Critical regions for the sweetness of brazzein

Brazzein is a small, heat-stable, intensely sweet protein consisting of 54 amino acid residues. Based on the wild-type brazzein, 25 brazzein mutants have been produced to identify critical regions important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29-33 and 39-43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness of brazzein. Second, charge plays an important role in the interaction between brazzein and the sweet taste receptor.     

Probing the sweet determinants of brazzein: wild-type brazzein and a tasteless variant, brazzein-ins(R18a-I18b), exhibit different pH-dependent NMR chemical shifts

Brazzein is a small, intensely sweet protein. As a probe of the functional properties of its solvent-exposed loop, two residues (Arg-Ile) were inserted between Leu18 and Ala19 of brazzein. Psychophysical testing demonstrated that this mutant is totally tasteless. NMR chemical shift mapping of differences between this mutant and brazzein indicated that residues affected by the insertion are localized to the mutated loop, the region of the single alpha-helix, and around the Cys16-Cys37 disulfide bond. Residues unaffected by this mutation included those near the C-terminus and in the loop connecting the alpha-helix and the second beta-strand. In particular, several residues of brazzein previously shown to be essential for its sweetness (His31, Arg33, Glu41, Arg43, Asp50, and Tyr54) exhibited negligible chemical shift changes. Moreover, the pH dependence of the chemical shifts of His31, Glu41, Asp50, and Tyr54 were unaltered by the insertion. The insertion led to large chemical shift and pKa perturbation of Glu36, a residue shown previously to be important for brazzein's sweetness. These results serve to refine the known sweetness determinants of brazzein and lend further support to the idea that the protein interacts with a sweet-taste receptor through a multi-site interaction mechanism, as has been postulated for brazzein and other sweet proteins (monellin and thaumatin).     

Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408-->Glu/Lys939-->Arg mutant retained significant activity, while an Asp407-->Glu/Lys939-->Arg mutant exhibited only marginal function. An Asp407-->Glu/Asp408-->Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407-->Glu/Asp408-->Glu/Lys939-->Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.     

Sweetness determinant sites of brazzein, a small, heat-stable, sweet-tasting protein

Brazzein, originally isolated from the fruit of the African plant Pentadiplandra brazzeana Baillon, is the smallest, most heat-stable and pH-stable member of the set of proteins known to have intrinsic sweetness. These properties make brazzein an ideal system for investigating the chemical and structural requirements of a sweet-tasting protein. We have used the three-dimensional structure of the protein (J. E. Caldwell et al. (1998) Nat. Struct. Biol. 5, 427-431) as a guide in designing 15 synthetic genes in expression constructs aimed at delineating the sweetness determinants of brazzein. Protein was produced heterologously in Escherichia coli, isolated, and purified as described in the companion paper (Assadi-Porter, F. M., Aceti, D., Cheng, H., and Markley, J. L., this issue). Analysis by one-dimensional (1)H NMR spectroscopy indicated that all but one of these variants had folded properly under the conditions used. A taste panel compared the gustatory properties of solutions of these proteins to those of sucrose and brazzein isolated from fruit. Of the 14 mutations in the des-pGlu1-brazzein background, four exhibited almost no sweetness, six had significantly reduced sweetness, two had taste properties equivalent to des-pGlu1-brazzein (two times as sweet as the major form of brazzein isolated from fruit which contains pGlu1), and two were about twice as sweet as des-pGlu1-brazzein. Overall, the results suggest that two regions of the protein are critical for the sweetness of brazzein: a region that includes the N- and C-termini of the protein, which are located close to one another, and a region that includes the flexible loop around Arg43.     

Introduction of a negative charge at Arg82 in thaumatin abolished responses to human T1R2-T1R3 sweet receptors

Thaumatin, an intensely sweet-tasting protein, elicits a sweet-taste sensation at a level as low as 50 nM. Although previous sensory analyses have suggested that Lys67 and Arg82 are important to the sweetness of thaumatin, the exact effects of each residue on sweet receptors are still unknown. In the present study, various mutants of thaumatin altered at Arg82 as well as Lys67 were prepared and their sweetness levels were quantitatively evaluated by cell-based assays using HEK293 cells expressing human sweet receptors. Mutations at Arg82 had a more deteriorative effect on sweetness than mutations at Lys67. Particularly, a charge inversion at Arg82 (R82E) resulted in an abolishment of the response to sweet receptors even at a concentration as high as 1 mM. These results indicate that Arg82 plays a central role in determining the sweetness of thaumatin. A strict spatial charge location at residue 82 appears to be required for interaction with sweet receptors.     

