Lipid‐like Peptides can Stabilize Integral Membrane Proteins for Biophysical and Structural Studies

Abstract

A crucial bottleneck in membrane protein structural biology is the difficulty in identifying a detergent that can maintain the stability and functionality of integral membrane proteins (IMPs). Detergents are poor membrane mimics, and their common use in membrane protein crystallography may be one reason for the challenges in obtaining high‐resolution crystal structures of many IMP families. Lipid‐like peptides (LLPs) have detergent‐like properties and have been proposed as alternatives for the solubilization of G protein‐coupled receptors and other membrane proteins. Here, we systematically analyzed the stabilizing effect of LLPs on integral membrane proteins of different families. We found that LLPs could significantly stabilize detergent‐solubilized IMPs in vitro. This stabilizing effect depended on the chemical nature of the LLP and the intrinsic stability of a particular IMP in the detergent. Our results suggest that screening a subset of LLPs is sufficient to stabilize a particular IMP, which can have a substantial impact on the crystallization and quality of the crystal.

Novel CaM-binding motif in its NudT9H domain contributes to temperature sensitivity of TRPM2

“…The stability and thermal unfolding behavior of purified NudT9H in the presence and absence of ligands was followed using a nanoDSF differential scanning fluorimeter (Prometheus, NanoTemper Technologies, Munich) as previously described [29]. The intrinsic fluorescence at 330 and 350 nm after excitation at 280 nm is used to monitor the fluorescence change upon heat unfolding.…”

High-throughput stability screening for detergent-solubilized membrane proteins

“…The inflection point in a standard denaturation curve, T m , has been a recurrent parameter to compare protein thermodynamic stability. Other studies have evaluated the stability of membrane proteins following the change of intrinsic fluorescence during thermal denaturation using a differential scanning fluorimetry device for the screening of lipid-like peptides 29 and ligands 30–33 . Recently, Nji et al .…”

Control over the fibrillization yield by varying the oligomeric nucleation propensities of self-assembling peptides

“…S9). For applications that require that SLPs stay in dynamic form, e.g., the solubilization of membrane proteins 44 , one should increase the charges and lower the β-sheet propensities of SLPs in order to prevent fibrillization, as we here demonstrate it for SLP4. In contrast, for applications that require the fibril infrastructure, e.g., for therapeutic scaffolds [2][3][4] , one should engineer the SLP with higher β-sheet propensities and lower number of charges to maximize the yield of fibril structures (like SLP1).…”

“…toward the size distribution of the oligomeric particles and the thermodynamic stability of the fibrils are also evaluated, which helps defining the mechanism leading to the differential polymerization yield. Thereby, our study enhances our understanding of the important role of oligomeric intermediates in defining the outcome of self-assembly systems and advances the rational design principles of self-assembling peptides that give different supramolecular properties for a wide latitude of applications [2][3][4]44,45 . Moreover, our findings suggest that charged protein gatekeeper sequences 46 can prevent amyloidosis by lowering the size of the oligomeric particles.…”