Lipid-Protein Interactions Revealed by Fluorescence Spectroscopy

The Edinburgh Instruments research team collaborated with Dr Tim Rasmussen, a Research Fellow at the University of Aberdeen to report on lipid-protein interaction using our Fluorescence Spectrometers.

INTRODUCTION

Interactions between lipid bilayers and membrane proteins can be revealed by recording the fluorescence of tryptophan residues and collisional quenching by bromine atoms bound to lipid fatty acid chains. These important interactions are far easier to detect using fluorescence quenching than with other biophysical methods as for example X-ray crystallography. By choosing the tryptophan site and location of bromine on the fatty acid chain, high structural specificity is obtained.

METHODS and MATERIALS

Emission spectra were recorded in an FLS980 Fluorescence Spectrometer equipped with double excitation and emission monochromators. Calcite polarisers were used in the excitation and emission, while a photomultiplier tube detector (Hamamatsu, R928P) with 1s dwell was utilised. The FLS980 provides a flexible and sensitive instrument to record the emission that allows for optimising the settings, while polarisers are convenient to repress effects interfering with the fluorescence of tryptophan. For this reason, the polarisers are set to 90° at the excitation and 0° at the emission to minimise the effect of light scattering.

RESULTS - DISCUSSION

The crystal structure of MscS shows unusual "paddles", see inset of Figure 3, that seem to be located in the membrane and are presumably important for tension sensing. MscS is a homoheptamer with "paddles" on the membrane domain and in addition a large cytosolic domain.

Control spectra of lipid samples without MscS were subtracted from the raw spectra and are displayed in Figure 2. Then, the fractional quenching was calculated over the whole spectrum, see Figure 3.