Functional role of putative critical residues in Mycobacterium tuberculosis RNase P protein

RNase P is involved in processing the 5⿲ end of pre-tRNA molecules. Bacterial RNase P contains a catalytic RNA subunit and a protein subunit. In this study, we have analyzed the residues in RNase P protein of M. tuberculosis that differ from the residues generally conserved in other bacterial RNase Ps. The residues investigated in the current study include the unique residues, Val27, Ala70, Arg72, Ala77, and Asp124, and also Phe23 and Arg93 which have been found to be important in the function of RNase P protein components of other bacteria. The selected residues were individually mutated either to those present in other bacterial RNase P protein components at respective positions or in some cases to alanine. The wild type and mutant M. tuberculosis RNase P proteins were expressed in E. coli, purified, used to reconstitute holoenzymes with wild type RNA component in vitro, and functionally characterized. The Phe23Ala and Arg93Ala mutants showed very poor catalytic activity when reconstituted with the RNA component. The catalytic activity of holoenzyme with Val27Phe, Ala70Lys, Arg72Leu and Arg72Ala was also significantly reduced, whereas with Ala77Phe and Asp124Ser the activity of holoenzyme was similar to that with the wild type protein. Although the mutants did not suffer from any binding defects, Val27Phe, Ala70Lys, Arg72Ala and Asp124Ser were less tolerant towards higher temperatures as compared to the wild type protein. The K_m of Val27Phe, Ala70Lys, Arg72Ala and Ala77Phe were >2-fold higher than that of the wild type, indicating the substituted residues to be involved in substrate interaction. The study demonstrates that residues Phe23, Val27 and Ala70 are involved in substrate interaction, while Arg72 and Arg93 interact with other residues within the protein to provide it a functional conformation.     

Site-directed mutants of Escherichia coli alpha-ketoglutarate permease (KgtP).

To investigate an active site(s) in the Escherichia coli alpha-ketoglutarate premease, 11 point mutants were made in the corresponding structural gene, kgtP, by oligonucleotide-directed mutagenesis and the polymerase chain reaction. On the basis of sequences conserved in KgtP and related members of a transporter superfamily [Henderson P. J. F., & Maiden, M. C. (1990) Philos. Trans. R. Soc. London B 326, 391], Arg76 was replaced with Ala, Asp, or Lys; Asp88 with Asn or Glu; His90 with Ala; Arg92 with Ala or Lys; and Arg198 with Ala, Asp, or Lys. Mutant proteins expressed using the T7 polymerase system were in each case shown to be membrane-associated. However, they differed in transport activity. Mutants H90A and R198K had activities similar to that of wild type, and R76K and R198A retained 10-60% of the wild-type activity. In all other mutants, alpha-ketoglutarate transport was abolished. The results suggest that Arg92, which is highly conserved among other members of the transporter superfamily, is necessary for activity and also that Asp88 is critical for function, as observed for the tetracycline transporter. These data show further that a positive charge is essential at position 76 and is also important, but not absolutely required, at position 198 for alpha-ketoglutarate transport. Unlike lacY permease which was inactivated by deleting the last helix [McKenna, E., Hardy, D., Pastore, J. C., & Kaback, H. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969], a KgtP truncation mutant missing the last putative membrane-spanning region was relatively stable and also retained 10-50% of the wild-type level of alpha-ketoglutarate transport activity.     

Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato.

The importance of two putative Zn2+-binding (Asp347, Glu429) and two catalytic (Arg431, Lys354) residues in the tomato leucine aminopeptidase (LAP-A) function was tested. The impact of substitutions at these positions, corresponding to the bovine LAP residues Asp255, Glu334, Arg336, and Lys262, was evaluated in His6-LAP-A fusion proteins expressed in Escherichia coli. Sixty-five percent of the mutant His6-LAP-A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His6-LAP-As on Xaa-Leu and Leu-Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His6-LAP-As that cleaved dipeptides at slower rates. The Glu429 mutants (a Zn2+-binding residue) had more diverse phenotypes. Some mutations abolished activity and others retained partial or complete activity. The E429D His6-LAP-A enzyme had Km and kcat values similar to the wild-type His6-LAP-A. One catalytic (Arg431) and one Zn-binding (Asp347) residue were essential for His6-LAP-A activity, as most R431 and D347 mutant His6-LAP-As did not hydrolyze dipeptides. The R431K His6-LAP-A that retained the positive charge had partial activity as reflected in the 4.8-fold decrease in kcat. Surprisingly, while the D347E mutant (that retained a negative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity. A model to explain these data is proposed.     

