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1. Efficient localization of a native metal ion within a protein by Cu ²⁺ -based EPR distance measurements

   https://doi.org/10.1039/c8cp07143h  

   Gamble Jarvi, Austin ; Cunningham, Timothy F. ; Saxena, Sunil ( May 2019 , Physical Chemistry Chemical Physics)

   Electron paramagnetic resonance (EPR) based distance measurements have been exploited to measure protein–protein docking, protein–DNA interactions, substrate binding and metal coordination sites. Here, we use EPR to locate a native paramagnetic metal binding site in a protein with less than 2 Å resolution. We employ a rigid Cu 2+ binding motif, the double histidine (dHis) motif, in conjunction with double electron electron resonance (DEER) spectroscopy. Specifically, we utilize a multilateration approach to elucidate the native Cu 2+ binding site in the immunoglobulin binding domain of protein G. Notably, multilateration performed with the dHis motif required only the minimum number of four distance constraints, whereas comparable studies using flexible nitroxide-based spin labels require many more for similar precision. This methodology demonstrates a significant increase in the efficiency of structural determinations via EPR distance measurements using the dHis motif.
2. Dynamic RNA Fitness Landscapes of a Group I Ribozyme during Changes to the Experimental Environment

   https://doi.org/10.1093/molbev/msab373  

   Peri, Gianluca ; Gibard, Clémentine ; Shults, Nicholas H ; Crossin, Kent ; Hayden, Eric J ( March 2022 , Molecular Biology and Evolution)

   Zhang, Jianzhi (Ed.)

   Abstract Fitness landscapes of protein and RNA molecules can be studied experimentally using high-throughput techniques to measure the functional effects of numerous combinations of mutations. The rugged topography of these molecular fitness landscapes is important for understanding and predicting natural and experimental evolution. Mutational effects are also dependent upon environmental conditions, but the effects of environmental changes on fitness landscapes remains poorly understood. Here, we investigate the changes to the fitness landscape of a catalytic RNA molecule while changing a single environmental variable that is critical for RNA structure and function. Using high-throughput sequencing of in vitro selections, we mapped a fitness landscape of the Azoarcus group I ribozyme under eight different concentrations of magnesium ions (1–48 mM MgCl2). The data revealed the magnesium dependence of 16,384 mutational neighbors, and from this, we investigated the magnesium induced changes to the topography of the fitness landscape. The results showed that increasing magnesium concentration improved the relative fitness of sequences at higher mutational distances while also reducing the ruggedness of the mutational trajectories on the landscape. As a result, as magnesium concentration was increased, simulated populations evolved toward higher fitness faster. Curve-fitting of the magnesium dependence of individual ribozymes demonstrated that deep sequencingmore »of in vitro reactions can be used to evaluate the structural stability of thousands of sequences in parallel. Overall, the results highlight how environmental changes that stabilize structures can also alter the ruggedness of fitness landscapes and alter evolutionary processes.« less
3. Efficient sampling of molecular orientations for Cu( ii )-based DEER on protein labels

   https://doi.org/10.1039/D3CP00404J  

   Hasanbasri, Zikri ; Moriglioni, Nicholas A. ; Saxena, Sunil ( July 2023 , Physical Chemistry Chemical Physics)

   Combining rigid Cu( ii ) labels and pulsed-EPR techniques enables distance constraint measurements that are incisive probes of protein structure and dynamics. However, the labels can lead to a dipolar signal that is biased by the relative orientation of the two spins, which is typically unknown a priori in a bilabeled protein. This effect, dubbed orientational selectivity, becomes a bottleneck in measuring distances. This phenomenon also applies to other pulsed-EPR techniques that probe electron–nucleus interactions. In this work, we dissect orientational selectivity by generating an in silico sample of Cu( ii )-labeled proteins to evaluate pulse excitation in the context of double electron–electron resonance (DEER) at Q-band frequencies. This approach enables the observation of the contribution of each protein orientation to the dipolar signal, which provides direct insights into optimizing acquisition schemes to mitigate orientational effects. Furthermore, we incorporate the excitation profile of realistic pulses to identify the excited spins. With this method, we show that rectangular pulses, despite their imperfect inversion capability, can sample similar spin orientations as other sophisticated pulses with the same bandwidth. Additionally, we reveal that the efficiency of exciting spin-pairs in DEER depends on the frequency offset of two pulses used in the experiment andmore »the relative orientation of the two spins. Therefore, we systematically examine the frequency offset of the two pulses used in this double resonance experiment to determine the optimal frequency offset for optimal distance measurements. This procedure leads to a protocol where two measurements are sufficient to acquire orientational-independent DEER at Q-band. Notably, this procedure is feasible with any commercial pulsed-EPR spectrometer. Furthermore, we experimentally validate the computational results using DEER experiments on two different proteins. Finally, we show that increasing the amplitude of the rectangular pulse can increase the efficiency of DEER experiments by almost threefold. Overall, this work provides an attractive new approach for analyzing pulsed-EPR spectroscopy to obtain microscopic nuances that cannot be easily discerned from analytical or numerical calculations.« less
4. The CHD Protein, Kismet, is Important for the Recycling of Synaptic Vesicles during Endocytosis

