such weak interactions is that only 10–20% of the recombinant proteins are properly folded, although this idea is not supported by our CD and gel filtration results (Figure 3). Alternatively, these experiments were performed at protein concentrations (1–5 mM) that may be substantially lower than the binding constants for the protein-protein interactions. We suggest that the exocyst complex is formed from a combination of many low affinity interactions, which would lead to cooperative assembly and disassembly of the complex at sites of secretion in vivo. Regulation of this process by Rho/Rab-GTP binding partners on the vesicle and plasma membranes may trigger conformational changes to activate assembly of the subunits [4]. Conclusions We used a profile HMM prediction algorithm to predict that all of the exocyst subunits will have similar helical bundle structures. This approach has been shown to be generally useful to examine other families of proteins without high sequence similarity [40]. The HMM predictions have also proven to be a sensitive computational tool for defining structural domains. It ultimately allowed us to express and purify a soluble domain of the yeast exocyst protein Sec10p that will enable further biochemical and structural analyses. Similar analyses are being used for the other exocyst subunits, which will significantly contribute to our ability to elucidate the structure of the entire exocyst complex and its function in exocytosis. Materials and Methods Prediction of protein structure homology We built hidden Markov models for each exocyst component starting from the yeast proteins. Homologous proteins were collected using PSI-Blast for a maximum of 2 iterations with default parameters. Secondary structure was predicted by PSIPRED [41] and added to the profiles. HMMs were built using the HHmake function of the HHpred suite [25]. HMMs were compared to each other and to a PDB HMM database using the HHSearch function of the HHpred suite. Models for each sequence were built using MODELLER [42], and evaluated for the general trend of atomic interaction by statistical potential [43] and a composite model evaluation criterion [44]. To create a soluble construct of Sec10p, the HMM profile of Sec10p was directly compared with the profiles for the known structures of Exo70p, Sec15CT, Sec6CT and Exo84CT. A structural domain in the range of residues 148–867 was predicted. Using this information and secondary structure predictions (http://npsa-pbil.ibcp.fr/ and [41], we chose the N- and C- terminal ends of the structural domain to be residues 145 and 827, residues predicted to be at the ends of helices. Protein Expression and Purification Genes encoding full-length Sec10p (residues 1–872), the various truncations [Sec10(1–589); Sec10(590–871); Sec10(75–859), and the predicted structural domain Sec10(145–827)] were amplified by polymerase chain reaction and cloned into the NdeI and BamHI restriction sites of the vector pET15b (Novagen), which introduces a 6-histidine tag (His6) at the N termini. All constructs were confirmed by sequencing. All proteins were expressed in E. coli BL21(DE3) cells. To maximize protein solubility, cells were grown in LB to an OD at 600 nm of ,0.4 at 37uC. Cells were shifted to 15uC until the OD600 reached between 0.6–0.9. Protein expression was induced with 0.1 mM isopropyl-b-D-thiogalactoside (IPTG), and cells were grown for an additional 14–18 h at 15uC. Cells were harvested and frozen at 280uC until lysis. His6-tagged Exocyst Helical Bundles Sec10(145–827) protein was purified using nickel-NTA resin (Qiagen). b-Mercaptoethanol (5 mM) or dithiothreitol (DTT, 1 mM) was used in all buffers. Sec10(145–827) was purified using a Superdex 200 16/60 gel filtration column (GE) in KPhos buffer (10 mM potassium phosphate at pH 7.4 containing 140 mM KCl and 1 mM DTT) plus 10% glycerol. Fractions were analyzed by SDS-PAGE and stained with Coomassie blue. Those fractions containing .90% pure protein were pooled. Protein was concentrated using a stirred cell concentrator (Millipore) to ,1 mg/ml, and the protein concentration was determined by measuring the absorbance at 280 nm and by a quantitative ninhydrin protein assay [45]. Protein was flash frozen in liquid nitrogen in KPhos buffer containing 10% glycerol and stored at 280uC. Circular Dichroism Spectroscopy CD spectra were recorded on a J810 spectropolarimeter (Jasco) fitted with a Peltier-type temperature controller set to 4uC. The Sec10(145–827) protein was at a concentration of 0.8 mM in KPhos buffer containing 1 mM DTT and 10% glycerol. The spectra were recorded as an average of three scans from 200 to 270 nm, in a 1 mm path-length quartz cuvette (Hellma). For each spectrum, the minimum at 222 nm was used to estimate the mean residue ellipticity and percent helicity [39]. Analytical Gel Filtration Sec10(145–827) was chromatographed on a Superdex 200 10/ 30 column (GE) at a protein concentration of 1 mM. The column was pre-equilibrated in sodium phosphate buffer containing 300 mM NaCl, 10% glyercol and 1 mM DTT; eluted peaks were observed by monitoring the absorbance at 280 nm. The gel filtration column was calibrated using standards (thyroglobulin, 670 kD; c-globulin, 158 kD; ovalbumin, 44 kD; myoglobin, 17 kD; Bio-Rad). MBP pull-down assays and Western blot analyses Sec6p (residues 1–805), Exo70p (residues 63–623; [12], and Exo84CT (residues 523–753; [12] were subcloned into pMALc2X (New England Biolabs). Maltose binding protein (MBP) was expressed at 37uC and MBP-tagged Sec6p, Exo70p, and Exo84CT were expressed at 20uC for 3 h after induction with 0.1 mM IPTG. They were purified using amylose resin affinity chromatography (New England Biolabs). The binding reactions contained purified MBP-tagged proteins immobilized on amylose resin in binding buffer (10 mM HEPES pH 7, 100 mM NaCl, 0.1% NP-40, and 1 mM DTT), and an equimolar amount of the purified Sec10(145–827) protein (1 mM) was added. The reactions were incubated for 1 h at 4uC with mixing to allow binding. Beads were centrifuged and washed three times in binding buffer and analyzed by SDS-PAGE. Proteins were transferred to nitrocellu- lose and probed with a-His5 (Qiagen) or a-MBP antibodies (Invitrogen). Western blots were developed using horseradish peroxidase-conjugated a-mouse IgG (Roche), followed by chemi- luminescent detection (ECL; Amersham) and luminescent image analysis (FUJIFILM LAS-3000). The blots shown are representa- tive of at least three separate experiments. Acknowledgments We are grateful to A. Malaby for technical assistance and to Dr. C. R. Matthews and his lab for use of the CD and advice. We thank members of the Munson lab for critical reading of this manuscript and suggestions. We dedicate this to Jen Songer (1979–2008), who inspired us all, in so many ways. PLoS ONE | www.plosone.org 7 February 2009 | Volume 4 | Issue 2 | e4443