Reversal of negative charges on the surface of Escherichia coli thioredoxin: pockets versus protrusions

Recent studies of proteins with reversed charged residues have demonstrated that electrostatic interactions on the surface can contribute significantly to protein stability. We have used the approach of reversing negatively charged residues using Arg to evaluate the effect of the electrostatics context on the transition temperature (T(m)), the unfolding Gibbs free energy change (DeltaG), and the unfolding enthalpy change (DeltaH). We have reversed negatively charged residues at a pocket (Asp9) and protrusions (Asp10, Asp20, Glu85), all located in interconnecting segments between elements of secondary structure on the surface of Arg73Ala Escherichia coli thioredoxin. DSC measurements indicate that reversal of Asp in a pocket (Asp9Arg/Arg73Ala, DeltaT(m) = -7.3 degrees C) produces a larger effect in thermal stability than reversal at protrusions: Asp10Arg/Arg73Ala, DeltaT(m) = -3.1 degrees C, Asp20Arg/Arg73Ala, DeltaT(m) = 2.0 degrees C, Glu85Arg/Arg73Ala, DeltaT(m) = 3.9 degrees ). The 3D structure of thioredoxin indicates that Asp20 and Glu85 have no nearby charges within 8 A, while Asp9 does not only have Asp10 as sequential neighbor, but it also forms a 5-A long-range ion pair with the solvent-exposed Lys69. Further DSC measurements indicate that neutralization of the individual charges of the ion pair Asp9-Lys69 with nonpolar residues produces a significant decrease in stability in both cases: Asp9Ala/Arg73Ala, DeltaT(m) = -3.7 degrees C, Asp9Met/Arg73Ala, DeltaT(m) = -5.5 degrees C, Lys69Leu/Arg73Ala, DeltaT(m) = -5.1 degrees C. However, thermodynamic analysis shows that reversal or neutralization of Asp9 produces a 9-15% decrease in DeltaH, while both reversal of Asp at protrusions and neutralization of Lys69 produce negligible changes. These results correlate well with the NMR analysis, which demonstrates that only the substitution of Asp9 produces extensive conformational changes and these changes occur in the surroundings of Lys69. Our results led us to suggest that reversal of a negative charge at a pocket has a larger effect on stability than a similar reversal at a protrusion and that this difference arises largely from short-range interactions with polar groups within the pocket, rather than long-range interactions with solvent-exposed charged groups.     

Correlation of the sweetness of variants of the protein brazzein with patterns of hydrogen bonds detected by NMR spectroscopy

In sequence-function investigations, approaches are needed for rapidly screening protein variants for possible changes in conformation. Recent NMR methods permit direct detection of hydrogen bonds through measurements of scalar couplings that traverse hydrogen bonds (trans-hydrogen bond couplings). We have applied this approach to screen a series of five single site mutants of the sweet protein brazzein with altered sweetness for possible changes in backbone hydrogen bonding with respect to wild-type. Long range, three-dimensional data correlating connectivities among backbone 1HN, 15N, and 13C' atoms were collected from the six brazzein proteins labeled uniformly with carbon-13 and nitrogen-15. In wild-type brazzein, this approach identified 17 backbone hydrogen bonds. In the mutants, altered magnitudes of the couplings identified hydrogen bonds that were strengthened or weakened; missing couplings identified hydrogen bonds that were broken, and new couplings indicated the presence of new hydrogen bonds. Within the series of brazzein mutants investigated, a pattern was observed between sweetness and the integrity of particular hydrogen bonds. All three "sweet" variants exhibited the same pattern of hydrogen bonds, whereas all three "non-sweet" variants lacked one hydrogen bond at the middle of the alpha-helix, where it is kinked, and one hydrogen bond in the middle of beta-strands II and III, where they are twisted. Two of the non-sweet variants lack the hydrogen bond connecting the N and C termini. These variants showed greater mobility in the N- and C-terminal regions than wild-type brazzein.     

