Two dimensional crowding effects on protein folding at interfaces observed by chiral vibrational sum frequency generation spectroscopy

Abstract

The crowding effect is prevalent in cellular environments due to high concentrations of biomacromolecules. It can alter the structures and dynamics of proteins and thus impact protein functions. The crowding effect is important not only in 3-dimensional cytoplasm but also for a 2-dimensional (2D) cell surface due to the presence of membrane proteins and glycosylation of membrane proteins and phospholipids. These proteins and phospholipids - with limited translational degrees of freedom along the surface normal - are confined in 2D space. Although the crowding effect at interfaces has been studied by adding crowding agents to bulk solution, the 2D crowding effect remains largely unexplored. This is mostly due to challenges in controlling 2D crowding and synergistic use of physical methods for in situ protein characterization. To address these challenges, we applied chiral vibrational sum frequency generation (SFG) spectroscopy to probe the sp1 zinc finger (ZnF), a 31-amino acid protein, folding into a β-hairpin/α-helix (ββα) motif upon binding to Zn2+. We anchored ZnF at the air/water interface via covalent linkage of ZnF to palmitic acid and controlled 2D crowding by introducing neutral lipid as a spacer. We obtained chiral amide I SFG spectra upon addition of Zn2+ and/or spacer lipid. The chiral SFG spectra show that interfacial crowding in the absence of spacer lipid hinders ZnF from folding into the ββα structure even in the presence of Zn2+. The results establish a paradigm for future quantitative, systematic studies of interfacial crowding effects.

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“…Another possible explanation can be given by considering molecular crowding (Liu et al, 2018 ) and induced changes in lipid composition and their asymmetry (Bigay and Antonny, 2012 ; McMahon and Boucrot, 2015 ) at the contact perimeter, which may even lead to local conformational switches (Aisenbrey et al, 2008 ) and nano-domain clustering, as observed for glycolipid GM3 and PIP2 in molecular simulations of complex lipid bilayers (Koldsø et al, 2014 ). The large headgroups in PIP3 confers an inverted conical shape to the lipids, thereby favoring the bending of the membrane into a positive curvature (Di Paolo and De Camilli, 2006 ; McMahon and Boucrot, 2015 ).…”

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“…The resulting partially folded conformation of the synuclein was resistant to aggregation, suggesting that the abundance of membrane‐bound proteins in living cells likely affects the conformational equilibrium of IDPs [151]. Somewhat differently to IDPs, folding of a small, 31 amino acid long zinc finger protein covalently anchored to a lipid monolayer at a high density could not be accomplished, likely due to steric repulsion between otherwise structured domains [152]. Under these crowded conditions and reduced degrees of freedom, the protein acquired partial α‐helical fold.…”

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“…This is most likely caused by the presence of the other ion channels whose interfacial ion populations all have to compete for the same space. This crowding effect could be important in regulatory structures where the ion channel density is high …”