Hydrogen exchange (HX) mass spectrometry (MS) is dear for providing conformational details for protein/peptides that have become difficult to investigate with other strategies such as for example peripheral membrane protein and peptides that connect to membranes. process was equal to deuterium recovery in conventional answer HX MS. The reproducibility of the measurements was high despite the requirement of generating a new Org 27569 monolayer for each deuterium labeling time. We validated that known conformational changes in the presence of a monolayer/membrane could be Org 27569 observed with the peptide melittin and the myristoylated protein Arf-1. Results in an accompanying paper show that the method can reveal details of conformational changes in a protein (HIV-1 Nef) which adopts a different conformation depending on if Rabbit Polyclonal to TNF14. it can insert into the lipid layer. Overall the HX MS Langmuir monolayer method provided new and meaningful conformational information for proteins that associate with lipid layers. The combination of HX MS results with neutron or X-ray reflection of the same proteins in Langmuir monolayers can be more useful than isolated use of either method. Keywords: Arf-1 deuterium melittin dynamics membrane Membrane proteins are involved in many cellular processes ranging from regulation recognition metabolism transport and signaling1. Recent accounts indicate that ～58% of utilized drug targets were membrane proteins2 3 Despite much effort focused on both transmembrane and peripheral membrane proteins it has been difficult to obtain high resolution structural information for many membrane proteins. Membrane protein structures account for ～2.5% of all coordinate files deposited in the Protein Data Bank4 (www.rcsb.org5). The major obstacle to structural characterization of membrane proteins is usually often the membrane itself which is generally not compatible with structural studies and many biophysical measurements. Solubilizing membrane proteins with detergents to make them compatible with aqueous buffers and methodologies is usually one alternative but this is not always successful and even when successful questions can linger as to how detergents may alter protein structure6. Analytical methods that make use of membrane mimetics (i.e. artificial membranes) are attractive alternatives because the structure of the membrane protein is usually more likely to be preserved in the mimetic. We have previously applied hydrogen exchange (HX) mass spectrometry (MS) to membrane proteins7-14 primarily using liposomes or nanodiscs as the membrane mimetic. Other groups have also used HX MS for membrane proteins utilizing detergents [e.g. 15-17] and liposomes [e.g. Org 27569 18-20]. Each membrane mimetic has advantages and disadvantages. Detergents may pressure the protein into a nonnative conformation6 and can suppress peptide ionization if not properly removed prior to electrospray7 21 Creation of both liposomes and nanodiscs can be challenging. For liposomes there can be issues with reproducibility lipid membrane curvature effects (especially in vesicles see Refs. 22 23 for review) and protein directionality while nanodiscs have a background undesirable protein component (the membrane stabilizing protein) and lipid packing density is not easily altered. Lipid packing density the number of lipid molecules per unit area is usually fluid in cellular membranes with some regions Org 27569 packed more tightly than other regions often dependent on the lipid composition degree of hydrocarbon chain saturation percentage of cholesterol and other factors24-27. Some proteins may alter the packing density of surrounding lipids perhaps as a result of conformational changes during function membrane insertion or other reasons. To study membrane protein/peptide conformational changes therefore control over lipid packing density can sometimes be critical28 including the ability to reproducibly produce measure and change lipid packing density. Nanodiscs have a fixed density and a fixed number of lipid molecules; packing density can be modulated during nanodisc creation but cannot be altered once the nanodisc is usually formed. As a result protein/peptide interactions with nanodiscs that require insertion or conformational changes may be restricted. Liposomes can change size and therefore offer the least.