Identification and sequence analysis of the gene mutated in the conditionally lethal outer membrane permeability mutant SS-C of Salmonella typhimurium.

The biosynthesis and structure-function relationships of the enterobacterial outer membrane are subjects of current intensive research. We have previously described the antibiotic supersensitive SS-C mutant (SH7622) of Salmonella typhimurium and shown that its outer membrane permeability barrier against hydrophobic antibiotics is severely defective. In this study, we show that this mutant is heat-sensitive, conditionally lethal, and carries a missense base-pair substitution in a novel gene which we have recently reported and now named the ssc gene. ssc encodes an earlier uncharacterized 36 kd protein (the Ssc protein) and the mutant expresses Ssc which has valine 291 changed to methionine in a methionine-rich region of Ssc. A plasmid containing the wild-type ssc allele completely reverts the antibiotic- and heat-sensitive phenotype of the SS-C mutant. Corresponding plasmids carrying the mutant allele, or an identical mutant allele prepared by localized mutagenesis, are inactive. The ssc gene is probably analogous to the firA locus of Escherichia coli which has earlier been implicated in a totally different function, mRNA synthesis. Furthermore, ssc apparently lies very close to the lpx genes involved in the thus far known steps of lipid A biosynthesis (the distance, approximately 560 bp). To conclude, our findings define a new essential gene involved in the generation of the outer membrane.     

Effects of the phenylalanine-22----leucine, glutamic acid-49----methionine, glycine-234----aspartic acid, and glycine-234----lysine mutations on the folding and stability of the alpha subunit of tryptophan synthase from Escherichia coli

The effects of four single amino acid replacements on the stability and folding of the alpha subunit of tryptophan synthase from Escherichia coli have been investigated by ultraviolet differences spectroscopy. In previous studies [Miles, E. W., Yutani, K., & Ogasahara, K. (1982) Biochemistry 21, 2586], it had been shown that the urea-induced unfolding at pH 7.8, 25 degrees C, proceeds by the initial unfolding of the less stable carboxyl domain (residues 189-268) followed by the unfolding of the more stable amino domain (residues 1-188). The effects of the Phe-22----Leu, Glu-49----Met, Gly-234----Asp, and Gly-234----Lys mutants on the equilibrium unfolding process can all be understood in terms of the domain unfolding model. With the exception of the Glu-49----Met replacement, the effects on stability are small. In contrast, the effects of three of the four mutations on the kinetics of interconversion of the native form and one of the stable partially folded intermediates are dramatic. The results for the Phe-22----Leu and Gly-234----Asp mutations indicate that these residues play a key role in the rate-limiting step. The Glu-49----Met mutation increases the stability of the native form with respect to that of the intermediate but does not affect the rate-limiting step. The Gly-234----Lys mutation does not affect either the stability or the kinetics of folding for the transition between native and intermediate forms. The changes in stability calculated from the unfolding and refolding rate constants agree quantitatively with those obtained from the equilibrium data. When considered with the results from a previous study on the Gly-211----Glu replacement [Matthews, C. R., Crisanti, M. M., Manz, J. T., & Gepner G. L. (1983) Biochemistry 22, 1445], it can be concluded that the rate-limiting step in the conversion of the intermediate to the native conformation involves either domain association or some other type of molecule-wide phenomenon.     

Eight bacterial proteins, including UDP-N-acetylglucosamine acyltransferase (LpxA) and three other transferases of Escherichia coli, consist of a six-residue periodicity theme

Only a few prokaryotic or eukaryotic enzymes are known to consist of a tandem-repeat structure. This report describes a common hexapeptide-repeat theme in four Escherichia coli transferases and in four less-characterized bacterial proteins. The proteins are the Ssc protein of Salmonella typhimurium (25), UDP-N-acetylglucosamine acyltransferase of E. coli (24), the hypothetical proteins Tms of Bacillus subtilis (23) and Yglm of E. coli (22), succinyldiaminopimelate aminotransferase of E. coli (14), serine acetyltransferase of E. coli (13), NodL of Rhizobium leguminasorum (13), and thiogalactoside acetyltransferase of E. coli (8) (number of repeats indicated in parentheses). In UDP-N-acetylglucosamine acyltransferase, the repeats constitute 55% of the total protein. Each hexapeptide repeat of the eight proteins starts with Ile, Leu, or Val. Position b is occupied by Gly, position d by Gly, Asn, or Asp, and position e by Val or Ala in 52%, 54%, and 56% of the hexapeptide repeats, respectively.     

