Energy-transducing Membrane Proteins 
Although site-directed mutagenesis is useful 
for delineating amino acid residues that are im- 
portant for lactose-H"^ symport and/or substrate 
binding and recognition, it has become apparent 
that high-resolution structure is required to be- 
gin to determine the role of these residues in the 
mechanism. Moreover, dynamic information at 
high resolution will also be required to solve the 
mechanism. In this respect, chemical labeling 
and spectroscopic approaches in w^hich reactive 
cysteinyl residues are tagged v^^ith radioactive 
sulfhydryl reagents, electron paramagnetic la- 
bels, or fluorescent probes are potentially power- 
ful means for examining static and dynamic 
aspects of protein structure-function relation- 
ships at high resolution. A principle difficulty 
with the general approach, however, is the com- 
plexity resulting from the presence of multiple 
cysteinyl residues in most proteins, eight in the 
case of lac permease. Thus, in addition to the im- 
portant conclusion that cysteinyl residues do not 
play an important role in the mechanism of lac 
permease, the construction of a functional per- 
mease molecule devoid of cysteinyl residues pro- 
vides the basis for an approach to the analysis of 
static and dynamic aspects of permease structure- 
function relationships. 
By using the lacY gtne encoding C-less per- 
mease, for instance, it is now possible to design 
mutants in which an individual amino acid resi- 
due in a putative hydrophilic or hydrophobic 
domain is replaced with a cysteinyl residue. 
This can then be reacted specifically with ei- 
ther permeant or impermeant sulfhydryl re- 
agents in right-side-out or inside-out membrane 
vesicles, followed by solubilization and im- 
munoprecipitation. In addition, single Cys mu- 
tants can be solubilized and purified, tagged 
with appropriate electron paramagnetic or fluo- 
rescent probes, then reconstituted and studied 
spectroscopically. 
Finally, it should be possible to study proxim- 
ity relationships between transmembrane do- 
mains by placing single cysteinyl residues in pairs 
of helical domains predicted to lie close to each 
other within the membrane. In these contexts, it 
is encouraging that more than 200 single Cys re- 
placements have been constructed in the C-less 
permease and that the great majority of the mu- 
tants exhibit highly significant transport activity. 
Lactose transport in Escherichia coli. A: 
Uphill lactose (Lac ) transport in response to 
generated either by respiration or A TP hy- 
drolysis. B: Uphill transport in response 
to an inwardly directed lactose gradient. C: 
Uphill transport in response to an out- 
wardly directed lactose gradient. 
From Kaback, H.R. 1989. Harvey Lect 
83:77-105. Copyright© 1989 Alan R. Liss, 
Inc. Reprinted by permission ofWiley-Liss, a 
division of John Wiley and Sons, Inc. 
214 
