Lipid interactions with bacterial channels: fluorescence studies

Abstract

Interactions between a membrane protein and the lipid molecules that surround it in the membrane are important in determining the structure and function of the protein. These interactions can be pictured at the molecular level using fluorescence spectroscopy, making use of the ability to introduce tryptophan residues into regions of interest in bacterial membrane proteins. Fluorescence quenching methods have been developed to study lipid binding separately on the two sides of the membrane. Lipid binding to the surface of the mechanosensitive channel MscL is heterogeneous, with a hot-spot for binding anionic lipid on the cytoplasmic side, associated with a cluster of three positively charged residues. The environmental sensitivity of tryptophan fluorescence emission has been used to identify the residues at the ends of the hydrophobic core of the second transmembrane alpha-helix in MscL. The efficiency of hydrophobic matching between MscL and the surrounding lipid bilayer is high. Fluorescence quenching methods can also be used to study binding of lipids to non-annular sites such as those between monomers in the homotetrameric potassium channel KcsA.

Figure 1. The structure of the mechanosensitive channel of large conductance, MscL

(A) A surface polarity plot for MscL showing the location of the hydrophobic domain defined by fluorescence studies. The positions of Leu⁶⁹ on the periplasmic side of the membrane and of Val⁹¹ and Tyr⁹⁴ on the cytoplasmic side are marked. For clarity, only one set of defining residues is shown. The hydrophobic thickness of the protein, defined by the positions of Asp⁶⁸ and Asp¹⁶ is 25 Å. (B) A similar view but showing the relative locations of residues 69 and 87, used to study binding on the periplasmic and cytoplasmic sides of the protein respectively and the location of the positively charged cluster Arg⁹⁸, Lys⁹⁹ and Lys¹⁰⁰ that gives rise to a hot-spot for binding anionic lipid on the cytoplasmic side of the membrane.

Figure 2. Lipid binding on the two sides of MscL

Binding constants for di(C_18:1)PE (PE), dioleoylphosphatidylserine (PS) and dioleoylphosphatidic acid (PA) are shown relative to that for di(C_18:1)PC (PC) on the periplasmic (filled bars) and cytoplasmic (hatched bars) sides of the protein.

Figure 3. Defining the hydrophobic thickness of MscL

A plot of the tryptophan fluorescence emission maxima (nm) against the position of the tryptophan residue introduced into the lipid-exposed second transmembrane α-helix of MscL allows the identification of residues defining the ends of the hydrophobic core-spanning region of the helix: ○, di(C_12:0)PC; □, di(C_14:1)PC; ●, di(C_18:1)PC; △, di(C_24:1)PC. The dotted line at 332.6 nm marks the expected fluorescence emission maximum for a tryptophan residue immediately below the glycerol backbone region of the bilayer. The lower panel shows the locations of the mutated residues in MscL.