Amino acid substitutions in the beta-subunit of RNA-polymerase from E. coli compensating for mutation-induced damage of the rho termination factors

Ts-phenotype of the E. coli rho-factor mutant rho 15 is suppressed by two rifampicin-resistance mutations, rhoB1019 resulting in a single amino acid substitution Val146----Phe and rhoB268 resulting in a single substitution Gln513----Leu in beta-subunit of the E. coli RNA polymerase. Rifampicin-resistance mutations rhoB255 (Asp516----Val), rhoB1016 (Asp516----Asn), rhoB1001 (His526----Tyr), rhoB1004 (Ser531----Phe), rhoB1005 (Pro564----Leu), and streptolydigin-resistance' mutation rhoB1018 (double substitution Gly544----Asp and Phe545----Ser) do not suppress the rho15 mutation.     

Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C-II, and C-III.

ApoA-I and apoC-II are eluted in two isoforms and apoC-III2 is eluted in three isoforms by reversed phase high performance liquid chromatography (HPLC). The structural basis of these nongenetic heterogeneities was unravelled using HPLC of proteolytic peptides and time-of-flight secondary ion mass spectrometry (TOF-SIMS). In apoA-I, the chromatographic microheterogeneity was caused by the formation of methionine sulfoxides (MetSO). However, only residues Met112 and Met148 were found oxidized, whereas Met86 was unaffected and also resistant towards artificial oxidation. To assess whether and to what extent amino acid substitutions in apoA-I might affect methionine sulfoxidation, the tryptic peptides of 13 different mutant apoA-I proteins from 24 heterozygous apoA-I variant carriers were analyzed by HPLC. In normal apoA-I, the ratios MetSO112/Met112 and MetSO148/Met148 were highly variable. By contrast, the relative ratio of oxidation of methionine residues 112 and 148 was constant. The amino acid changes Lys107----Met, Lys107----O, Glu139----Gly, Glu147----Val, and Pro165----Arg resulted in the preferential oxidation of Met112, and Asp103----Asn resulted in a preferential oxidation of Met148; whereas Pro3----Arg, Pro3----His, Pro4----Arg, Asp89----Glu, Ala158----Asp, Glu198----Lys, and Asp213----Gly had no impact. ApoC-II and apoC-III isoforms differed by the oxidation of the two methionine residues in these proteins. Whereas in apoC-II both methionine residues were oxidized in parallel, in apoC-III the two methionine residues differed in their susceptibility towards oxidation. We conclude that the formation of MetSO depends on the molecular microenvironment within a protein.     

The influence of amino substitutions within the mature part of an Escherichia coli outer membrane protein (OmpA) on assembly of the polypeptide into its membrane

