The role of helix VIII in the lactose permease of Escherichia coli: II. Site-directed sulfhydryl modification.

Cys-scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S. Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431-437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport. In this study, the rate of inactivation of each N-ethylmaleimide (NEM)-sensitive mutant is examined in the absence or presence of beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). Remarkably, the analogue affords protection against inactivation with mutants Val 264-->Cys, Gly 268-->Cys, and Asn 272-->Cys, and alkylation of these single-Cys mutants in right-side-out membrane vesicles with [14C]NEM is attenuated by TDG. In contrast, alkylation of Thr 265-->Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue. Furthermore, NEM-labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent. Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262-->Cys, Glu 269-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, or Ala 279-->Cys. The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand-induced conformational change.     

Fluorescence of native single-Trp mutants in the lactose permease from Escherichia coli: structural properties and evidence for a substrate-induced conformational change.

Six single-Trp mutants were engineered by individually reintroducing each of the native Trp residues into a functional lactose permease mutant devoid of Trp (Trp-less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Natl Acad Sci USA 87:1638-1642), and fluorescent properties were studied with respect to solvent accessibility, as well as alterations produced by ligand binding. The emission of Trp 33, Trp 78, Trp 171, and Trp 233 is strongly quenched by both acrylamide and iodide, whereas Trp 151 and Trp 10 display a decrease in fluorescence in the presence of acrylamide only and no quenching by iodide. Of the six single-Trp mutants, only Trp 33 exhibits a significant change in fluorescence (ca. 30% enhancement) in the presence of the substrate analog beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). This effect was further characterized by site-directed fluorescent studies with purified single-Cys W33-->C permease labeled with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS). Titration of the change in the fluorescence spectrum reveals a 30% enhancement accompanied with a 5-nm blue shift in the emission maximum, and single exponential behavior with an apparent KD of 71 microM. The effect of substrate binding on the rate of MIANS labeling of single-Cys 33 permease was measured in addition to iodide and acrylamide quenching of the MIANS-labeled protein. Complete blockade of labeling is observed in the presence of TDG, as well as a 30% decrease in accessibility to iodide with no change in acrylamide quenching. Overall, the findings are consistent with the proposal (Wu J, Frillingos S, Kaback HR, 1995a, Biochemistry 34:8257-8263) that ligand binding induces a conformational change at the C-terminus of helix I such that Pro 28 and Pro 31, which are on one face, become more accessible to solvent, whereas Trp 33, which is on the opposite face, becomes less accessible to the aqueous phase. The findings regarding accessibility to collisional quenchers are also consistent with the predicted topology of the six native Trp residues in the permease.     

A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential.

Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N-ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (delta microH+; Sahin-Tóth M, Kaback HR, 1993, Protein Sci 2:1024-1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315-->Cys permease reacts with N-ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and beta-galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315-->Cys permease is enhanced by imposition of a membrane potential (delta psi, interior negative). The results indicate that either ligand binding or delta psi induces a conformational change in the permease that brings the N-terminus of helix X into an environment that is more accessible from the lipid phase.     

The role of helix VIII in the lactose permease of Escherichia coli: I. Cys-scanning mutagenesis.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII 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-->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 expected (Ujwal ML, Sahin-Tóth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys. Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys). The results indicate that transmembrane domain VIII is in alpha-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.     

A mutation in the lactose permease of Escherichia coli that decreases conformational flexibility and increases protein stability