   https://doi.org/10.1038/s41598-019-55900-6  

   Latcheva, Nina K. ; Delaney, Taylor L. ; Viveiros, Jennifer M. ; Smith, Rachel A. ; Bernard, Kelsey M. ; Harsin, Benjamin ; Marenda, Daniel R. ; Liebl, Faith L. W. ( December 2019 , Scientific Reports)

   Chromatin remodeling proteins of the chromodomain DNA-binding protein family, CHD7 and CHD8, mediate early neurodevelopmental events including neural migration and differentiation. As such, mutations in either protein can lead to neurodevelopmental disorders. How chromatin remodeling proteins influence the activity of mature synapses, however, is relatively unexplored. A critical feature of mature neurons is well-regulated endocytosis, which is vital for synaptic function to recycle membrane and synaptic proteins enabling the continued release of synaptic vesicles. Here we show that Kismet, theDrosophilahomolog of CHD7 and CHD8, regulates endocytosis. Kismet positively influenced transcript levels and bound todap160andendophilin Btranscription start sites and promoters in whole nervous systems and influenced the synaptic localization of Dynamin/Shibire. In addition,kismetmutants exhibit reduced VGLUT, a synaptic vesicle marker, at stimulated but not resting synapses and reduced levels of synaptic Rab11. Endocytosis is restored atkismetmutant synapses by pharmacologically inhibiting the function of histone deacetyltransferases (HDACs). These data suggest that HDAC activity may oppose Kismet to promote synaptic vesicle endocytosis. A deeper understanding of how CHD proteins regulate the function of mature neurons will help better understand neurodevelopmental disorders.
5. Orientation and dynamics of Cu ²⁺ based DNA labels from force field parameterized MD elucidates the relationship between EPR distance constraints and DNA backbone distances

   https://doi.org/10.1039/d0cp05016d  

   Ghosh, Shreya ; Casto, Joshua ; Bogetti, Xiaowei ; Arora, Charu ; Wang, Junmei ; Saxena, Sunil ( December 2020 , Physical Chemistry Chemical Physics)

   Pulsed electron paramagnetic resonance (EPR) based distance measurements using the recently developed Cu 2+ -DPA label present a promising strategy for measuring DNA backbone distance constraints. Herein we develop force field parameters for Cu 2+ -DPA in order to understand the features of this label at an atomic level. We perform molecular dynamics (MD) simulations using the force field parameters of Cu 2+ -DPA on four different DNA duplexes. The distance between the Cu 2+ centers, extracted from the 2 μs MD trajectories, agrees well with the experimental distance for all the duplexes. Further analyses of the trajectory provide insight into the orientation of the Cu 2+ -DPA inside the duplex that leads to such agreement with experiments. The MD results also illustrate the ability of the Cu 2+ -DPA to report on the DNA backbone distance constraints. Furthermore, measurement of fluctuations of individual residues showed that the flexibility of Cu 2+ -DPA in a DNA depends on the position of the label in the duplex, and a 2 μs MD simulation is not sufficient to fully capture the experimental distribution in some cases. Finally, the MD trajectories were utilized to understand the key aspects of the double electron electronmore »resonance (DEER) results. The lack of orientational selectivity effects of the Cu 2+ -DPA at Q-band frequency is rationalized in terms of fluctuations in the Cu 2+ coordination environment and rotameric fluctuations of the label linker. Overall, a combination of EPR and MD simulations based on the Cu 2+ -DPA labelling strategy can contribute towards understanding changes in DNA backbone conformations during protein–DNA interactions.« less