Protein Conformational Dynamics upon Association with the Surfaces of Lipid Membranes and Engineered Nanoparticles: Insights from Electron Paramagnetic Resonance Spectroscopy

Abstract

Detailed study of conformational rearrangements and dynamics of proteins is central to our understanding of their physiological functions and the loss of function. This review outlines the applications of the electron paramagnetic resonance (EPR) technique to study the structural aspects of proteins transitioning from a solution environment to the states in which they are associated with the surfaces of biological membranes or engineered nanoobjects. In the former case these structural transitions generally underlie functional protein states. The latter case is mostly relevant to the application of protein immobilization in biotechnological industries, developing methods for protein purification, etc. Therefore, evaluating the stability of the protein functional state is particularly important. EPR spectroscopy in the form of continuous-wave EPR or pulse EPR distance measurements in conjunction with protein spin labeling provides highly versatile and sensitive tools to characterize the changes in protein local dynamics as well as large conformational rearrangements. The technique can be widely utilized in studies of both protein-membrane and engineered nanoobject-protein complexes.

Keywords: EPR spectroscopy; lipid membranes; protein conformation; protein-surface association; surface-immobilized protein.

Figure 1

Spin labeling of proteins. (A–C) show nitroxide spin labels widely used for study of proteins by EPR spectroscopy. (D) emphasizes the residue 65 in T4 lysozyme spin-labeled with MTSSL. (PDB accession code 3LZM). Spin-label side chains were generated using the MMM program (Jeschke and Polyhach) [42].

Figure 2

Continuous-wave (CW) electron paramagnetic resonance (EPR) spectra of the nitroxide spin label. The spectra correspond to different ranges of spin-label and protein motion. The coarse parameters, ΔH, and splitting of the outer hyperfine lines are shown in the bottom spectrum. Both parameters depend on the extent of spin label immobilization. The increase in the outer line splitting is due to the contribution from the A_zz hyperfine tensor component. Thus, less mobile spin labels lead to broader spectrum.

Figure 3

Double electron–electron resonance (DEER) distance measurements in doubly spin-labeled protein molecules. (A) The method is illustrated with T4 lysozyme (PDB accession code 3LZM) doubly spin-labeled at positions 65C and 135C. The spin label side-chains were generated with the program MMM. The measured inter-spin distance is indicated by the line. (B) The four-pulse microwave (MW) sequence that is used most often in the DEER experiment. The electron spins corresponding to different spin-labeled sites are frequency-selected for detection or pumping, respectively. The three MW pulses separated by fixed intervals select one of the spins to contribute to the detected spin-echo signal. The 4th (pump) pulse applied at a different frequency may select a coupled spin within the respective region of the spectrum. The pump pulse delay is advanced to give the spin-echo amplitude modulation envelope caused by dipolar coupling between the paired electron spins. (C) The recorded time-domain DEER signal (left) and the respective distances (right) between the MTSSL spins at residues 65C and 135C are shown. In general, a decaying oscillating time-domain signal is obtained from which the inter-spin distances and distance distributions are reconstructed.

Figure 4

The structure of membrane-bound tau protein as revealed by EPR spectroscopy is shown [45]. (A) Long inter-spin distances were measured by DEER spectroscopy for spin-labeled residues located in microtubule binding domain (MBD) repeats. The spin-labeled residues and their locations are indicated in the second row of the table. The lipid membrane mimetic (environment) in which the distances were obtained is indicated in the first column. The obtained distances (r) suggest that tau MBD has distinct structures when bound to LPPG micelles and 1-palmitoyl−2-oleoyl-glycero−3-phosphocholine/1-palmitoyl−2-oleoyl-sn-glycero−3-phospho-L-serine (POPC/POPS) liposomes. Longer distances in liposomes suggest more extended conformation. The distance distributions with relatively broad full widths at half maximum Δr indicated highly heterogeneous conformation of membrane-bound tau. (B) The binding of tau to lipid membranes was confirmed by CW EPR measurements. Nitroxide EPR lineshape broadening was observed for liposome-bound proteins (red spectra) compared to protein in solution (green spectra). The spin-labeled residues are indicated. (C) Model of tau-lipid membrane interaction based on the results from CW and DEER experiments was proposed—tau MBD structure when bound to micelles (left) and liposomes (right) is shown.

Figure 5

Conformations of liver alcohol dehydrogenase (LADH). (A) CW EPR spectra of spin-labeled OPSL bound to LADH in solution (bottom) and immobilized on two different carriers (mid and top). Tree sharp lines in each spectrum are from OPSL in solution with the rest of each spectrum is due to enzyme-bound OPSL. The numbers on the right represent the percentages of catalytic zinc ions and consequently of active sites available to OPSL. A_MAX corresponds to the outer spectral splitting, as indicated. (This figure from Ref. [126] is reproduced with permission from John Wiley; Sons) (B) A close-up view of 1,10-Phenanthroline bound to LADH at a putative binding site for OPSL, (PDB accession code 5VJ5).