University, College Station, TX 77843, + Texas A&M System HSC, Houston, TX 77030 GroEL, a chaperonin from E. coli, is responsible for folding and refolding globular proteins in vitro [1] . It has also been reported that GroEL improves the ability of a membrane protein synthesized in vitro to insert post-translationally into liposomes [2] . Here we investigate the behavior of GroEL towards membrane proteins. One of the membrane proteins studied in this respect is the 105aa S protein, a prototype

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... holin from bacteriophage λ. S accumulates in the cytoplasmic membrane during late gene expression until, at a time programmed into its primary structure, it disrupts the membrane and allows the λ lysozyme, R, to attack the cell wall [3] . In order to characterize how S may be affected by GroEL, solubilized S protein was subjected to detergent removal by dialysis. Interestingly, in the presence of GroEL, S remained in solution after the detergent had been removed while in the control sample (without GroEL) S precipitated. Concerning the question how GroEL interacts with membrane proteins, GroEL was examined in the electron microscope in the absence and presence of S. Briefly, GroEL and GroEL plus S at 0.1 mg protein/ml were negatively stained using a 2% (w/v) aqueous solution of uranyl acetate and omitting any fixation steps [4] . Specimens were observed in a Zeiss 10C operated at 80 kV and images recorded at calibrated magnifications (35,500x and 27,000x). Selected micrographs were digitized using a Leafscan 45 at 20 µm increments corresponding to 0.56 and 0.74 nm/pixel, respectively, and processed using the IMAGIC 5 [5] and EMAN [6] software packages. Fig.1 depicts the results obtained after single particle analysis demonstrating central as well as peripheral protein densities in the samples that contained GroEL+ S. This finding indicates that GroEL may be providing a hydrophobic surface that can bind and prevent the membrane protein from precipitating. It is furthermore conceivable that other S molecules may be recruited to the core of GroEL-bound S giving rise to the peripheral densities. As the potential for high-resolution data retrieval is considerably enhanced using an electron crystallographic approach, the growth of 2-D crystals was attempted. Using the lipid-monolayer technique in the presence of charged nickel-chelating lipids, His-tagged GroEL assembled into 2-D crystals. Crystals were harvested onto holy Formvar/carbon grids and stained using 1% (w/v) uranyl acetate [4] . Micrographs were recorded and digitized as described above. Crystallographic processing was carried out using the software package CRISP [7] . Two different crystals forms were found, one consisting of GroEL in a side-on projection (Fig. 2a) , and the other comprising molecules viewed end-on (Fig. 2b) . The corresponding projection maps ( Fig. 2c and d, respectively) echo the structural features found by single particle averaging. However, in the end-on view ( Fig. 2b and d) the central protein deficit is more pronounced compared to Fig. 2a . This difference seems to be due to a more effective stain retention in the densely packed protein crystals compared to single molecules protruding from the support film (compare e.g. single end-on projections in Fig. 2a with b) . Co-crystallization trials of GroEL + S are currently under way. The ability of GroEL to solubilize membrane proteins allows the use of assays that are sensitive to detergent and may make it feasible to attempt 2-D and 3-D crystallography studies. Moreover, this approach may be applicable to other systems requiring detergent-free preparation of membrane