Expression and purification of membrane proteins: Initial screening of Thermotoga maritima α-helical membrane proteins for NMR structural studies This morning I would like to talk about a structure initiative in which we target membrane proteins of T. maritima for solution NMR structure determination. Most of our efforts have been in sample preparation and utilizing 1H 1D NMR spectroscopy to evaluate the integrity of the samples and we are just heading into the structure determination stages of the project.

The membrane proteome of T. maritima Electron micrograph of T. maritima. The arrow points to the outer membrane, the “toga”, for which the organism is named. Percentage of Membrane Proteins in T. maritima 24% 76% Membrane proteins Cytosolic proteins I would like to begin with a brief introduction to the membrane proteome T. maritima. The thermophilic bacteria has an inner and outer membrane. The outer membrane, for which the “toga” portion is named for is attached to the innermembrane at the center of the cell and forms ballon-like structures at the poles of the cell. The outer membrane is made up prodominately of one protein; in fact the outer membrane is a 2D array of a trimeric beta-barrel (one of maybe four of the whole proteome). Therefore, it is highly likely that most of the membrane proteome is alpha-helical and localized to the inner membrane. Membrane proteins comprise 24% of the proteome and of that 24%; 20% are ABC transporters and another 25% have been functionally annotated leaving 55% of which we haven’t a clue to what they do. As far as structures go, there are a few permeases that probably resemble the recent structure of that bacterial transporters that were solved recently, some of the ABC transporters are of the family that was determined by Doug Rees, a glycerol transporter similar to the GlpF structure. For a structural biologist and a biochemist, the membrane proteome is a hidden treasure of sorts, and we need a map, to help us, because as of yet there is no paradigm to determining the structure of a membrane protein. 55% 20% 25% Proteins with unknown function Other functionally annotated proteins ABC transporters Functional Annotations of the Membrane Proteome

Overall Approach to Preparing Membrane Protein Samples for NMR Studies So our initial map, which is subject to change and revisions along the way, is as follows. We select our targets, we assay expression of the targets in E. coli, then we make sure, since they are putative membrane proteins, that they are localizing to the membrane, or at least are not expressed in a soluble form. If they express in the membrane fraction than the targets are extracted from the membrane usually with DM, and purified. For each target, eleven detergents are screened in parallel top optimize the solubility over a period of approximately a week. The fold of the proteins that remain soluble are assayed using 1D 1H NMR spectroscopy and if that looks promising the samples are 15N labeled and are further assessed and optimized using 2D spectroscopy. I will not talk about proteins expressed predominately to the insoluble fraction.

Target Selection Size distribution of the membrane proteome # of NMR sample requirements 600 mL of 1 mM protein uniform 15N and 13C-labeling monodisperse MW < 30 kD (for standard NMR experiments, development of TROSY has extended the MW limit for NMR studies) Size distribution of the membrane proteome # of proteins 0 200 400 600 800 1000 1200 1400 1600 Protein length (amino acids) Selected fifty targets less than 16 KD (~130 aa) that contained one to four predicted transmembrane segments. Target selection was mainly dictated by our requirements for an NMR sample specificly the molecular weight. NMR spectroscopy can be applied to very large complexes with the development of TROSY; however, given the difficulty of the protein samples we wanted to keep the complexity of the investigation to tackling one problem at a time. So we chose to target fifty membrane proteins less than 16kD, which is the majority of the proteins in this light-weight class as you can see from this histogram, which plots the number of proteins versus protein length. The bar indicates approximately the 16 kD mark. If we take into account the detergent micelle MW, than the protein/micelle complex is likely to reach 30 kD.

Expression Assay 96 x 65 mL Fermentor Expression Vector Para 6-aa Pml I 6-his CAC GTG TM gene TTA ATT AA Pac I Pme I Nco I RBS Sma I PT7 21.5 14.4 6.0 kD Eleven out of fifty targets overexpressed using arabinose induction.

Localization of target membrane proteins IB S M IB S M In order to verify that the target proteins are membrane proteins, the E. coli membranes were isolated using ultracentrifugation and extracted with n-decyl--D-maltoside . Of the eleven expressing targets, two expressed exclusively in the insoluble fraction (IB) and nine expressed to both the insoluble fraction and the n-decyl--D-maltoside solubilized membrane (M) fraction. None of the proteins were found in the soluble (S) fraction.

Soluble, monodisperse, and folded Is that too much to ask? Which detergent will do all this? For now, there is no paradigm. There isn’t one detergent that works for all membrane proteins; therefore, for each protein, many detergents need to be screened.

Detergent Screen W indole side chain protons TM0361 Aromatic side OG NG DG OM DM P S P S P S P S P S TM0361 Aromatic side chain protons Backbone amide protons DoDM DHPC CHAPS LDAO DPC W indole side chain protons P S P S P S P S P S 1H (ppm)

Soluble ≠ folded! 9.5 9.0 8.5 8.0 7.5 7.0 6.5 1H (ppm)

Optimization of solution conditions for structure determination: 2D spectroscopy of 15N-labeled protein TM0361 TM1634 105 110 110 115 115 15N (ppm) 120 15N (ppm) 120 125 125 130 130 11.0 10.0 9.0 8.0 7.0 9.0 8.0 8.5 7.5 7.0 1H (ppm) 1H (ppm) Using the 96 x 65 mL fermentor, these samples were prepared with less than 1L of commercial media.

Summary Approximately 20% of the membrane protein targets express in HK100 E. coli cells with arabinose induction. 2. Detergent screening has been successful in preparing solubilized membrane protein samples, but will be revised as we gain experience. 3. Similar to soluble proteins, 1H 1D NMR spectroscopy is suitable for evaluating the overall fold of the protein. 4. TM1634 has been optimized and NMR structure determination is in progress.

Acknowledgements Kurt Wüthrich Scott Lesley Heath Klock Bernhard Geierstanger Joanna Hale This work is funded by the NIH protein structure initiative grant P50 GM62411. LC is funded by NIH grant 1F32GM068286. KW is funded by the endowment of Cecil H. and Ida M. Green (TSRI).