52 research outputs found
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 <sup>2</sup>H‑Labeled <i>d</i><sub>10</sub>-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 <sup>2</sup>H 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 <sup>2</sup>H-labeled, such as <sup>2</sup>H-labeled <i>d</i><sub>10</sub>-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 <sup>2</sup>H-labeled <i>d</i><sub>10</sub>-Leu, four Leu residues within the AChR M2δ peptide
were fully mapped out using this ESEEM method. Unique <sup>2</sup>H-ESEEM patterns were observed with the <sup>2</sup>H-labeled <i>d</i><sub>10</sub>-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 <sup>2</sup>H 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 3<sub>10</sub>-helix. Moreover, <sup>2</sup>H 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 3<sub>10</sub>-Helical Peptide
Electron
spin echo envelope modulation (ESEEM) spectroscopy was
used to distinguish between the local secondary structures of an α-helix
and a 3<sub>10</sub>-helix. Previously, we have shown that ESEEM spectroscopy
in combination with site-directed spin labeling (SDSL) and <sup>2</sup>H-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 <sup>2</sup>H 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 3<sub>10</sub>-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 3<sub>10</sub>-helix. In this study, <sup>2</sup>H-labeled <i>d</i><sub>10</sub>-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,
LRL<sub>8</sub>. When LRL<sub>8</sub> is solubilized in trifluoroethanol
(TFE), the peptide adopts an α-helical structure, and alternatively,
forms a 3<sub>10</sub>-helical secondary structure when incorporated
into liposomes. Larger <sup>2</sup>H 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 3<sub>10</sub>-helix.
These unique patterns provide pertinent local secondary structural
information to distinguish between the α-helical and 3<sub>10</sub>-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
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