Phedra Marius

Lipid interactions with bacterial channels: fluorescence studies

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.

Probing the interaction of lipids with the non-annular binding sites of the potassium channel KcsA by magic-angle spinning NMR

The activity of the potassium channel KcsA is tightly regulated through the interactions of anionic lipids with high-affinity non-annular lipid binding sites located at the interface between the channels subunits. Here we present solid-state phosphorous NMR studies that resolve the negatively charged lipid phosphatidylglycerol within the non-annular lipid-binding site. Perturbations in chemical shift observed upon the binding of phosphatidylglycerol are indicative of the interaction of positively charged sidechains within the non-annular binding site and the negatively charged lipid headgroup. Site directed mutagenesis studies have attributed these charge interactions to R64 and R89. Functionally the removal of the positive charges from R64 and R89 appears to act synergistically to reduce the probability of channel opening.

Stability and membrane orientation of the fukutin transmembrane domain: a combined multiscale molecular dynamics and circular dichroism study.

The N-terminal domain of fukutin-I has been implicated in the localization of the protein in the endoplasmic reticulum and Golgi Apparatus. It has been proposed to mediate this through its interaction with the thinner lipid bilayers found in these compartments. Here we have employed multiscale molecular dynamics simulations and circular dichroism spectroscopy to explore the structure, stability, and orientation of the short 36-residue N-terminus of fukutin-I (FK1TMD) in lipids with differing tail lengths. Our results show that FK1TMD adopts a stable helical conformation in phosphatidylcholine lipids when oriented with its principal axis perpendicular to the bilayer plane. The stability of the helix is largely insensitive to the lipid tail length, preventing hydrophobic mismatch by virtue of its mobility and ability to tilt within the lipid bilayers. This suggests that changes in FK1TMD tilt in response to bilayer properties may be implicated in the regulation of its trafficking. Coarse-grained simulations of the complex Golgi membrane suggest the N-terminal domain may induce the formation of microdomains in the surrounding membrane through its preferential interaction with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol 4,5-bisphosphate lipids.

Probing the oligomeric state and interaction surfaces of Fukutin-I in dilauroylphosphatidylcholine bilayers

Fukutin-I is localised to the endoplasmic reticulum or Golgi apparatus within the cell, where it is believed to function as a glycosyltransferase. Its localisation within the cell is thought to to be mediated by the interaction of its N-terminal transmembrane domain with the lipid bilayers surrounding these compartments, each of which possesses a distinctive lipid composition. However, it remains unclear at the molecular level how the interaction between the transmembrane domains of this protein and the surrounding lipid bilayer drives its retention within these compartments. In this work, we employed chemical cross-linking and fluorescence resonance energy transfer measurements in conjunction with multiscale molecular dynamics simulations to determine the oligomeric state of the protein within dilauroylphosphatidylcholine bilayers to identify interactions between the transmembrane domains and to ascertain any role these interactions may play in protein localisation. Our studies reveal that the N-terminal transmembrane domain of Fukutin-I exists as dimer within dilauroylphosphatidylcholine bilayers and that this interaction is driven by interactions between a characteristic TXXSS motif. Furthermore residues close to the N-terminus that have previously been shown to play a key role in the clustering of lipids are shown to also play a major role in anchoring the protein in the membrane.

Fluorescence Quenching Methods to Study Lipid‐Protein Interactions

This unit describes how fluorescence quenching methods can be used to determine binding constants for phospholipids binding to intrinsic membrane proteins. Reconstitution of a Trp‐containing intrinsic membrane protein with bromine‐containing phospholipids leads to quenching of the Trp fluorescence of the protein; the extent of quenching depends on the strength of binding of the phospholipid to the protein. Protocols are included for the synthesis of bromine‐containing phospholipids from phospholipids containing carbon‐carbon double bonds in their fatty acyl chains and for the reconstitution of membrane proteins into bilayers containing bromine‐containing phospholipids. Details are included on data analysis, including equations and software that can be used for fitting the fluorescence quenching data.

Expression and purification of the transmembrane domain of Fukutin-I for biophysical studies.

Fukutin-I is a member of a family of putative O-linked glycosyltransferases linked to the glycosylation of the dystrophin complex. Mutations in this family of proteins have been linked to a number of congenital muscular dystrophies that arise from the hypoglycosylation of α-dystroglycan. Critical to the function of Fukutin and other members of this family is their localisation within the cell, which has been shown to depend critically on the interactions between the N-terminal transmembrane domain of these proteins and the lipid bilayer within the ER/Golgi. To investigate how the interactions between the N-terminal transmembrane domain and the lipid bilayer regulate the localisation of Fukutin-I, we have developed an efficient expression and purification protocol in Escherichia coli to allow biophysical studies to be performed. Expressing the N-terminal domain of Fukutin-1 fused to a His6 tag resulted in the localisation of the protein to the bacterial membrane. A purification strategy has been developed to isolate the highly hydrophobic transmembrane domain of Fukutin-1 from the membrane with yields of approximately 4 mg per litre of minimal media. Preliminary biophysical analyses have confirmed the identity of the peptide and revealed that in hydrophobic solvents mimicking the bilayer, the peptide adopts a well-structured α-helix as predicted from the sequence.

The Fukutin Transmembrane Domain: Capturing the Complexity of the Golgi Apparatus Membrane via Multiscale MD Simulations

The membrane of the Golgi apparatus is a complex mixture of lipids and proteins. In the present work we describe multiscale molecular dynamics simulations of transmembrane protein domains in model membranes that represent the in vivo Golgi environment. The transmembrane domain of glycosyltransferases is required for their correct sorting within the Golgi apparatus. The hydrophobic thickness and oligomerization state of the transmembrane domains have been proposed to mediate this sorting. Fukutin, is a putative Golgi glycosyltransferase implicated in muscular dystrophy.We employ atomistic and coarse-grained molecular dynamics simulations to investigate the stability and membrane interactions of the fukutin transmembrane domain, using various models of the membrane. Our atomistic simulations reveal that the fukutin transmembrane domain can exist as a stable α helix irrespective of the headgroup charge, fatty acid saturation or hydrophobic thickness of the lipid bilayer. Coarse-grained simulations reveal that the tilt angle of the fukutin transmembrane domain is highly variable and dependent upon its local environment; both the hydrophobic thickness and the headgroup charge of the lipid bilayer can alter the tilt angle of the protein. Lastly, we study the dynamics of the fukutin transmembrane domain in a mixed lipid bilayer whose composition closely mimics the complex lipid headgroup composition of the Golgi apparatus.