Lactose permease with Cys154 --> Gly (helix V) binds substrate with high affinity but catalyzes little or no transport. The purified, detergent-solubilized mutant protein exhibits much greater thermal stability than the wild type and little tendency to aggregate. Stabilization is also observed in vivo with an unstable mutant that is expressed at significantly higher levels when the Cys154 --> Gly mutation is introduced. In addition, ligand-induced conformational changes are markedly reduced or abolished by the Cys154 --> Gly mutation: (i) Although the fluorescence of purified single Trp33 (helix I) permease is enhanced by ligand binding, introduction of the Cys154 --> Gly mutation abolishes the effect. (ii) The rate of 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) labeling of permease with a single Cys residue in place of Val331 (helix X) is increased in the presence of ligand but reduced when the Cys154 --> Gly mutation is present. (iii) Fluorescence emission intensity of MIANS-labeled single Cys331 permease is enhanced and blue shifted in the Cys154 --> Gly mutant background, indicating that the latter mutation causes position 331 to become exposed to a less polar environment. The results indicate that the Cys154 --> Gly mutation causes a more compact structure and decreased conformational flexibility, an alteration that specifically blocks the structural changes necessary for substrate translocation with little or no effect on ligand binding.     

Cysteine scanning mutagenesis of putative transmembrane helices IX and X in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino-acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn-290 to Lys-335). Thirty-four of 46 mutants accumulate lactose to high levels (70-100% or more of C-less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation. As expected (see Kaback, H.R., 1992, Int. Rev. Cytol. 137A, 97-125), Cys substitution for Arg-302, His-322, or Glu-325 results in inactive permease molecules. Although Cys replacement for Lys-319 or Phe-334 also inactivates lactose accumulation, Lys-319 is not essential for active lactose transport (Sahin-Tóth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc. Natl. Acad. Sci. USA 89, 10547-10551), and replacement of Phe-334 with leucine yields permease with considerable activity. All single-Cys mutants except Gly-296 --> Cys are present in the membrane in amounts comparable to C-less permease, as judged by immunological techniques. In contrast, mutant Gly-296 --> Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from T7 promoter. Finally, studies with N-ethylmaleimide (NEM) show that only a few mutants are inactivated significantly. Remarkably, the rate of inactivation of Val-315 --> Cys permease is enhanced at least 10-fold in the presence of beta-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or an H+ electrochemical gradient (delta mu-H+). The results demonstrate that only three residues in this region of the permease -Arg-302, His-322, and Glu-325-are essential for active lactose transport. Furthermore, the enhanced reactivity of the Val-315 --> Cys mutant toward NEM in the presence of TDG or delta mu-H+ probably reflects a conformational alteration induced by either substrate binding or delta mu-H+.     

The relationship between MRP1 activities and its NBD conformational changes

MIANS, a sulfhydryl-reactive fluorescence, was used to label the cysteines of MRP1 (multidrug resistance protein), and the results indicated that an increase in fluorescence intensity and a large emission blue shift took place after two Cys residues of MRP1 reacted with MIANS, which demonstrated that labeled Cys residues in MRP1 reside in a relatively hydrophobic environment. The experimental results obtained from fluorescence resonance energy transfer further uncover that two Cys residues of MRP1 modified by MIANS located in the vicinity of its NBDs, of which one lies close to NBD1, and the other near NBD2. ATP, ADP and anticancer drugs can all reduce the rate of reaction of MRP1 with MIANS. The collisional quenchers, acrylamide, I^-1, and Cs⁺ were used to assess local environments of MIANS bound to MRP1 and the results showed that the region around the MIANS-labeled cysteine is positively charged. Both MIANS and NEM, which are sulfhydryl-reactive reagents, inhibited MRP1 ATPase activity, whereas anticancer drugs activated it. These results demonstrated that all nucleotides and drugs could induce changes in conformation of the NBDs in MRP1. Nucleotides can bind directly to NBDs, but drugs may react first with TMDs, which in turn alters the accessibility of the two Cys residues bound by MIANS and affects MRP1 ATPase activity, which is coupled with the transport of its substrates. Taken together, the above experimental results provide direct evidence for further study on the coupling of translocation of the transported species to hydrolysis of ATP in MRP1.     

The relationship between MRP1 activities and its NBD conformational changes

The term multidrug resistance (MDR) is classi-cally used to define a resistance phenotype where cells become resistant simultaneously to different drugs with no obvious structural resemblance and with dif-ferent cellular targets. For some time after P-gp, the first membrane protein related with MDR, was dis-covered in 1976[1], it was widely believed that P-gp was the exclusive cause of multidrug resistance. However, increasing evidence afterward suggested that this was not the case. Several…     

Suppressor analysis of mutations in the loop 2-3 motif of lactose permease: evidence that glycine-64 is an important residue for conformational changes.

