COMMUNICATION Voltage and pH-induced Channel Closure of Porin OmpF Visualized by Atomic Force Microscopy Daniel J. Mu È ller and Andreas Engel* M. E. Mu È ller-Institute for Microscopy, Biozentrum University of Basel Klingelbergstrasse 70 CH-4056 Basel, Switzerland Gram-negative bacteria are protected by an outer membrane in which tri- meric channels, the porins, facilitate the passage of small solutes. The pores are formed by membrane-spanning antiparallel b-strands, which are connected by short turns on the periplasmic side and long loops on the extracellular side. Voltage and pH-dependent conformational changes of these extracellular loops have now been visualized by atomic force microscopy of two-dimensional crystals of Escherichia coli porin OmpF. The observed conformational changes accompany the closure of the chan- nel entrance, and suggest that this is a mechanism that the cells have evolved to protect themselves from drastic changes of the environment. # 1999 Academic Press Keywords: atomic force microscopy; conformation change; Escherichia coli porin OmpF; voltage gating *Corresponding author An E. coli outer membrane contains approxi- mately 10 5 porins to allow the passage of solutes <600 Da (Nikaido & Vaara, 1985). In addition to nutrients, antibiotics that bind to their periplasmic targets must diffuse through these aqueous pores (Nikaido, 1989). Conductance measurements have shown that porin OmpF trimers exist in either open or closed states, depending on the transmem- brane potential (Schindler & Rosenbusch, 1978; Lakey, 1987). The critical voltage above which channels close is V c > 90 mV for OmpF, depending on the ionic strength (Brunen & Engelhardt, 1993). As this voltage is larger than that expected for the outer membrane, the physiological relevance of voltage gating has been questioned (Sen et al., 1988). However, there is evidence that V C is affected by pH (Todt et al., 1992), and lowered by membrane-derived oligosaccharides (Delcour et al., 1992), polysaccharides (Schindler & Rosenbusch, 1981), polycations (dela Vega & Delcour, 1995), low ionic strength buffer (Brunen & Engelhardt, 1993) and pressure (Le Dain et al., 1996). Hence, voltage gating may be important under certain cir- cumstances. Although the structures of several por- ins have been solved (Weiss et al., 1991; Cowan et al., 1992; Schirmer, 1998), the mechanism of channel closure is not understood. A previous study of two-dimensional (2D) porin OmpF crystals with the atomic force microscope (AFM; Binnig et al., 1986) revealed two confor- mations of the extracellular surface (Schabert et al., 1995). We have since optimized the conditions to monitor conformational changes of proteins with the AFM at subnanometer resolution (Mu È ller et al., 1995, 1996, 1997b), and have analyzed several fac- tors that could induce the observed conformational transitions of porin OmpF. Figure 1 compares the atomic model of a rectangular porin crystal with the raw topographs recorded in buffer solution, operating the AFM in contact mode at minimal tip- loading force. Such crystals often consisted of two layers with the extracellular surfaces facing each other (Schabert et al., 1995). Topographs of the periplasmic OmpF surface displayed features that correlated directly with the periplasmic aspect of the atomic model rendered at 3 A Ê (Figure 1(a) arrows). To image the extracellular surface, the upper crystalline layer was removed with the sty- lus. Extracellular domains protruded 13(2) A Ê (n  78) from the lipid bilayer and revealed a sub- structure consistent with the extracellular aspect of the atomic model (Figure 1(b)). To explore a possible relationship between the conformational changes of the extracellular loops that had been observed (Schabert et al., 1995), and voltage gating (Schindler & Rosenbusch, 1978; Lakey, 1987; Todt et al., 1992; Buehler et al., 1991), E-mail address of the corresponding author: aengel@ubaclu.unibas.ch Abbreviations used: 2D, two-dimensional; AFM, atomic force microscope; HOPG, highly oriented pyrolytic graphite. Article No. jmbi.1998.2359 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 285, 1347±1351 0022-2836/99/041347±05 $30.00/0 # 1999 Academic Press