Photo-Crosslinking Analysis of Preferential Interactions between a Transmembrane Peptide and Matching Lipids

Abstract

In this study, a novel method is presented by which the molecular environment of a transmembrane peptide can be investigated directly. This was achieved by incorporating a photoactivatable crosslinking probe in the hydrophobic segment of a model transmembrane peptide. When this peptide was incorporated into lipid bilayers and irradiated with UV light, a covalent bond was formed between the crosslinking probe and a lipid. This crosslinking reaction could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the resulting product could be characterized by mass spectrometry. By use of phospholipases, it was demonstrated that the peptide crosslinks to both acyl chains of the lipids. The peptide showed a clear preference to partition into fluid lipids and was excluded from lipids in the gel phase. However, when the peptide was incorporated into bilayers containing two lipid species with different acyl chain lengths, molecular sorting of the lipids around the peptide based on hydrophobic matching was not observed. It is proposed that the size of the transmembrane part plays an important role in the dynamic interactions of membrane proteins with the surrounding lipids and hence in determining whether molecular sorting can occur.Proteins in biological membranes reside in a complex environment in which they are surrounded by many different lipid species. Transmembrane proteins can have different affinities for different lipids, and as a result particular lipids or members of particular lipid classes may become enriched around a certain protein. Such differences in affinity may be due to specific interactions with the lipid headgroups or the lipid acyl chains. An example of the latter would be the enrichment of matching lipids around a protein, which could be a mechanism by which mismatches between hydrophobic transmembrane segments and the hydrophobic thickness of the lipid bilayer can be relieved. Such a mechanism of molecular sorting by transmembrane proteins has been predicted from theoretical calculations (1, 2; reviewed in refs 3 and 4) and has indeed been shown to occur for several transmembrane proteins (5-8).To gain insight into the role of protein-lipid interactions in the sorting of lipids around specific proteins, tools must be available to determine the lipid environment of membrane proteins. Previously, this has been probed mainly by fluorescence and ESR 1 methods using labeled lipids (5-11). In this study, we explore a more direct method to investigate the immediate surroundings of membrane proteins. For this purpose we make use of a synthetic model peptide, which contains a photoactivatable probe that is able to form covalent bonds with the surrounding lipids upon UV irradiation. The crosslinking reaction can be visualized by SDS-PAGE analysis and the identity of the lipid that is crosslinked to the peptide can subsequently be characterized by mass spectrometry.

Low membrane fluidity triggers lipid phase separation and protein segregation in vivo

“…Small hydrophobic dyes may themselves alter the composition and ordering of coexisting phases, even when used in trace amounts (Veatch, 2007). However, we are convinced that (i) by using the combination of two chemically distinct fluorescent membrane dyes Laurdan and FM 5-95 exhibiting opposing phase preferences, (ii) combining dye-based approaches with localization of WALP23 peptide previously shown to exhibit liquid-disordered phase preference both in vivo, in vitro, and in silico (Ridder et al, 2004;Schäfer et al, 2011;Scheinpflug et al, 2017b), and (iii) by following the reversible phase separation through its consequences on lateral membrane protein diffusion, we have exhausted the possibility that the observed phase separation is an artefact caused by the labeling techniques used. Importantly, by demonstrating lipid liquid-gel phase separation and the associated membrane protein segregation occurring in protein-crowded, native membranes of living cells, our results are fully consistent with comparable phenomena observed in simplified in vitro and in silico model systems (Domański et al, 2012;Picas et al, 2010;Suárez-Germà et al, 2011), thus providing strong, complementary in vivo support for the general validity of the respective membrane models.…”

“…In addition, Laurdan also showed a clear preferred accumulation in membrane areas of lower fluidity ( Figures 5B and 5C). To verify these findings with an independent method, and to address the common concerns regarding the specificity of chemical dyes in context of lipid domains, we repeated the experiments with cells expressing helical transmembrane peptide WALP23, which has been shown to preferentially accumulate in fluid membrane areas (Ridder et al, 2004;Schäfer et al, 2011;Scheinpflug et al, 2017b). Co-labeling of cells with Laurdan clearly demonstrated that the observed lipid phase separation results in segregation of WALP23 in membrane areas of low Laurdan fluorescence, thus indicating partitioning into the more fluid membrane areas ( Figures 5D, 5E, S4A, and S4B).…”

Exploiting Benzophenone Photoreactivity To Probe the Phospholipid Environment and Insertion Depth of the Cell‐Penetrating Peptide Penetratin in Model Membranes

“…They do not ascertain whether the peptide interacts directly with specific phospholipids, nor do they provide information about the location at the level of the lipid bilayer of such interactions. It was previously reported that the environment of a photoactivatable transmembrane diazirin‐modified peptide preinserted in the membrane bilayer could be probed . In this study, we probed the lipid environment of a CPP that dynamically inserts by itself into the membrane bilayer.…”

Mesoscopic models of biological membranes

“…It was found that the smaller the protein, the more pronounced is the tilt. There are few experimental attempts to estimate the extent of the protein-induced bilayer perturbation; nevertheless, the existing ones confirm a qualitative mismatch dependence of the extent of the perturbation [204,207,[226][227][228][229]. Nielsen et al [123] carried out MD simulations on a coarse-grained model to analyze the lipid bilayer perturbation around a transmembrane hydrophobic nanotube.…”