Methodology and Fundamentals
EPR method development and spin labeling are at the heart of the Saxena group’s research efforts. We develop EPR methods to measure biomolecular structure, large-scale conformational changes, and site-specific dynamics. Much of this work is backed by use of first principle theory and computations. From a biochemical perspective, we have recently pioneered the Cu(II)-based EPR spin labeling method, which is an incisive probe of biophysical information that is inaccessible to traditional EPR spin labels. Examples include an enhancement in resolution to conformational dynamics, elucidation of interactions between biomolecules, and the measurement of fast site-specific dynamics. In addition, we are developing foundational methodology to couple Cu(II) labeling based EPR information with molecular dynamics simulations.
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Advancements in Distance Measurements
EPR has emerged as an important method for the measurement of structure-function relationships in proteins and nucleic acids. In this arena our group has pioneered the development of EPR methods to measure the dipolar interaction between two electron spins. In early work, we established the conceptual basis for double quantum and double resonance-based methods for the case of Cu(II) spins, and for experiments involving orthogonal labeling schemes (primarily Cu(II) and nitroxides). In addition, we created experimental and analysis protocols to interpret experimental data in terms of distances without the complications from electron-nuclear interactions. More recently, we have developed computational means to advantageously relate Cu(II) spin label positioning to elucidate protein subunit orientations to add an additional dimension to Cu(II) distance measurements. We continue to incorporate new isotopic substitution schemes such as deuteration and pulse shaping to enhance signal sensitivity and distance range of the Cu(II) method. The close coupling between development of conceptual methodology to experimentation is in our recent work where we addressed the limitations of pulses used in EPR. In this effort we leveraged a in silico simulation technique to learn about molecular orientations that are excited by a given pulse. This effort lead to the realization of an experimental scheme by which excitation of only 10% spins was sufficient to adequately measure the distance. As a result, we can acquire data up to six times faster than previously possible.
Site-Specific Protein Dynamics
Protein dynamics are central in every biophysical process. By understanding protein dynamics, we then can understand and describe the transitions between functional states, protein oligomerization, ligand recognition, etc. For years, EPR line shape analysis on nitroxide labeled proteins proved useful in measuring site-specific protein dynamics. However, this technique has largely been limited to solvent exposed α-helices and flexible loops. The flexibility of nitroxide labels causes them to sterically clash with neighboring residues when on β-sheet sites. Additionally, the cysteine dependence with nitroxide labeling can be limiting if the protein contains other functional cysteines.
In light of these limitations, our group developed methodology to measure site-specific dynamics on a protein using the site directed Cu(II) labeling method developed in our group. We show that Cu(II) EPR lineshapes at room temperature are gorgeous signatures of site-specific reorientational dynamics. The rigid binding of Cu(II) to native amino acid residues alleviates uncertainties from sidechain motions and better reflects backbone dynamics. Secondly, Cu(II) having much larger anisotropy than other common labels, i.e., a much broader spectrum, means that observed line shape changes due to dynamics are amplified. Consequently, Cu(II)-based dynamics measurements are more sensitive to smaller changes in dynamics and to faster dynamics than other traditional labels. Through this method, our group direction now looks to understand functional dynamics in enzymes and protein-DNA interactions.
In-Cell EPR
EPR-based distance constraints have been used in many publications to answer many important biological questions including ligand-binding, protein-protein or protein-DNA interactions, conformational changes. These systems of interest were primarily observed under test tube conditions but require complementary experiments in cells. Specifically, cellular environment has been shown to affect protein behavior due to the crowdedness within the cellular space. The crowding effects lead to changes in binding affinities, protein stability, and function. Unfortunately, the crowded environment is not easily replicated in test tubes. Therefore, performing in-cell measurements are crucial for validating test tube observations.
Despite the need for in-cell experiments, obtaining EPR results are challenging due to other factors in the cellular environment. First, the cytosolic environment is highly reductive. As a result, spin-labels that provide EPR signals can be reduced, leading to loss of signal. Additionally, the reducing environment can lead to detachment between the spin-label and the protein. Finally, highly crowded environments surrounding the spin-label lead to large amounts of protons that can amplify the loss of EPR signal for distance measurements.
As a response, our lab has explored the time requirement for a protein to be fully equilibrated inside Xenopus laevis eggs and how we can extend the lifetime of our EPR signal of commercial spin-labels for in-cell distance measurements. This understanding allows for in-cell distance measurements to be more accessible using commercially available resources. Additionally, working with the Benoit Driesschaert laboratory (WVU), we have developed a new trityl-based spin-label that provides high signal intensity even at temperatures of 150 K and is sterically protected in cell. Furthermore, the spin-label can be incorporated into proteins with high efficiency without being cleaved in cell. Consequently, this new spin-label allows for in-cell distance measurements with high sensitivity compared to commercial spin-labels. By further developing the methodology for in-cell measurements, the field of structural biology can further expand in a biologically relevant context.
Collaborator: Benoit Driesschaert