UC Santa Barbara

Transmembrane proteins are essential biomolecules that reside in cell membranes and play important roles in biological functions including ion transport, signal transductions, and enzymatic reactions. These proteins are surrounded by a highly heterogeneous environment. They have hydrophilic domains that are exposed to water molecules and solutes in solution and hydrophobic membrane-spanning domains that are embedded within a lipophilic environment. This unique combination of environment around the transmembrane protein is not only required to preserve a proper folding of its structure but can also take part in regulating its functional activity. Due to the complexity of the environment, biophysicists are still developing approaches to elucidate the role of different factors in modulating the function of transmembrane proteins.

This dissertation aims to investigate the influence of environmental factors on the structure and function of transmembrane proteins by studies of Proteorhodopsin (PR), a light-activated proton pump that originated from marine bacteria. PR is an excellent model for studying transmembrane protein function as its proton transport function can be directly assessed by optical absorbance spectroscopic techniques. PR also shares the same seven-helical transmembrane (7TM) structure with a large family of human receptors, which makes the structural knowledge learned from PR studies potentially transferable to other physiologically important transmembrane proteins (e.g. G protein-coupled receptors). On the other hand, PR has excellent robustness and is found to be stable in a variety of environments. This allows it to be studied in environments not limited to native-like bacteria membrane but also other biomimetic platforms with different factors that can be independently controlled. PR also has its significance in protein engineering applications. The proton motive force generated by PR through its vectorial proton transport can be utilized as an approach to harvest solar energy. All these together make the studies of PR appeal to objectives of both fundamental biophysical understanding and bioengineering applications on transmembrane proteins.

To achieve our objectives, the functions of PR under the influences of different modulators are examined in biomimetic environments including synthetic liposomes, lipid nanodiscs, and synthetic host materials. With controls over the surrounding lipid composition and the oligomeric distribution of PR between its monomeric and oligomeric forms, we identify that protein-protein interaction (i.e. the formation of oligomers) is a key factor to determine the proton transport kinetic of PR. Nevertheless, in the lipid membrane environment, the protein-protein interaction shows no impact on the protonation behavior of PR’s embedded ionizable amino acid D97, a switch that controls the population of active PR with proton transport capability. Instead, we find that the electrostatic environment around PR (e.g. the ion type or concentration in the buffer and the net charge of lipid headgroups) can significantly modulate the pKa of this embedded D97 (pKaD97), and in turn affects the population of active PR. Most importantly, the same concept is found to be transferable to PR reconstituted in synthetic host materials with increased thermal and mechanical stability for bioengineering applications.

To further understand the correlation between the structure and function of transmembrane proteins, complementary magnetic resonance spectroscopic tools were used to acquire the structure of PR with different functional outcomes. This dissertation interrogates the structural rearrangement of PR associated with the pKaD97 modulation by different electrostatic environments. Local conformation changes at the third intracellular loop of PR are uncovered by using electron paramagnetic resonance (EPR) analysis and Overhauser dynamic nuclear polarization (ODNP) relaxometry through a change of local environment experienced by a site-directly introduced spin label on the loop. Moreover, by conducting dynamic nuclear polarization (DNP)-enhanced solid-state nuclear magnetic resonance (ssNMR) measurements on PR either site-specifically or uniformly labeled with NMR active nuclei, we identify the residues that rearranged while PR is in different electrostatic environments. On the other hand, to have a structural-based understanding of the functional impact of oligomerization, this dissertation also seeks to develop a novel approach to map out the structural rearrangement of PR activation by high field EPR spectroscopy with Gadolinium-based spin labels. The study of PR provides an opportunity of directly correlating the measured structural dynamics with the kinetics of proton transport function measured by its transient absorbance change.

Altogether, PR studies presented here elucidate the roles of oligomerization in modulating its proton transport function and underscore the importance of the biomimetic environment on affecting the function of transmembrane proteins. For bioengineering and biomedical applications relying on transmembrane protein systems, this dissertation offers a guideline for optimizing functions of proteins that are controlled by a similar mechanism as PR. Furthermore, the change of structural properties observed here by magnetic resonance tools from PR with different functional outcomes adds value to biophysical understandings on the relationship between structure and function of transmembrane proteins that share a similar structure as PR.