Analysis of side chain rotational restrictions of membrane-embedded proteins by spin-label ESR spectroscopy (original) (raw)
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Biochemistry, 2011
In this work, electron paramagnetic resonance (EPR) spectroscopy and X-ray crystallography were used to examine the origins of EPR line shapes from spin-labels at the protein−lipid interface on the β-barrel membrane protein BtuB. Two atomic-resolution structures were obtained for the methanethiosulfonate spin-label derivatized to cysteines on the membrane-facing surface of BtuB. At one of these sites, position 156, the label side chain resides in a pocket formed by neighboring residues; however, it extends from the protein surface and yields a single-component EPR spectrum in the crystal that results primarily from fast rotation about the fourth and fifth bonds linking the spin-label to the protein backbone. In lipid bilayers, site 156 yields a multicomponent spectrum resulting from different rotameric states of the labeled side chain. Moreover, changes in the lipid environment, such as variations in bilayer thickness, modulate the EPR spectrum by modulating label rotamer populations. At a second site, position 371, the labeled side chain interacts with a pocket on the protein surface, leading to a highly immobilized single-component EPR spectrum that is not sensitive to hydrocarbon thickness. This spectrum is similar to that seen at other sites that are deep in the hydrocarbon, such as position 170. This work indicates that the rotameric states of spin-labels on exposed hydrocarbon sites are sensitive to the environment at the protein−hydrocarbon interface, and that this environment may modulate weak interactions between the labeled side chain and the protein surface. In the case of BtuB, lipid acyl chain packing is not symmetric around the β-barrel, and EPR spectra from labeled hydrocarbon-facing sites in BtuB may reflect this asymmetry. In addition to facilitating the interpretation of EPR spectra of membrane proteins, these results have important implications for the use of long-range distance restraints in protein structure refinement that are obtained from spin-labels. S pin-labels have proven to be powerful tools for probing protein structure and dynamics. In the EPR-based technique site-directed spin-labeling (SDSL), a spin-labeled side chain is used to probe the local structure and dynamics at the labeled site and to provide distance restraints between pairs of labeled side chains. 1−5 This approach is particularly valuable in the case of large protein complexes or membrane proteins, where other approaches may have limited utility. Spin-labels have also been used extensively to refine structures using high-resolution nuclear magnetic resonance, where paramagnetic enhancements of nuclear relaxation provide long-range distance restraints between nuclei and spin-labeled side chains. 6,7 Although there are several approaches that can be used to covalently attach spin-labels to proteins, the ease of attachment of labels based upon cysteine chemistry has made the methanethiosulfonate-derivatized cysteine side chain, R1, the most popular spin-labeled side chain for protein labeling (Figure 1a). An important aspect of interpreting EPR spectra and longrange distances from spin-labeled sites is knowledge of the configuration of the label side chain. The configuration of the R1 side chain at labeled sites has been determined experimentally by examining the modes of motion that
Structural studies on membrane proteins using non-linear spin label EPR spectroscopy
Cellular & molecular biology letters, 2002
Non-linear electron spin resonance (EPR) techniques suitable for measuring proximity relationships in membranes are reviewed. These were developed during the past decade in order to measure changes sensitively in the spin-lattice relaxation time (T1) of nitroxyl spin labels covalently attached to membrane lipids or proteins. In combination with paramagnetic quenching agents and double spin-labelling, the methods were further developed for distance measurements. Selected examples are given to illustrate different methods, and types of data obtained for both integral and peripheral membrane proteins.
Peptide–Membrane Interactions by Spin-Labeling EPR
Methods in Enzymology, 2015
Site-directed spin labeling (SDSL) in combination with Electron Paramagnetic Resonance (EPR) spectroscopy is a well-established method that has recently grown in popularity as an experimental technique, with multiple applications in protein and peptide science. The growth is driven by development of labeling strategies, as well as by considerable technical advances in the field, that are paralleled by an increased availability of EPR instrumentation. While the method requires an introduction of a paramagnetic probe at a well-defined position in a peptide sequence, it has been shown to be minimally destructive to the peptide structure and energetics of the peptide-membrane interactions. In this chapter, we describe basic approaches for using SDSL EPR spectroscopy to study interactions between small peptides and biological membranes or membrane mimetic systems. We focus on experimental approaches to quantify peptide-membrane binding, topology of bound peptides, and characterize peptide aggregation. Sample preparation protocols including spinlabeling methods and preparation of membrane mimetic systems are also described.