A superfamily of transport proteins, which includes the lactose permease of Escherichia coli, contains a highly conserved motif, G-X-X-X-D/E-R/K-X-G-R/K-R/K, in the loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. Previous analysis of this motif in the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995) has shown that the conserved glycine residue found at the first position in the motif (i.e., Gly-64) is important for transport function. Every substitution at this site, with the exception of alanine, greatly diminished lactose transport activity. In this study, three mutants in which glycine-64 was changed to cysteine, serine, and valine were used as parental strains to isolate 64 independent suppressor mutations that restored transport function. Of these 64 isolates, 39 were first-site revertants to glycine or alanine, while 25 were second-site mutations that restored transport activity yet retained a cysteine, serine, or valine at position 64. The second-site mutations were found to be located at several sites within the lactose permease (Pro-28 --> Ser, Leu, or Thr; Phe-29 --> Ser; Ala-50 --> Thr, Cys-154 --> Gly; Cys-234 --> Phe; Gln-241 --> Leu; Phe-261 --> Val; Thr-266 --> Iso; Val-367 --> Glu; and Ala-369 --> Pro). A kinetic analysis was conducted which compared lactose uptake in the three parental strains and several suppressor strains. The apparent Km values of the Cys-64, Ser-64, and Val-64 parental strains were 0.8 mM, 0.7 mM, and 4.6 mM, respectively, which was similar to the apparent Km of the wild-type permease (1.4 mM). In contrast, the Vmax values of the Cys-64, Ser-64, and Val-64 strains were sharply reduced (3.9, 10.1, and 13.2 nmol of lactose/min x mg of protein, respectively) compared with the wild-type strain (676 nmol of lactose/min x mg of protein). The primary effect of the second-site suppressor mutations was to restore the maximal rate of lactose transport to levels that were similar to the wild-type strains. Taken together, these results support the notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes that are necessary for lactose translocation across the membrane. According to our tertiary model, this site is at an interface between the two halves of the protein.     

The role of transmembrane domain III in the lactose permease of Escherichia coli.

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty-one of the 25 mutants accumulate lactose to > 70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport to lower, but significant levels (40-60% of C-less). Cys replacement for Leu 76 results in low transport activity (18% of C-less). However, when placed in the wild-type background, mutant Leu 76-->Cys exhibits highly significant rates of transport (55% of wild type) and steady-state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that mutant Gly 96-->Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96-->Cys permease is enhanced at least 2-fold in the presence of beta-galactopyranosyl 1-thio-beta, D-galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.     

Cysteine scanning mutagenesis of the N-terminal 32 amino acid residues in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in the hydrophilic N-terminus and the first putative transmembrane helix was systematically replaced with Cys (from Tyr-2 to Trp-33). Twenty-three of 32 mutants exhibit high lactose accumulation (70-100% or more of C-less), and an additional 8 mutants accumulate to lower but highly significant levels. Surprisingly, Cys replacement for Gly-24 or Tyr-26 yields fully active permease molecules, and permease with Cys in place of Pro-28 also exhibits significant transport activity, although previous mutagenesis studies on these residues suggested that they may be required for lactose transport. As expected, Cys replacement for Pro-31 completely inactivates, in agreement with previous findings indicating that "helix-breaking" propensity at this position is necessary for full activity (Consler TG, Tsolas O, Kaback HR, 1991, Biochemistry 30:1291-1297). Twenty-nine mutants are present in the membrane in amounts comparable to C-less permease, whereas membrane levels of mutants Tyr-3-->Cys and Phe-12-->Cys are slightly reduced, as judged by immunological techniques. Dramatically, mutant Phe-9-->Cys is hardly detectable when expressed from the lac promoter/operator at a relatively low rate, but is present in the membrane in a stable form when expressed at a high rate from the T7 promoter. Finally, studies with N-ethylmalemide show that 6 Cys-replacement mutants that cluster at the C-terminal end of putative helix I are inactivated significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution 1H nuclear magnetic resonance determination of the distal pocket structure of cyanomet complexes of genetically engineered sperm whale myoglobin His64 (E7)-->Val, Thr67 (E10)-->Arg. The role of distal hydrogen bonding by Arg67 (E10) in modulating ligand tilt.