Structure-sweetness relationship in egg white lysozyme: role of lysine and arginine residues on the elicitation of lysozyme sweetness

Lysozyme is one of the sweet-tasting proteins. To clarify the structure-sweetness relationship and the basicity-sweetness relationship in lysozyme, we have generated lysozyme mutants with Pichia systems. Alanine substitution of lysine residues demonstrated that two out of six lysine residues, Lys13 and Lys96, are required for lysozyme sweetness, while the remaining four lysine residues do not play a significant role in the perception of sweetness. Arginine substitution of lysine residues revealed that the basicity, but not the shape, of the side chain plays a significant role in sweetness. Single alanine substitutions of arginine residues showed that three arginine residues, Arg14, Arg21, and Arg73, play significant roles in lysozyme sweetness, whereas Arg45, Arg68, Arg125 and chemical modification by 1,2-cyclohexanedione did not affect sweetness. From investigation of the charge-specific mutations, we found that the basicity of a broad surface region formed by five positively charged residues, Lys13, Lys96, Arg14, Arg21, and Arg73, is required for lysozyme sweetness. Differences in the threshold values among sweet-tasting proteins might be caused by the broadness and/or the density of charged residues on the protein surface.     

The region from phenylalanine-17 to phenylalanine-28 of a yeast mitochondrial ATPase inhibitor is essential for its ATPase inhibitory activity.

Mitochondrial ATP synthase (F(1)F(o)-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, we investigated the structure-function relationship of the yeast ATPase inhibitor by amino acid replacement. A total of 22 mutants were isolated and characterized. Five mutants (F17S, R20G, R22G, E25A, and F28S) were entirely inactive, indicating that the residues, Phe17, Arg20, Arg22, Glu25, and Phe28, are essential for the ATPase inhibitory activity of the protein. The activity of 7 mutants (A23G, R30G, R32G, Q36G, L37G, L40S, and L44G) decreased, indicating that the residues, Ala23, Arg30, Arg32, Gln36, Leu37, Leu40, and Leu44, are also involved in the activity. Three mutants, V29G, K34Q, and K41Q, retained normal activity at pH 6.5, but were less active at pH 7.2, indicating that the residues, Val29, Lys34, and Lys41, are required for the protein's action at higher pH. The effects of 6 mutants (D26A, E35V, H39N, H39R, K46Q, and K49Q) were slight or undetectable, and the residues Asp26, Glu35, His39, Lys46, and Lys49 thus appear to be dispensable. The mutant E21A retained normal ATPase inhibitory activity but lacked pH-sensitivity. Competition experiments suggested that the 5 inactivated mutants (F17S, R20G, R22G, E25A, and F28S) could still bind to the inhibitory site on F(1)F(o)-ATPase. These results show that the region from the position 17 to 28 of the yeast inhibitor is the most important for its activity and is required for the inhibition of F(1), rather than binding to the enzyme.     

Lys691 and Asp714 of the Na+/K+-ATPase alpha subunit are essential for phosphorylation, dephosphorylation, and enzyme turnover

P-type ATPases such as the sodium pump appear to be members of a superfamily of hydrolases structurally typified by the L-2-haloacid dehalogenases. In the dehalogenase L-DEX-ps, Lys151 serves to stabilize the excess negative charge in the substrate/reaction intermediates and Asp180 coordinates a water molecule that is directly involved in ester intermediate hydrolysis. To investigate the importance of the corresponding Lys691 and Asp714 of the sodium pump alpha subunit, sodium pump mutants were expressed in yeast and analyzed for their properties. Lys691Ala, Lys691Asp, Asp714Ala, and Asp714Arg mutants were inactive, not only with respect to ATPase activity but also to interaction with the highly sodium pump-specific inhibitors ouabain or palytoxin (PTX). In contrast, conservative mutants Lys691Arg and Asp714Glu retained some of the partial activities of the wild-type enzyme, although they completely failed to display any ATPase activity. Yeast cells expressing Lys691Arg and Asp714Glu mutants are sensitive to the sodium pump-specific inhibitor PTX and lose intracellular K+. Their sensitivity to PTX, with EC50 values of 118 +/- 24 and 76.5 +/- 3.6 nM, respectively, was clearly reduced by almost 7- or 4-fold below that of the native sodium pump (17.8 +/- 2.7 nM). Ouabain was recognized under these conditions with low affinity by the mutants and inhibited the PTX-induced K+ efflux from the yeast cells. The EC50 for the ouabain effect was 183 +/- 20 microM for Lys691Arg and 2.3 +/- 0.08 mM for the Asp714Glu mutant. The corresponding value obtained with cells expressing the native sodium pump was 69 +/- 18 microM. In the presence of Pi and Mg2+, none of the mutant sodium pumps were able to bind ouabain. When Mg2+ was omitted, however, both Lys691Asp and Asp714Glu mutants displayed ouabain binding that was reduced by Mg2+ with an EC50 of 0.76 +/- 0.11 and 2.3 +/- 0.2 mM, respectively. In the absence of Mg2+, ouabain binding was also reduced by K+. The EC50 values were 1.33 +/- 0.23 mM for the wild-type enzyme, 0.93 +/- 0.2 mM for the Lys691Arg mutant, and 1.02 +/- 0.24 mM for the Asp714Glu enzyme. None of the neutral or nonconservative mutants displayed any ouabain-sensitive ATPase activity. Ouabain-sensitive phosphatase activity, however, was present in membranes containing either the wild-type (1105 +/- 100 micromol of p-nitrophenol phosphate hydrolyzed min(-1) mg of protein(-1)) or the Asp714Glu mutant (575 +/- 75 micromol min(-1) mg(-1)) sodium pump. Some phosphatase activity was also associated with the Lys691Arg mutant (195 +/- 63 micromol min(-1) mg(-1)). The results are consistent with Lys691 and Asp714 being essential for the phosphorylation/dephosphorylation process that allows the sodium pump to accomplish the catalytic cycle.     