Journal of Peptide Science, 2011
EPR spectroscopy is a technique that specifically detects unpaired electrons. EPR-sensitive reporter groups (spin labels or spin probes) can be introduced into biological systems via site-directed spin-labeling (SDSL). The basic strategy of SDSL involves the introduction of a paramagnetic group at a selected protein site. This is usually accomplished by cysteine-substitution mutagenesis, followed by covalent modification of the unique sulfydryl group with a selective reagent bearing a nitroxide radical. In this review we briefly describe the theoretical principles of this well-established approach and illustrate how we successfully applied it to investigate structural transitions in both human pancreatic lipase (HPL), a protein with a well-defined α/β hydrolase fold, and the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N TAIL ) upon addition of ligands and/or protein partners. In both cases, SDSL EPR spectroscopy allowed us to document protein conformational changes at the residue level. The studies herein summarized show that this approach is not only particularly well-suited to study IDPs that inherently escape atomistic description by X-ray crystallography but also provides dynamic information on structural transitions occurring within well-characterized structured proteins for which X-ray crystallography can only provide snapshots of the initial and final stages.
Motional Restrictions of Membrane Proteins: A Site-Directed Spin Labeling Study
Biophysical Journal, 2006
Site-directed mutagenesis was used to produce 27 single cysteine mutants of bacteriophage M13 major coat protein spanning the whole primary sequence of the protein. Single-cysteine mutants were labeled with nitroxide spin labels and incorporated into phospholipid bilayers with increasing acyl chain length. The SDSL is combined with ESR and CD spectroscopy. CD spectroscopy provided information about the overall protein conformation in different mismatching lipids. The spin label ESR spectra were analyzed in terms of a new spectral simulation approach based on hybrid evolutionary optimization and solution condensation. This method gives the residue-level free rotational space (i.e., the effective space within which the spin label can wobble) and the diffusion constant of the spin label attached to the protein. The results suggest that the coat protein has a large structural flexibility, which facilitates a stable protein-to-membrane association in lipid bilayers with various degrees of hydrophobic mismatch.
EPR spectroscopy is a technique that specifically detects unpaired electrons. EPR-sensitive reporter groups (spin labels or spin probes) can be introduced into biological systems via site-directed spin-labeling (SDSL). The basic strategy of SDSL involves the introduction of a paramagnetic group at a selected protein site. This is usually accomplished by cysteine-substitution mutagenesis, followed by covalent modification of the unique sulfydryl group with a selective reagent bearing a nitroxide radical. In this review we briefly describe the theoretical principles of this well-established approach and illustrate how we successfully applied it to investigate structural transitions in both human pancreatic lipase (HPL), a protein with a well-defined α/β hydrolase fold, and the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N TAIL ) upon addition of ligands and/or protein partners. In both cases, SDSL EPR spectroscopy allowed us to document protein conformational changes at the residue level. The studies herein summarized show that this approach is not only particularly well-suited to study IDPs that inherently escape atomistic description by X-ray crystallography but also provides dynamic information on structural transitions occurring within well-characterized structured proteins for which X-ray crystallography can only provide snapshots of the initial and final stages.
Biophysica
Membrane proteins are essential for the survival of living organisms. They are involved in important biological functions including transportation of ions and molecules across the cell membrane and triggering the signaling pathways. They are targets of more than half of the modern medical drugs. Despite their biological significance, information about the structural dynamics of membrane proteins is lagging when compared to that of globular proteins. The major challenges with these systems are low expression yields and lack of appropriate solubilizing medium required for biophysical techniques. Electron paramagnetic resonance (EPR) spectroscopy coupled with site directed spin labeling (SDSL) is a rapidly growing powerful biophysical technique that can be used to obtain pertinent structural and dynamic information on membrane proteins. In this brief review, we will focus on the overview of the widely used EPR approaches and their emerging applications to answer structural and conforma...