Sequence-specific 2D methodology has been used to assign the 1H NMR signals for all active site residues in the paramagnetic cyano-met complexes of sperm whale synthetic double mutant His64[E7]-->Val/Thr67[E10]-->Arg (VR-met-MbCN) and triple mutant His64[E7]-->Val/Thr67[E10]-->Arg/Arg45[CD3]-->Asn (VRN-metMbCN). The resulting dipolar shifts for noncoordinated proximal side residues were used to quantitatively determine the orientation of the paramagnetic susceptibility tensor in the molecular framework for the two mutants, which were found indistinguishable but distinct from those of both wild-type and the His64[E7]-->Val single point mutant (V-metMbCN). The observed dipolar shifts for the E helix backbone protons and Phe43[CD1], together with steady-state nuclear Overhauser effect between the E helix and the heme, were analyzed to show that both the E helix and Phe43[CD1] move slightly closer to the iron to minimize the vacancy resulting from the His64[E7]-->Val substitution, as found in V-metMbCN (Rajarathnam, K., J. Qin, G.N. LaMar, M. L. Chiu, and S. G. Sligar. 1993. Biochemistry. 32:5670-5680). The dipolar shifts of the mutated Val64[E7] and Arg67[E10] allow the determination of their orientations relative to the heme, and the latter residue is shown to insert into the pocket and provide a hydrogen bond to the coordinated ligand, as found in the naturally occurring ValE7/ArgE10 genetic variant, Aplysia limacina Mb. The oxy-complex of both A. limacina Mb and VR-Mb, VRN-Mb have been proposed to be stabilized by this hydrogen bonding interaction (Travaglini Allocatelli, C. et al. 1993. Biochemistry. 32:6041-6049). The magnitude of the tilt of the major magnetic axes from the heme normal in VR-metMbCN and VRN-metMbCN, which is related to the tilt of the ligand, is the same as in wild-type or V-metMbCN, but the direction of tilt is altered from that in V-metMbCN. It is concluded that the change in the direction of the ligand tilt in both the double and triple mutants, as compared to WT metMbCN and V-metMbCN single mutant, is due to the attractive hydrogen-bonding between ArgE10 and the bound cyanide.     

The sucrose permease of Escherichia coli: functional significance of cysteine residues and properties of a cysteine-less transporter

The sucrose (CscB) permease belongs to the oligosaccharide:H(+) symporter family of the Major Facilitator Superfamily and is homologous to the lactose permease from Escherichia coli. Sucrose transport in cells expressing sucrose permease is completely inhibited by N-ethylmaleimide (NEM), suggesting that one or more of the seven native Cys residues may be important for transport. In this paper, each Cys residue was individually replaced with Ser, and transport activity, membrane expression, and NEM sensitivity are documented. All seven single Cys-->Ser mutants are expressed normally in the membrane and catalyze sucrose transport with activities ranging from 80% to 180% of wild type. Six of the seven Ser mutants are completely inactivated by NEM, while Cys122-->Ser permease is insensitive to the sulfhydryl reagent, indicating that NEM inhibition results from alkylation of Cys122. Subsequently, a sucrose permease devoid of Cys residues (Cys-less) was constructed in which all Cys residues were replaced with Ser simultaneously by using a series of overlap-extension PCRs. Membrane expression and kinetic parameters for Cys-less [K(m) 4.8 mM, V(max) 192 nmol min(-1) (mg of protein)(-1)] are essentially identical to those of wild type [K(m) 5.4 mM, V(max) 196 nmol min(-1) (mg of protein)(-1)]. However, Cys-less permease catalyzes sucrose accumulation to steady-state levels that are approximately 2-fold higher than those of wild type. As anticipated, Cys-less permease is completely resistant to NEM inhibition. The observations demonstrate that Cys residues play no functional role in sucrose permease. Furthermore, the approach described to create the Cys-less transporter is generally applicable to other proteins. An application of Cys-less permease in the study of the substrate binding site is presented in the accompanying paper.     