The membrane part of the 325-residue outer membrane protein OmpA of Escherichia coli encompasses residues 1-177. This part is thought to cross the membrane eight times in antiparallel beta-strands, forming four loops of an amphipathic beta-barrel. With the aim of gaining some insight into the mechanism of sorting, i.e. the way the protein recognizes and assembles into its membrane, a set of point mutants in the ompA gene has been generated. Selection for toxicity of ompA expression following mutagenesis with sodium bisulfite yielded genes with multiple base pair substitutions, the majority of which resulted in amino acid substitutions in the membrane moiety of the protein. None of the altered proteins was blocked in membrane incorporation. A proline residue exists at or near each of the presumed turns at the inner side of the outer membrane. Using oligonucleotide-directed mutagenesis, each of them was replaced by a leucine residue which is thought to be a turn blocking residue. None of these proteins had lost the ability to be incorporated into the membrane. Apparently, leucine residues are tolerated at turns in this protein. To interfere with the formation of antiparallel beta-strands, four double mutants were prepared: ompA-ON3 (Ala11----Pro, Leu13----Pro), -ON4 (Ala11----Asp, Leu13----Pro), -ON5 (Gly160----Val, Leu162----Arg), and -ON6 (Leu164----Pro, Val166----Asp). The former three proteins and even quadruple mutants consisting of a combination of ompA-ON2 or -ON4 with -ON5 were not defective in membrane assembly. In contrast, the OmpA-ON6 protein was translocated across the plasma membrane but could not be incorporated into the outer membrane. It is concluded that at least one rather small area of the polypeptide is of crucial importance for the assembly of OmpA into the outer membrane.     

Easy identification of cDNA clones

A set of six cloning vectors, pUR 278, 288, 289, 290, 291, 292 is presented. These vectors have the cloning sites, BamHI, SalI, PstI, XbaI and HindIII, in all frames at the 3' end of the lacZ gene. Insertion of cDNA in the proper cloning sites leads to a fusion protein of active beta-galactosidase and the peptide encoded by the cDNA. A simple immunoenzymatic assay can be used to identify clones in such a cDNA library.     

Impact of aromatic residues within transmembrane helix 6 of the human gonadotropin-releasing hormone receptor upon agonist and antagonist binding.

To investigate the impact of aromatic residues within transmembrane helix 6 (TMH6) of the human gonadotropin-releasing hormone receptor (GnRH-R) on agonist and antagonist binding, residues Y(283), Y(284), W(289), Y(290), W(291), and F(292) were exchanged to alanine and analyzed comprehensively in functional reporter gene and ligand binding assays. Whereas receptor mutants Y(283)A, Y(284)A, and W(291)A were capable of neither ligand binding nor signal transduction, mutants W(289)A, Y(290)A, and F(292)A were functional: the F(292)A mutant behaved like wild-type receptor, while mutants W(289)A and Y(290)A differentiated between agonistic and antagonistic ligands. On the basis of the high-resolution X-ray structure of bovine rhodopsin as well as available data on GnRH-R mutants, models for ligand-receptor interactions are proposed. The model for D-Trp(6)-GnRH (Triptorelin) binding, representing a superagonistic ligand, is in full accordance to available data. Furthermore, new interactions are proposed: pGlu(1) interacts with N(212) in transmembrane helix 5, Tyr(5) with Y(290), and D-Trp(6) with W(289). The binding behavior of mutants W(289)A and Y(290)A corresponds to the proposed binding model for the antagonist Cetrorelix. In summary, our data as presented indicate that Y(290) plays a key function in agonist but not antagonist binding.     

Biogenesis of mitochondria: DNA sequence analysis of mit- mutations in the mitochondrial oli1 gene coding for mitochondrial ATPase subunit 9 in Saccharomyces cerevisiae.

The nucleotide sequence of the yeast mitochondrial olil gene has been obtained in a series of mit- mutants with mutations in this gene, which codes for subunit 9 of of the mitochondrial ATPase complex. Subunit 9 is the proteolipid, 76 amino acids in length, necessary for the proton translocation function of the membrane Fo-sector. These mutants were classified on the basis of their rescue by a petite strain shown here to retain the entire wild-type olil gene. The mutation in one mit- strain removes a positively charged residue (Arg39----Met) which is likely to be located in a segment of subunit 9 that protrudes from the inner mitochondrial membrane. In a second mit- mutant, a negatively charged residue replaces a conserved glycine residue (Gly18----Asp) in a glycine-rich segment of the protein that is most likely embedded within the membrane. Other mit- mutations result in frameshifts with predicted products 7, 65 and 68 amino acid residues long. In each mit- mutant, there is the loss of one or more of the amino acid residues that are highly conserved among diverse species. The location and nature of specific changes pinpoint amino acid residues in subunit 9 essential to the activity of the mitochondrial ATPase complex.     

