A mutant of Escherichia coli lacking phosphatidylethanolamine (PE) and a monoclonal antibody (mAb 4B1) directed against a conformationally sensitive epitope (4B1) of lactose permease were used to establish a novel role for a phospholipid in the assembly of a membrane protein. Epitope 4B1 is readily detectable in spheroplasts and right-side-out membrane vesicles from PEcontaining but not from PE-deficient cells expressing lactose permease. Lactose permease from PE-containing membranes, but not from PE-deficient membranes, subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis is also recognized by mAb 4B1. If total E. coli phospholipids or PE (but not phosphatidylcholine, phosphatidylglycerol, or cardiolipin) are blotted on nitrocellulose sheets (Eastern blot) prior to transfer of proteins from SDSpolyacrylamide gels, the permease from PE-deficient cells regains its recognition by mAb 4B1. Therefore, PE is required during assembly to form epitope 4B1, but, once formed, sufficient "conformational memory" is retained in the permease to either retain or reform this epitope in the absence of PE. Lactose permease lacking epitope 4B1 can be induced to form the epitope if partially denatured and then renatured in the presence of PE specifically. These results establish for the first time a role for PE as a molecular chaperone in the assembly of the lactose permease.Only a limited number of reports address the role of the native phospholipid environment in the function and assembly of membrane proteins. Clearly, the amphipathic environment of the membrane is an important determinant in the folding and maintenance of membrane protein structure. However, what has not been widely considered is a role for individual phospholipids in determining the folding pathway for membrane proteins independent from the maintenance of the final structure, i.e. to act in the capacity of a non-protein molecular chaperone.Mutants of Escherichia coli are available in which membrane phospholipid composition can be varied in a way that is difficult to achieve in vitro (DeChavigny et al., 1991;Dowhan, 1992). Such "phospholipid mutants" were used to study the in vivo role of PE 1 in lactose permease (lacY gene product) function (Bogdanov and Dowhan, 1995). The lactose permease of E. coli is an extensively studied prototype of most secondary transport systems found in both prokaryotic and eukaryotic organisms . PE is not required for energyindependent downhill translocation of substrate mediated by lactose permease, but appears to be essential for H ϩ -coupled active lactose accumulation in vivo (Bogdanov and Dowhan, 1995). These results parallel the earlier observation (Chen and Wilson, 1984;Page et al., 1988) with purified permease reconstituted into proteoliposomes where PE was also found to be required specifically for active transport.How phospholipids affect membrane protein assembly and function is still largely unknown. Do the properties of the target membrane phospholipids affect assembly an...

Monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic surface of the lactose permease of Escherichia coli, uncoupling lactose and H+ translocation in a manner indicating that it blocks deprotonation [Carrasco, N., Viitanen, P., Herzlinger, D., & Kaback, H. R. (1984) Biochemistry 23, 3681; Herzlinger, D., Viitanen, P., Carrasco, N., & Kaback, H. R. (1984) Biochemistry 23, 3688]. In this paper, 4B1 binding to purified lactose permease is shown to exhibit a KD of about 5 x 10(-10) M by surface plasmon resonance. Furthermore, the combined use of mutants containing 6 contiguous His residues in each periplasmic loop in the permease and Cys-scanning mutagenesis in conjunction with chemical labeling demonstrates that 4B1 binds specifically to the periplasmic loop between helices VII and VIII and that Phe247 and Gly254 are the primary determinants. Remarkably, although 4B1 binding uncouples lactose and H+ translocation, none of the amino acid residues in periplasmic loops, particularly Phe247 or Gly254, play an important role in the transport mechanism. Moreover, binding of avidin to biotinylated Glu255-->Cys in the loop containing the epitope has no effect on transport activity. Therefore, the uncoupling effect of 4B1 involves highly specific interactions which in all likelihood exert a torsional effect on the loop, resulting in a conformational change in helix VII and/or VIII that alters the pKas of residues involved in lactose-coupled H+ translocation.

A series of site-directed mutants, E35Q, E39Q, and E35Q-D179N, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium, was created by overlap extension, using the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidases (MnPs) were purified and characterized. The molecular masses of the mutant proteins, as well as UV-vis spectral features of their oxidized states, were very similar to those of the wild-type enzyme. Resonance Raman spectral results indicated that the heme environment of the mutant MnP proteins also was similar to that of the wild-type protein. Steady-state kinetic analyses of the E35Q and E39Q mutant MnPs yielded K(m) values for the substrate MnII that were approximately 50-fold greater than the corresponding K(m) value for the wild-type enzyme. Likewise, the kcat values for MnII oxidation were approximately 300-fold lower than that for wild-type MnP. With the E35Q-D179N double mutant, the K(m) value for MnII was approximately 120-fold greater, and the kcat value was approximately 1000-fold less than that for the wild-type MnP1. Transient-state kinetic analysis of the reduction of MnP compound II by MnII allowed the determination of the equilibrium dissociation constants (KD) and first- order rate constants for the mutant proteins. The KD values were approximately 100-fold higher for the single mutants and approximately 200-fold higher for the double mutant, as compared with the wild-type enzyme. The first-order rate constants for the single and double mutants were approximately 200-fold and approximately 4000-fold less, respectively, than that of the wild-type enzyme. In contrast, the K(m) values for H2O2 and the rates of compound I formation were similar for the mutant and wild-type MnPs. The second-order rate constants for p-cresol and ferrocyanide reduction of the mutant compounds II also were similar to those of the wild-type enzyme.