Myoglobin mutants giving the largest geminate yield in CO rebinding in the nanosecond time domain.

We have measured the rebinding of carbon monoxide (CO) to some distal mutants of myoglobin (Mb) in the time range from 10(-8) to 10(-1) s by flash photolysis, in which the photodissociated CO rebinds to the heme iron without escaping to the solvent water from the protein matrix. We have found that the double mutants [His64-->Val/Val68-->Thr (H64V/V68T) and His64-->Val/Val68-->Ser (H64V/V68S)] have an extremely large geminate yield (70-80%) in water at 5 degreesC, in contrast to the 7% of the geminate yield of wild-type Mb. The CO geminate yields for these two mutants are the largest in those of Mb mutants reported so far, showing that the two mutants have a unique heme environment that favors CO geminate rebinding. Comparing the crystal structures and 1H-NMR and vibrational spectral data of H64V/V68T and H64V/V68S with those of other mutants, we discuss factors that may control the nanosecond geminate CO rebinding and CO migration in the protein matrix.     

Cys(577) is a conformationally mobile residue in the ATP-binding domain of the Na,K-ATPase alpha-subunit.

2-[4'-Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mM ATP or low K(+) (<1 mM); the protective effect K(+) is reversed at higher concentrations. This biphasic effect was also observed with K(+) congeners. In contrast, Na(+) ions did not protect. MIANS inactivation disrupted high affinity ATP binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography. ATP clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of approximately 5 kDa. N-terminal amino acid sequencing identified the peptide (V(545)LGFCH...). This hydrophobic peptide contains only two Cys residues in all sodium pump alpha-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with ATP binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys(577). The cation-dependent change in reactivity of Cys(577) implies structural alterations in the ATP-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-ATPase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the ATP hydrolysis domain.     

The response of T4 lysozyme to large-to-small substitutions within the core and its relation to the hydrophobic effect.

To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the "large-to-small" substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts < or = 0.3 A); in other cases mainchain atoms moved up to 2 A. In the case of Ile 29 --> Ala the structure collapsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 --> Ala (V149A) and Met 6 --> Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the cavity. The Val 149 --> Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 --> Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., -2.1 kcal/mol for all Leu --> Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol(-1) A(-3). Physically, this term is due to the loss of van der Waal''s interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu --> Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255:178-183).     

Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor.

Most G protein-coupled receptors contain a series of highly conserved proline and tryptophan residues within their hydrophobic transmembrane domains (TMD I-VII). To study their potential role in ligand binding and receptor function, the rat m3 muscarinic acetylcholine receptor was used as a model system. A series of mutant receptors in which the conserved proline and tryptophan residues were individually replaced with alanine and phenylalanine, respectively, was created and transiently expressed in COS-7 cells. [3H]N-methylscopolamine ([3H]NMS) saturation binding studies showed that three of the seven mutant receptors studied (Pro242-->Ala, TMD V; Pro505-->Ala, TMD VI; Pro540-->Ala, TMD VII) were expressed at 35-100 times lower levels than the wild-type receptor while displaying 'm3-like' antagonist binding affinities. Pro201-->Ala (TMD IV) showed drastically reduced binding affinities (up to 450-fold) for both muscarinic agonists and antagonists. Whereas most mutant receptors retained strong functional activity, Pro540-->Ala (TMD VII) was found to be severely impaired in its ability to stimulate carbachol-induced phosphatidyl inositol hydrolysis (Emax approximately 25% of wild type m3). Interestingly, this mutant receptor bound muscarinic agonists with 7- to 19-fold higher affinities than the wild type receptor. The Trp-->Phe substitutions (Trp192-->Phe, TMD IV; Trp503-->Phe, TMD VI; Trp530-->Phe, TMD VII) resulted in less pronounced changes (compared with the Pro-->Ala mutant receptors) in both ligand binding and receptor function. Our data indicate that the proline residues that are highly conserved across the entire superfamily of G protein-coupled receptors play key roles in receptor expression, ligand binding and receptor activation.     