Site-directed mutagenesis of GLUT1 in helix 7 residue 282 results in perturbation of exofacial ligand binding.

The structure-function relationship of the HepG2/erythrocyte-type glucose transporter (GLUT1) has been studied by in vitro site-directed mutagenesis. Chinese hamster ovary clones in which glucose transporters were transfected were shown by Western blotting with a GLUT1 anti-COOH-terminal peptide antibody to have expression levels of Gln282----Leu, Asn288----Ile, and Asn317----Ile mutations that were comparable with the wild type. All three mutant GLUT1 clones had high 2-deoxy-D-glucose transport activity compared with a nontransfected clone, suggesting that these residues are not absolutely required for the transport function. We have examined the possibility that the inner and outer portions of the transport pathway are structurally separate by measuring the interaction of the mutant transporters with the inside site-specific ligand cytochalasin B and the outside site-specific ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA). All three mutant GLUT1 clones showed high levels of cytochalasin B labeling, and the N288I and N317I mutants showed high levels of ATB-BMPA labeling. In contrast to the transport and cytochalasin B labeling results, the transmembrane helix 7 Gln282----Leu mutant was labeled by ATB-BMPA to a level that was only 5% of the level observed in the wild type. We have confirmed that this mutant was defective in the outer site by comparing the inhibition of wild-type and mutant 2-deoxy-D-glucose transport by the outside site-specific ligand 4,6-O-ethylidene-D-glucose. 4,6-O-Ethylidene-D-glucose inhibited wild-type transport with a Ki of approximately 12 mM, but this was increased to greater than 120 mM in the Gln282----Leu mutant. Thus, of the 3 residues mutated in this study, only glutamine 282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the outside site-specific ligands ATB-BMPA and 4,6-O-ethylidene-D-glucose.     

Phage lambda repressor revertants. Amino acid substitutions that restore activity to mutant proteins

We have isolated same-site and second-site revertants that restore partial activity, wild-type activity, or greater than wild-type activity, to lambda repressor proteins bearing different mutations in the DNA binding domain. In some cases the revertant repressors contain same-site substitutions that are similar to the wild-type side-chain (e.g. Tyr22----Phe, Ser77----Thr). The activity of these revertants makes it possible to assess the role of specific hydrogen bonds and/or packing interactions in repressor structure and function. In other same-site revertants, a very different type of residue is introduced (e.g. Ser35----Leu, Gly48----Asn). This indicates that the chemical and steric requirements at these side-chain positions are relaxed. Two of the second-site revertants, Glu34----Lys and Gly48----Ser, restore activity to more than one primary mutant. Both substitutions apparently increase the affinity of the repressor-operator interaction by introducing new contacts with operator DNA. These results suggest that reversion may be a generally applicable method for identifying sequence changes that increase the activity of a protein to greater than wild-type levels.     

Mitochondrial Hsp70 Ssc1: role in protein folding

Ssc1, the major Hsp70 of the mitochondrial matrix, is involved in the translocation of proteins from the cytosol into the matrix and their subsequent folding. To better understand the physiological mechanism of action of this Hsp70, we have undertaken a biochemical analysis of Ssc1 and two mutant proteins, Ssc1--2 and Ssc1--201. ssc1--2 is a temperature-sensitive mutant defective in both translocation and folding; ssc1--201 contains a second mutation in this ssc1 gene that suppresses the temperature-sensitive growth defect of ssc1--2, correcting the translocation but not the folding defect. We found that although Ssc1 was competent to facilitate the refolding of denatured luciferase in vitro, both Ssc1--2 and Ssc1--201 showed significant defects, consistent with the data obtained with isolated mitochondria. Purified Ssc1--2 had a lowered affinity for a peptide substrate compared with wild-type Ssc1 but only in the ADP-bound state. This peptide binding defect was reversed in the suppressor protein Ssc1--201. However, a defect in the ability of Hsp40 to stimulate the ATPase activity of Ssc1--2 was not corrected in Ssc1--201. Thus, the inability of these two mutant proteins to efficiently facilitate luciferase refolding correlates with their defect in stimulation of ATPase activity by Hsp40s, indicating that this interaction is critical for protein folding in mitochondria.     