The proximal iron ligand in horseradish peroxidase (HRP) is His-170. The H170A mutant of polyhistidine-tagged HRP (hHRP) has been expressed in a baculovirus system and has been purified and characterized. At pH 7, the Soret maximum of the mutant is at 414 nm rather than 403 nm. Resonance Raman spectra indicate that the protein is primarily 6-coordinate low-spin in the ferric state with a band in the ferrous state at 212 cm-1 indicative of distal histidine coordination to the iron. Exogenous imidazole (Im) binds to the enzyme with Kd = 22 +/- 4 mM. Reaction of H170A hHRP with H2O2 does not give spectroscopically detectable compound I or compound II intermediates but results in gradual degradation of the heme group. Nevertheless, H170A hHRP is catalytically active, and its guaiacol and ABTS peroxidase activities are improved 260- and 125-fold, respectively, in the presence of saturating concentrations of Im. The Km for the stimulatory effect of Im is 24 mM for both guaiacol and ABTS. The pH profile of H170A hHRP differs from that of wild-type hHRP, but the differences are essentially eliminated by Im. The rate of formation of "compound I" for H170A hHRP, determined by steady state kinetic methods, is k1 = 16 M-1 s-1 without Im and k1 = 2.4 x 10(4) M-1 s-1 with Im. The corresponding rate for wild-type hHRP is k1 = 4.4 x 10(6) M-1 s-1. The results indicate that Im binds in the cavity created by the H170A mutation, coordinates to the heme iron atom, and restores a large part of the catalytic activity by rescuing the rate of compound I formation. However, this rescue of the catalytic activity by Im is possibly limited by coordination of the heme to the distal histidine (His-42) in the H170A mutant. Thus, a primary function of the proximal histidine is to tether the iron atom to disfavor sixth ligand binding, particularly coordination of the iron to the distal histidine. In addition, strong hydrogen bonding of the proximal ligand may be critical for facilitating O-O bond cleavage in the formation of compound I.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VI11 and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to >70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262 4 Cys, Gly 268 + Cys, Asn 272 + Cys, Pro 280 + Cys, Asn 284 + Cys, Gly 287 + Cys, and Gly 288 + Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As a-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S , Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ. Keywords: active transport; bioenergetics; Cys modification; Cys replacementThe lactose permease of Escherichin coli, which catalyzes the coupled stoichiometric translocation of P-galactosides and H+ as a monomer, is an integral membrane protein with 12-transmembrane domains that are most probably in a-helical conformation (reviewed in . Site-directed mutagenesis of wild-type permease and Cys-scanning mutagenesis of a functional mutant devoid of Cys residues (C-less permease) have provided important insights into the structure and mechanism of the permease. Although as few as four amino acid residues [Glu 269 (helix VIII), Arg 302 (helix IX), His 322 (helix X), and Glu 325 (helix X)] appear to be critically involved in the symport mechanism, the activity of various active Cys-replacement mutants is altered by alkylation, and these mutants appear in clusters, suggesting that helical interfaces within the permease are important for turnover. Moreover, site-directed fluorescence spectroscopy (Jung et al

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The human C3a anaphylatoxin receptor~C3aR! is a G protein-coupled receptor~GPCR! composed of seven transmembrane a-helices connected by hydrophilic loops. Previous studies of chimeric C3aR0C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A~helix IV!, R340A~helix V!, and D417Ã helix VII! showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81Ã helix II! and K96A~helix III! showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and0or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.

Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIA glc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIA glc with membrane-associated lactose permease was used to characterize the binding reaction. The N-and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIA glc . Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV͞V and VI͞VII and periplasmic loop VII͞VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIA glc , indicating that these regions of lactose permease may be involved in recognition of enzyme IIA glc . Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIA glc . A topological map of the proposed interaction is presented.Bacteria use a wide range of carbon sources that are transported across the cytoplasmic membrane by a variety of specific transport systems. The phosphoenolpyruvate:sugar phosphotransferase system (PTS), a multifunctional system consisting of several protein components, is widespread in bacteria. The primary function of the PTS is the concomitant phosphorylation and translocation of a variety of sugar substrates from the medium (1, 2). In the case of glucose transport in Gram-negative bacteria, three soluble proteins (enzyme I, HPr, and enzyme IIA glc ) and one membrane-bound protein (enzyme IIB, C glc ) are required for transport. In Escherichia coli, the PTS also regulates uptake of various non-PTS sugars such as lactose, maltose, melibiose, galactose, and raffinose (inhibition of non-PTS inducer transport by PTS sugars is termed ''inducer exclusion''), as well as the phosphorylation of glycerol and the synthesis of cyclic AMP (catabolite repression) (2-6). These regulatory roles of the PTS have been proposed to be mediated by the glucosespecific enzyme IIA glc , and the currently accepted model suggests that the phosphorylation state of IIA glc determines whether or not it binds to a target protein (2).E. coli lactose permease (lac permease) is an important model for the study of membrane transport proteins. Lac permease is one of the most extensively investigated secondary active transport proteins that transduce free energy stored in an electrochemical ion gradient into a solute concentration gradient (7-9). It is a hydrophobic, polytopic membrane protein that catalyzes the coupled stoichiometric translocation of ␤-galactosides and H ϩ . All available evidence indicates strongly that lac permease is comprised of a bundle of 12 transmembrane ␣-h...