CW-EPR studies revealed different motional properties and oligomeric states of the integrin β1a transmembrane domain in detergent micelles or liposomes

Abstract

Integrins are heterodimeric membrane proteins that regulate essential processes: cell migration, cell growth, extracellular matrix assembly and tumor metastasis. Each integrin α or β subunit contains a large extracellular domain, a single transmembrane (TM) domain, and a short cytoplasmic tail. The integrin TM domains are important for heterodimeric association and dissociation during the conversion from inactive to active states. Moreover, integrin clustering occurs by homo-oligomeric interactions between the TM helices. Here, the transmembrane and cytoplasmic (TMC) domains of integrin β1a were overexpressed, and the protein was purified in detergent micelles and/or reconstituted in liposomes. To investigate the TM domain conformational properties of integrin β1a, 26 consecutive single cysteine mutants were generated for site-directed spin labeling and continuous-wave electron paramagnetic resonance (CW-EPR) mobility and accessibility analyses. The mobility analysis identified two integrin β1a-TM regions with different motional properties in micelles and a non-continuous integrin β1a-TM helix with high immobility in liposomes. The accessibility analysis verified the TM range (Val737-Lys752) of the integrin β1a-TMC in micelles. Further mobility and accessibility comparisons of the integrin β1a-TMC domains in micelles or liposomes identified distinctively different oligomeric states of integrin β1a-TM, namely a monomer embedded in detergent micelles and leucine-zipper-like homo-oligomeric clusters in liposomes.

Probing Structural Dynamics and Topology of the KCNE1 Membrane Protein in Lipid Bilayers via Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy

“…36 The KCNE1 CW-EPR spectra are consistent with previously published CW-EPR data for a variety of different membrane proteins in proteoliposomes. 37–41 Inspection of the EPR spectral line-shapes indicates that different segments of KCNE1 interact differently with the lipid bilayers as observed by the variation in line broadenings for different spin-labeling positions. The two motional components are easily discerned in the EPR spectra for sites located in the extracellular (N-terminal) region of the KCNE1 sequence (Figure 2A).…”

Biophysical EPR Studies Applied to Membrane Proteins

“…Unique structural and dynamic information on membrane proteins can be gleaned using these powerful techniques. Table 1 shows recent examples of some biologically important membrane protein systems studied by using EPR spectroscopy [43,44,49,64,74,75,78,79]. EPR spectroscopy has become complementary to existing biophysical methods to study membrane proteins.…”

“…Membrane protein topology can be probed with respect to the membrane using nitroxide based SDSL EPR [10,49,50]. Power saturation EPR experiments can be used to determine a residue’s accessibility to different paramagnetic reagents [50].…”

“…A recent example of using nitroxide based site-directed spin labeling EPR is a study of the integrin β 1a [49]. Integrins are heterodimeric membrane proteins that regulate essential process related to cell-cell and cell-matrix interactions, i.e., cell migration, cell growth, extracellular matrix assembly and tumor metastasis.…”

“…Integrins are heterodimeric membrane proteins that regulate essential process related to cell-cell and cell-matrix interactions, i.e., cell migration, cell growth, extracellular matrix assembly and tumor metastasis. Yu et al used CW-EPR lineshape analysis of 26 consecutive single spin-labeled mutants on the transmembrane (TM) domain of integrin β 1a to determine the side-chain mobility and solvent accessibility in detergent micelles and liposomes [49]. The mobility data identified two integrin β 1a -TM regions with different motional properties in micelles and a highly immobile non-continuous integrin β 1a -TM helix in liposomes.…”