Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments1-7 and that lipids can bind to specific sites, for example in potassium channels8. Fundamental questions remain however regarding the extent of membrane protein selectivity toward lipids. Here we report a mass spectrometry (MS) approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL), aquaporin Z (AqpZ), and the ammonia channel (AmtB) using ion mobility MS (IM-MS), which reports gas-phase collision cross sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas-phase. By resolving lipid-bound states we then rank bound lipids based on their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Results show that lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability, the highest-ranking lipid however is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation9. AqpZ is also stabilized by many lipids with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays, we discover that cardiolipin modulates AqpZ function. Analogous experiments identify AmtB as being highly selective for phosphatidylglycerol prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that reposition AmtB residues to interact with the lipid bilayer. Overall our results demonstrate that resistance to unfolding correlates with specific lipid-binding events enabling distinction of lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be influential not only for defining the selectivity of membrane proteins toward lipids but also for understanding the role of lipids in modulating function or drug binding.

Advances in structure determination of membrane proteins enable analysis of the propensities of amino acids in extramembrane versus transmembrane locations to be performed on the basis of structure rather than of sequence and predicted topology. Using 29 available structures of integral membrane proteins with resolutions better than 4 A the distributions of amino acids in the transmembrane domains were calculated. The results were compared to analysis based on just the sequences of the same transmembrane alpha-helices and significant differences were found. The distribution of residues between transmembrane alpha-helices and beta-strands was also compared. Large hydrophobic (Phe, Leu, Ile, Val) residues showed a clear preference for the protein surfaces facing the lipids for beta-barrels, but in alpha-helical proteins no such preference was seen, with these residues equally distributed between the interior and the surface of the protein. A notable exception to this was alanine, which showed a slight preference for the interior of alpha-helical membrane proteins. Aromatic residues were found to follow saddle-like distributions preferring to be located in the lipid/water interfaces. The resultant 'aromatic belts' were spaced more closely for beta-barrel than for alpha-helical membrane proteins. Charged residues could be shown to generally avoid surfaces facing the bilayer although they were found to occur frequently in the transmembrane region of beta-barrels. Indeed detailed comparison between alpha-helical and beta-barrel proteins showed many qualitative differences in residue distributions. This suggests that there may be subtle differences in the factors stabilising beta-barrels in bacterial outer membranes and alpha-helix bundles in all other membranes.

The crystal structure of the open conformation of a bacterial voltage-gated sodium channel pore from Magnetococcus sp.(NaVMs) has provided the basis for a molecular dynamics study defining the channel's full ion translocation pathway and conductance process, selectivity, electrophysiological characteristics, and ion-binding sites. Microsecond molecular dynamics simulations permitted a complete time-course characterization of the protein in a membrane system, capturing the plethora of conductance events and revealing a complex mixture of single and multiion phenomena with decoupled rapid bidirectional water transport. The simulations suggest specific localization sites for the sodium ions, which correspond with experimentally determined electron density found in the selectivity filter of the crystal structure. These studies have also allowed us to identify the ion conductance mechanism and its relation to water movement for the NavMs channel pore and to make realistic predictions of its conductance properties. The calculated single-channel conductance and selectivity ratio correspond closely with the electrophysiology measurements of the NavMs channel expressed in HEK 293 cells. The ion translocation process seen in this voltage-gated sodium channel is clearly different from that exhibited by members of the closely related family of voltage-gated potassium channels and also differs considerably from existing proposals for the conductance process in sodium channels. These studies simulate sodium channel conductance based on an experimentally determined structure of a sodium channel pore that has a completely open transmembrane pathway and activation gate. V oltage-gated cation channels are proteins that produce electrical signals in neurons and other excitable cells to regulate muscle contraction, gene expression, and release of hormones and neurotransmitters among other functions. In response to a change in transmembrane electrical potential, the channels open pores through which ions move passively across the membrane. The large family of cation channels includes those selective for sodium, potassium, or calcium. The opening and closing of these ion-specific channels is carefully choreographed to produce the electrical signals required by the nervous system for rapid signal transduction (1).Voltage-gated sodium channels have been causally linked with a wide range of neurological and cardiovascular diseases and hence are important pharmaceutical drug-development targets (2, 3). Eukaryotic voltage-gated sodium channels are large, singlechain polypeptides, consisting of 24 transmembrane (TM) helices that form four homologous repeats, each contributing both a voltage sensor and a pore domain; the latter are arranged to form a central Na + -selective transmembrane pathway. Bacterial voltagegated sodium channels are far simpler, consisting of four polypeptide chains, each of which is composed of six TM segments, with segments TM1-TM4 forming the voltage sensors and TM5-TM6 forming the pore domains. The TM5-TM6 segment...

Distributions of each amino acid in the trans‐membrane domain were calculated as a function of the membrane normal using all currently available α‐helical membrane protein structures with resolutions better than 4 Å. The results were compared with previous sequence‐ and structure‐based analyses. Calculation of the average hydrophobicity along the membrane normal demonstrated that the protein surface in the membrane domain is in fact much more hydrophobic than the protein core. While hydrophobic residues dominate the membrane domain, the interfacial regions of membrane proteins were found to be abundant in the small residues glycine, alanine, and serine, consistent with previous studies on membrane protein packing. Charged residues displayed nonsymmetric distributions with a preference for the intracellular interface. This effect was more prominent for Arg and Lys resulting in a direct confirmation of the positive inside rule. Potentials of mean force along the membrane normal were derived for each amino acid by fitting Gaussian functions to the residue distributions. The individual potentials agree well with experimental and theoretical considerations. The resulting implicit membrane potential was tested on various membrane proteins as well as single trans‐membrane α‐helices. All membrane proteins were found to be at an energy minimum when correctly inserted into the membrane. For α‐helices both interfacial (i.e. surface bound) and inserted configurations were found to correspond to energy minima. The results demonstrate that the use of trans‐membrane amino acid distributions to derive an implicit membrane representation yields meaningful residue potentials. Proteins 2005. © 2005 Wiley‐Liss, Inc.