Light-stable rhodopsin. II. An opsin mutant (TRP-265----Phe) and a retinal analog with a nonisomerizable 11-cis configuration form a photostable chromophore.

In order to prepare a completely light-stable rhodopsin, we have synthesized an analog, II, of 11-cis retinal in which isomerization at the C11-C12 cis-double bond is blocked by formation of a cyclohexene ring from the C10 to C13-methyl. We used this analog to generate a rhodopsin-like pigment from opsin expressed in COS-1 cells and opsin from rod outer segments (Bhattacharya, S., Ridge, K.D., Knox, B.E., and Khorana, H. G. (1992) J. Biol. Chem. 267, 6763-6769). The pigment (lambda max, 512 nm) formed from opsin and analog II (rhodospin-II) showed ground state properties very similar to those of rhodopsin, but was not entirely stable to light. In the present work, 12 opsin mutants (Ala-117----Phe, Glu-122----Gln(Ala, Asp), Trp-126----Phe(Leu, Ala), Trp-265----Ala(Tyr, Phe), Tyr-268----Phe, and Ala-292----Asp), where the mutations were presumed to be in the retinal binding pocket, were reconstituted with analog II. While all mutants formed rhodopsin-like pigments with II, blue-shifted (12-30 nm) chromophores were obtained with Ala-117----Phe, Glu-122----Gln(Ala), Trp-126----Leu(Ala), and Trp-265----Ala(Tyr, Phe) opsins. The extent of chromophore formation was markedly reduced in the mutants Ala-117----Phe and Trp-126----Ala. Upon illumination, the reconstituted pigments showed varying degrees of light sensitivity; the mutants Trp-126----Phe(Leu) showed light sensitivity similar to wild-type. Continuous illumination of the mutants Glu-122----Asp, Trp-265----Ala, Tyr-268----Phe, and Ala-292----Asp resulted in hydrolysis of the retinyl Schiff base. Markedly reduced light sensitivity was observed with the mutant Trp-265----Tyr, while the mutant Trp-265----Phe was light-insensitive. Consistent with this result, the mutant Trp-265----Phe showed no detectable light-dependent activation of transducin or phosphorylation by rhodopsin kinase.     

Restoration of membrane incorporation of an Escherichia coli outer membrane protein (OmpA) defective in membrane insertion

The mechanism of sorting, to the outer membrane, of the 325-residue Escherichia coli protein OmpA has been investigated. It is thought to traverse the membrane eight times in antiparallel beta-strands, forming an amphiphilic beta-barrel which encompasses residues 1 to about 170; the COOH-terminal moiety is periplasmic. A mutant, carrying the substitutions Leu164----Pro and Val166----Asp within the last beta-strand (residues 160-170), has been described which was unable to assemble in the membrane (Klose, M., MacIntyre, S., Schwarz, H., and Henning, U. (1988) J. Biol. Chem. 263, 13297-13302). Linkers were inserted between the codons for residues 164 and 165 of the mutant protein. Of 13 different genes recovered, five encoded proteins which had regained the ability to assemble in the membrane. The properties of the mutant proteins, together with a structure prediction method, indicate the following rules for the final beta-strand to be compatible with, or possibly initiate, membrane insertion: (i) it must be amphiphilic or hydrophobic while its primary structure as such is fairly unimportant, (ii) it must extend over at least 9 residues, and (iii) it must not contain a proline residue around its center. One of the genes recovered coded for OmpA up to residue 164 and then followed by 10 linker-encoded residues. This 174-residue polypeptide was assembled in the membrane but did not, in contrast to all other proteins, expose sites sensitive to trypsin at the inner face of the membrane. This behavior agrees perfectly well with the OmpA model.     

