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

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 ß-hairpin/α-helix (ßßα) 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 ßßα structure even in the presence of Zn2+. The results establish a paradigm for future quantitative, systematic studies of interfacial crowding effects.

Frequency comb SFG: a new approach to multiplex detection.

Determination of molecular orientation at interfaces by vibrational sum frequency generation spectroscopy (VSFG) requires measurements using at least two different polarization combinations of the incoming visible, IR, and generated SFG beams. We present a new method for the simultaneous collection of different VSFG polarization outputs by use of a modified 4f pulseshaper to create a simple frequency comb. Via the frequency comb, two visible pulses are separated spectrally but aligned in space and time to interact at the sample with mixed polarization IR light. This produces two different VSFG outputs that are separated by their frequencies at the monochromator rather than their polarizations. Spectra were collected from organic thin films with different polarization combinations to show the reliability of the method. The results show that the optical arrangement is immune to fluctuations in laser power, beam pointing, and IR spectral shape.