Conformational Dynamics of metallo-β-lactamase CcrA during Catalysis Investigated by Using DEER Spectroscopy

Previous crystallographic and mutagenesis studies have implicated the role of a position-conserved hairpin loop in the metallo-β-lactamases in substrate binding and catalysis. In an effort to probe the motion of that loop during catalysis, rapid-freeze-quench double electron–electron resonance (RFQ-DEER) spectroscopy was used to interrogate metallo-β-lactamase CcrA, which had a spin label at position 49 on the loop and spin labels (at positions 82, 126, or 233) 20–35 Å away from residue 49, during catalysis. At 10 ms after mixing, the DEER spectra show distance increases of 7, 10, and 13 Å between the spin label at position 49 and the spin labels at positions 82, 126, and 233, respectively. In contrast to previous hypotheses, these data suggest that the loop moves nearly 10 Å away from the metal center during catalysis and that the loop does not clamp down on the substrate during catalysis. This study demonstrates that loop motion during catalysis can be interrogated on the millisecond time scale

Site-Directed Spin Labeling EPR for Studying Membrane Proteins

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy is a rapidly expanding powerful biophysical technique to study the structural and dynamic properties of membrane proteins in a native environment. Membrane proteins are responsible for performing important functions in a wide variety of complicated biological systems that are responsible for the survival of living organisms. In this review, a brief introduction of the most popular SDSL EPR techniques and illustrations of recent applications for studying pertinent structural and dynamic properties on membrane proteins will be discussed

Probing the Secondary Structure of Membrane Peptides Using ²H‑Labeled <i>d</i>₁₀-Leucine via Site-Directed Spin-Labeling and Electron Spin Echo Envelope Modulation Spectroscopy

Previously, we reported an electron spin echo envelope modulation (ESEEM) spectroscopic approach for probing the local secondary structure of membrane proteins and peptides utilizing ²H isotopic labeling and site-directed spin-labeling (SDSL). In order to probe the secondary structure of a peptide sequence, an amino acid residue (i) side chain was ²H-labeled, such as ²H-labeled <i>d</i>₁₀-Leucine, and a cysteine residue was strategically placed at a subsequent nearby position (denoted as <i>i +</i> 1 to <i>i +</i> 4) to which a nitroxide spin label was attached. In order to fully access and demonstrate the feasibility of this new ESEEM approach with ²H-labeled <i>d</i>₁₀-Leu, four Leu residues within the AChR M2δ peptide were fully mapped out using this ESEEM method. Unique ²H-ESEEM patterns were observed with the ²H-labeled <i>d</i>₁₀-Leu for the AChR M2δ α-helical model peptide. For proteins and peptides with an α-helical secondary structure, deuterium modulation can be clearly observed for <i>i</i> ± 3 and <i>i</i> ± 4 samples, but not for <i>i</i> ± 2 samples. Also, a deuterium peak centered at the ²H Larmor frequency of each <i>i</i> ± 4 sample always had a significantly higher intensity than the corresponding <i>i</i> + 3 sample. This unique feature can be potentially used to distinguish an α-helix from a π-helix or 3₁₀-helix. Moreover, ²H modulation depth for ESEEM samples on Leu10 were significantly enhanced which was consistent with a kinked or curved structural model of the AChR M2δ peptide as suggested by previous MD simulations and NMR experiments

Utilizing Electron Spin Echo Envelope Modulation To Distinguish between the Local Secondary Structures of an α‑Helix and an Amphipathic 3₁₀-Helical Peptide

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to distinguish between the local secondary structures of an α-helix and a 3₁₀-helix. Previously, we have shown that ESEEM spectroscopy in combination with site-directed spin labeling (SDSL) and ²H-labeled amino acids (<i>i</i>) can probe the local secondary structure of α-helices, resulting in an obvious deuterium modulation pattern, where <i>i</i>+4 positions generally show larger ²H ESEEM peak intensities than <i>i</i>+3 positions. Here, we have hypothesized that due to the unique turn periodicities of an α-helix (3.6 residues per turn with a pitch of 5.4 Å) and a 3₁₀-helix (3.1 residues per turn with a pitch of 5.8–6.0 Å), the opposite deuterium modulation pattern would be observed for a 3₁₀-helix. In this study, ²H-labeled <i>d</i>₁₀-leucine (Leu) was substituted at a specific Leu residue (<i>i</i>) and a nitroxide spin label was positioned 2, 3, and 4 residues away (denoted <i>i</i>+2 to <i>i</i>+4) on an amphipathic model peptide, LRL₈. When LRL₈ is solubilized in trifluoroethanol (TFE), the peptide adopts an α-helical structure, and alternatively, forms a 3₁₀-helical secondary structure when incorporated into liposomes. Larger ²H ESEEM peaks in the FT frequency domain data were observed for the <i>i</i>+4 samples when compared to the <i>i</i>+3 samples for the α-helix whereas the opposite pattern was revealed for the 3₁₀-helix. These unique patterns provide pertinent local secondary structural information to distinguish between the α-helical and 3₁₀-helical structural motifs for the first time using this ESEEM spectroscopic approach with short data acquisition times (∼30 min) and small sample concentrations (∼100 μM) as well as providing more site-specific secondary structural information compared to other common biophysical approaches, such as CD