The tonB gene of Serratia marcescens: sequence, activity and partial complementation of Escherichia coli tonB mutants

The TonB protein plays a key role in the energy-coupled transport of iron siderophores, of vitamin B12, and of colicins of the B-group across the outer membrane of Escherichia coli. In order to obtain more data about which of its particular amino acid sequences are necessary for TonB function, we have cloned and sequenced the tonB gene of Serratia marcescens. The nucleotide sequence predicts an amino acid sequence of 247 residues (Mr 27,389), which is unusually proline-rich and contains the tandem sequences (Glu-Pro)5 and (Lys-Pro)5. In contrast to the TonB proteins of E. coli and Salmonella typhimurium, translation of the S. marcescens TonB protein starts at the first methionine residue of the open reading frame, which is the only amino acid removed during TonB maturation and export. Only the N-terminal sequence is hydrophobic, suggesting its involvement in anchoring the TonB protein to the cytoplasmic membrane. The S. marcescens tonB gene complemented an E. coli tonB mutant with regard to uptake of iron siderophores, and sensitivity to phages T1 and phi 80, and to colicins B and M. However, an E. coli tonB mutant transformed with the S. marcescens tonB gene remained resistant to colicins Ia and Ib, to colicin B derivatives carrying the amino acid replacements Val/Ala and Val/Gly at position 20 in the TonB box, and they exhibited a tenfold lower activity with colicin D. In addition, the S. marcescens TonB protein did not restore T1 sensitivity of an E. coli exbB tolQ double mutant, as has been found for the overexpressed E. coli TonB protein, indicating a lower activity of the S. marcescens TonB protein. Although the S. marcescens TonB protein was less prone to proteolytic degradation, it was stabilized in E. coli by the ExbBD proteins. In E. coli, TonB activity of S. marcescens depended either on the ExbBD or the TolQR activities.     

Mutagenesis of the NH2-terminal domain of elongation factor Tu

Mutagenesis was carried out in the N-terminal domain of elongation factor Tu (EF-Tu) to characterize the structure-function relationships of this model GTP binding protein with respect to stability, the interaction with GTP and GDP, and the catalytic activity. The substitutions were introduced in elements around the guanine nucleotide binding site or in the loops defining this site, in the intact molecule or in the isolated N-terminal domain (G domain). The double substitution Val88----Asp and Leu121----Lys, two residues situated on two vicinal alpha-helices, influences the basic activities of the truncated factor to a limited extent, probably via long-range interactions, and induces a destabilisation of the G domain structure. The functional alterations brought about by substitutions on the consensus sequences 18-24 and 80-83 highlight the importance of these residues for the interaction with GTP/GDP and the GTPase activity. Mutations concerning residues interacting with the guanine base lead to proteins in large part insoluble and inactive. In one case, the mutated protein (EF-TuAsn135----Asp) inhibited the growth of the host cell. This demonstrates the crucial role of the base specificity for the active conformation of EF-Tu. The obtained results are discussed in the light of the three-dimensional structure of EF-Tu.     

Escherichia coli K-12 outer membrane protein (OmpA) as a bacteriophage receptor: analysis of mutant genes expressing altered proteins.

