The role of subunit a in promoting proton translocation and rotary motion in the Escherichia coli F 1Fo ATP synthase is poorly understood. In the membrane-bound F o sector of the enzyme, H ؉ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp-61 at the center of the membrane via aqueous channels formed at least in part by one or more of the five TMHs of subunit a. Aqueous access pathways have previously been mapped to surfaces of aTMH4. Here we have substituted Cys into the second and fifth TMHs of subunit a and carried out chemical modification with Ag ؉ and N-ethylmaleimide to define the aqueous accessibility of residues along these helices. Access to cAsp-61 at the center of the membrane may be mediated in part by Ag ؉ -sensitive residues 248, 249, 251, and 252 in aTMH5. From the periplasmic surface, aqueous access to cAsp-61 may be mediated by silver-sensitive residues 115, 116, 119, 120, 122, and 126 in aTMH2. The Ag ؉ -sensitive residues in TMH2, -4, and -5 form a continuum extending from the periplasmic to the cytoplasmic side of the membrane. In an arrangement of helices supported by second-site revertant and crosslinking analyses, these residues cluster at the interior of a four-helix bundle formed by TMH2-5. The aqueous access pathways at the interior of subunit a may be gated by a swiveling of helices in this bundle, alternately exposing cytoplasmic and periplasmic half channels to cAsp-61 during the H ؉ transport cycle. H ϩ transporting F 1 F o ATP synthases consist of two structurally and functionally distinct sectors termed F 1 and F o . In the intact enzyme, ATP synthesis or hydrolysis takes place in the F 1 sector and is coupled to active H ϩ transport through the F o sector. Structurally similar F 1 F o ATP synthases are present in mitochondria, chloroplasts, and most eubacteria (1). The F 1 sector lies at the surface of the membrane and in Escherichia coli consists of five subunits in an ␣ 3 ␤ 3 ␥ 1 ␦ 1 1 stoichiometry. The F o sector spans the membrane and in E. coli consists of three subunits in an a 1 b 2 c 10 stoichiometry (2). In the complete membranous enzyme, the rotation of subunit ␥ is proposed to be driven by H ϩ transport-coupled rotation of a connected ring of c subunits in the F o sector of the enzyme. The c subunit spans the membrane as a hairpin of two ␣-helices, and in the case of E. coli, contains the essential Asp-61 residue at the center of the second transmembrane helix (TMH). Asp-61 is thought to undergo protonation and deprotonation as each subunit of the oligomeric ring moves past a stationary subunit a. Subunit a is believed to provide access channels to the proton-binding Asp-61 residue, but the actual proton translocation pathway is only partially defined (3-5).Subunit a is known to fold with five TMHs (6-8), with aTMH4 packing in parallel to cTMH2, i.e., the helix to which Asp-61 is anchored (9). The interaction of the conserved Arg-210 residue in aTMH4 with cTMH2 is thought to be cri...

؉ -and NEM-sensitive residues were found at the cytoplasmic end of TMH5 and suggest a possible connection of this region to the NEM-and Ag ؉ -sensitive region of TMH4 described previously. From the now complete pattern of TMH residue reactivity, we conclude that aqueous access from the periplasmic side of F 0 to cAsp-61 at the center of the membrane is likely to be mediated by residues of TMHs 2, 3, 4, and 5 at the center of a four-helix bundle. Further, aqueous access between cAsp-61 and the cytoplasmic surface is likely to be mediated by residues in TMH4 and TMH5 at the exterior of the four-helix bundle that are in contact with the c-ring.

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Introduction: With the constant evolution of science and advancing technology, future physicians must learn to navigate an ever-changing health care environment by continuous learning throughout their professional careers. Lifelong, self-directed learning is a critical component of medical education to ensure future physicians are adept at identifying knowledge gaps and seeking, analyzing, and communicating new information. To train faculty who teach case-based, self-directed learning, we designed the Clumsy Horse Case. Methods: The Clumsy Horse Case was created as part of a faculty development program for facilitators of a new case-based, self-directed curriculum known as Patient-Centered Education (PaCE) Cases. An unfamiliar veterinary medicine case was designed to level the playing field for faculty from different specialty areas in order to provide an authentic self-directed learning experience. To determine effectiveness, faculty participants completed a standardized eight-question evaluation survey after the Clumsy Horse Case session, and facilitators received student feedback at the end of each semester via a standard faculty evaluation form. Results: Student ratings indicated that faculty were adequately prepared to be effective facilitators. The Clumsy Horse Case was an integral part of facilitator preparation and provided an engaging learning experience for over 60 faculty. Survey ratings and comments from faculty participants indicated a high level of engagement and satisfaction with the learning experience. Discussion: The Clumsy Horse Case is generalizable for developing faculty in any curriculum with a case-based, self-directed learning component. It can be modified to fit any school's curriculum and integrated into a professional development program.