany’s ubiquitously excellent bread proved to be true. 

My time in Freiburg was incredibly special and I’m grateful for the opportunity to have been immersed in German culture. I plan to carry many lessons home with me and look forward to my return!