The outer membrane protein OmpA of Escherichia coli K-12 serves as a receptor for a number of T-even-like phages. We have isolated a series of ompA mutants which are resistant to such phages but which still produce the OmpA protein. None of the mutants was able to either irreversibly or reversibly bind the phage with which they had been selected. Also, the OmpA protein is required for the action of colicins K and L and for the stabilization of mating aggregates in conjugation. Conjugal proficiency was unaltered in all cases. Various degrees of colicin resistance was found; however, the resistance pattern did not correlate with the phage resistance pattern. DNA sequence analyses revealed that, in the mutants, the 325-residue OmpA protein had suffered the following alterations: Gly-65----Asp, Gly-65----Arg, Glu-68----Gly, Glu-68----Lys (two isolates), Gly-70----Asp (four isolates), Gly-70----Val, Ala-Asp-Thr-Lys-107----Ala-Lys (caused by a 6-base-pair deletion), Val-110----Asp, and Gly-154----Ser. These mutants exhibited a complex pattern of resistance-sensitivity to 14 different OmpA-specific phages, suggesting that they recognize different areas of the protein. In addition to the three clusters of mutational alterations around residues 68, 110, and 154, a site around residue 25 has been predicted to be involved in conjugation and in binding of a phage and a bacteriocin (R. Freudl, and S. T. Cole, Eur. J. Biochem, 134:497-502, 1983; G. Braun and S. T. Cole, Mol. Gen. Genet, in press). These four areas are regularly spaced, being about 40 residues apart from each other. A model is suggested in which the OmpA polypeptide repeatedly traverses the outer membrane in cross-beta structure, exposing the four areas to the outside.     

Role of surface hydrophobic residues in the conformational stability of human lysozyme at three different positions

To evaluate the contribution of the amino acid residues on the surface of a protein to its stability, a series of hydrophobic mutant human lysozymes (Val to Gly, Ala, Leu, Ile, Met, and Phe) modified at three different positions on the surface, which are located in the alpha-helix (Val 110), the beta-sheet (Val 2), and the loop (Val 74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by X-ray crystallography at 100 K, respectively. Differences in the denaturation Gibbs energy change between the wild-type and the hydrophobic mutant proteins ranged from 4.6 to -9.6 kJ/mol, 2.7 to -1.5 kJ/mol, and 3.6 to -0.2 kJ/mol at positions 2, 74, and 110, respectively. The identical substitution at different positions and different substitutions at the same position resulted in different degrees of stabilization. Changes in the stability of the mutant proteins could be evaluated by a unique equation considering the conformational changes due to the substitutions [Funahashi et al. (1999) Protein Eng. 12, 841-850]. For this calculation, secondary structural propensities were newly considered. However, some mutant proteins were not adapted to the equation. The hydration structures around the mutation sites of the exceptional mutant proteins were affected due to the substitutions. The stability changes in the exceptional mutant proteins could be explained by the formation or destruction of the hydration structures. These results suggest that the hydration structure mediated via hydrogen bonds covering the protein surface plays an important role in the conformational stability of the protein.     

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.     

Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme.

To determine the energetic and structural consequences of placing a charged group within the core of a protein, two "buried charge" mutants, Met 102----Lys (M102K) and Leu 133----Asp (L133D) were constructed in phage T4 lysozyme. Both proteins fold at neutral pH, although they are substantially less stable than wild type. The activity of M102K is about 35% that of wild type, while that of L133D is about 4%. M102K could be crystallized, and its structure was determined at high resolution. The crystal structure (at pH 6.8) of the mutant is very similar to that of wild type except for the alpha-helix that includes residues 108-113. In wild-type lysozyme, one side of this helix is exposed to solvent and the other contacts Met 102. In the M102K structure this alpha-helix becomes much more mobile, possibly allowing partial access of Lys 102 to solvent. The stability of M102K, determined by monitoring the unfolding of the protein with CD, is pH-dependent, consistent with the charged form of the substituted amino acid being more destabilizing than the uncharged form. The pKa of Lys 102 was estimated to be 6.5 both by differential titration and also by NMR analysis of isotopically labeled protein with 13C incorporated at the C epsilon position of all lysines. As the pH is lowered below pH 6.5, the overall three-dimensional structure of M102K at room temperature appears to be maintained to pH 3 or so, although there is evidence for some structural adjustment possibly allowing solvent accessibility to the protonated form of Lys 102.