comparable to residues bl-34, which contains the membrane-spanning domain, dissolved

in a 4:4: 1 v/v mixture of chloroform/methanol/water previously used for solving the

structure of subunit c, has been solved by NMR (136). The data revealed an a-helical

monomeric structure with a 20o bend at residues 23-26 (KYVW) (Figure 1-5). The

hydrophobic residues, b4-22, formed an a-helix, followed by the 200 bend, and then

resumed with a-helical structure from residues b27-34. The bend was proposed to be

positioned as the b subunit exits the membrane. A series of cysteine substitutions

resulted in a high yield of crosslinks formed at residues b2, b6, and blo (Figure 1-5). A

lower yield of crosslinks were observed to form at residues b3, bs, b9 and bll (Figure 1-5).

No crosslinks were observed when cysteines were substituted for residues 12-21. The

observance of continuous crosslinks between residues b6-11 WeTO Suggested to indicate a

dynamic interaction between the contacting faces of the two b subunits in this region

(132). These observations led to a dimeric model in which the extreme amino-termini of

the b subunits crossed each other in close proximity at an angle of about 350 in the region

of residues b4-11, and then the two b subunits angled apart as they traverse the membrane

towards the cytoplasmic side (Figure 1-5) (136). The region of the 200 bend, b23-26, WAS

suggested to change the direction of the second a-helix, b27-33, Such that it would extend

into the cytoplasm at an angle perpendicular to the plan of the membrane. This model

was confirmed by a systematic mutational analysis of the membrane-spanning domain

performed by Hardy et al. (202).

 The tether domain of the b subunit, roughly b24-60, iS the least defined part of the

subunit domains from a structural point of view. It corresponds to the portion of the

peripheral stalk often seen in electron micrographs and is called "tether" simply because