Molecular analysis of Hurler syndrome in Druze and Muslim Arab patients in Israel: multiple allelic mutations of the IDUA gene in a small geographic area.

The mutations underlying Hurler syndrome (mucopolysaccharidosis IH) in Druze and Muslim Israeli Arab patients have been characterized. Four alleles were identified, using a combination of (a) PCR amplification of reverse-transcribed RNA or genomic DNA segments, (b) cycle sequencing of PCR products, and (c) restriction-enzyme analysis. One allele has two amino acid substitutions, Gly409-->Arg in exon 9 and Ter-->Cys in exon 14. The other three alleles have mutations in exon 2 (Tyr64-->Ter), exon 7 (Gln310-->Ter), or exon 8 (Thr366-->Pro). Transfection of mutagenized cDNAs into Cos-1 cells showed that two missense mutations, Thr366-->Pro and Ter-->Cys, permitted the expression of only trace amounts of alpha-L-iduronidase activity, whereas Gly409-->Arg permitted the expression of 60% as much enzyme as did the normal cDNA. The nonsense mutations were associated with abnormalities of RNA processing: (1) both a very low level of mRNA and skipping of exon 2 for Tyr64-->Ter and (2) utilization of a cryptic splice site for Gln310-->Ter. In all instances, the probands were found homozygous, and the parents heterozygous, for the mutant alleles, as anticipated from the consanguinity in each family. The two-mutation allele was identified in a family from Gaza; the other three alleles were found in seven families, five of them Druze, residing in a very small area of northern Israel. Since such clustering suggests a classic founder effect, the presence of three mutant alleles of the IDUA gene was unexpected.     

Conservative and nonconservative mutations in proteins: anomalous mutations in a transport receptor analyzed by free energy and quantum chemical calculations.

Experimental studies on a bacterial sulfate receptor have indicated anomalous relative binding affinities for the mutations Ser130-->Cys,Ser130-->Gly, and Ser130-->Ala. The loss of affinity for sulfate in the former mutation was previously attributed to a greater steric effect on the part of the Cys side chain relative to the Ser side chain, whereas the relatively small loss of binding affinity for the latter two mutations was attributed to the loss of a single hydrogen bond. In this report we present quantum chemical and statistical thermodynamic studies of these mutations. Qualitative results from these studies indicate that for the Ser130-->Cys mutation the large decrease in binding affinity is in part caused by steric effects, but also significantly by the differential work required to polarize the Cys thiol group relative to the Ser hydroxyl group. The Gly mutant cobinds a water molecule in the same location as the Ser side chain resulting in a relatively small decrease in binding affinity. Results for the Ala mutant are in disagreement with experimental results but are likely to be limited by insufficient sampling of configuration space due to physical constraints applied during the simulation.     

Purification and some properties of alpha-L-arabinofuranosidase from Bacillus subtilis 3-6.

alpha-L-Arabinofuranosidase (EC 3.2.1.55) was purified from culture supernatant of Bacillus subtilis 3-6. The enzyme had a molecular weight of 61,000 and displayed maximum activity at pH 7.0 and 60 degrees C. It released arabinose from O-alpha-L-arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-x ylopyranos e (A1X2), O-beta-D-xylopyranosyl-(1-->4)-[O-alpha-L-arabinofuranosyl-(1-->3)]- O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (A1X3), and arabinan, but not from O-beta-D-xylopyranosyl-(1-->2)-O-alpha-L- arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-O-beta-D-xylopyr anosyl- (1-->4)-D-xylopyranose (A1X4), arabinoxylan, gum arabic, or arabinogalactan.