Functional conservation in the putative substrate binding site of the sucrose permease from Escherichia coli

The sucrose (CscB) permease is the only member of the oligosaccharide:H(+) symporter family in the Major Facilitator Superfamily that transports sucrose but not lactose or other galactosides. In lactose permease (lac permease), the most studied member of the family, three residues have been shown to participate in galactoside binding: Cys148 hydrophobically interacts with the galactosyl ring, while Glu126 and Arg144 are charge paired and form H-bonds with specific galactosyl OH groups. In the present study, the role of the corresponding residues in sucrose permease, Asp126, Arg144, and Ser148, is investigated using a functional Cys-less mutant (see preceding paper). Replacement of Ser148 with Cys has no significant effect on transport activity or expression, but transport becomes highly sensitive to the sulfhydryl reagent N-ethylmaleimide (NEM) in a manner similar to that of lac permease. However, in contrast to lac permease, substrate affords no protection whatsoever against NEM inactivation of transport or alkylation with [(14)C]NEM. Neutral (Ala, Cys) mutations of Asp126 and Arg144 abolish sucrose transport, while membrane expression is not affected. Similarly, combination of two Ala mutations within the same molecule (Asp126-->Ala/Arg144-->Ala) yields normally expressed, but completely inactive permease. Conservative replacements result in highly active molecules: Asp126-->Glu permease catalyzes sucrose transport comparable to Cys-less permease, while mutant Arg144-->Lys exhibits decreased but significant activity. The observations demonstrate that charge pair Asp126-Arg144 plays an essential role in sucrose transport and suggest that the overall architecture of the substrate binding sites is conserved between sucrose and lac permeases.     

Characterization of yeast seryl-tRNA synthetase active site mutants with improved discrimination against substrate analogues

The involvement of amino acids within the motif 2 loop of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) in serine and ATP binding was demonstrated previously [B. Lenhard et al., J. Biol. Chem. 272 (1997) 1136-1141]. In our attempt to analyze the structural basis for the substrate specificity and to explore further the catalytic mechanism employed by S. cerevisiae SerRS, two new active site mutants, SerRS11 and SerRS12, were constructed. The catalytic effects of amino acid replacement at positions Lys287, Asp288 and Ala289 with purified wild-type and mutant seryl-tRNA synthetases were tested. The alteration of these semi-conserved amino acids interferes with tRNA-dependent optimization of serine recognition. Additionally, mutated enzymes SerRS11 (Lys287Thr, Asp288Tyr, Ala289Val) and SerRS12 (Lys287Arg) are less sensitive to inhibition by two competitive inhibitors: serine hydroxamate, an analogue of serine, and 5'-O-[N-(L-seryl)-sulfamoyl]adenosine, a stable analogue of aminoacyl adenylate, than the wild-type enzyme. SerRS mutants also display different activation kinetics for serine and serine hydroxamate, indicating that specificity toward the substrates is modulated by amino acid replacement in the motif 2 loop.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

大肠杆菌精氨酰－tRNA合成酶的Lys378和381的点突变研究

用点突变的方法将大肠杆菌精氨酰—ｔＲＮＡ合成酶（ＡｒｇＲＳ）的基因ａｒｇｓ上相应于Ｌｙｓ３７８和Ｌｙｓ３８１的密码ＡＡＡ分别变为两氨酸的密码ＧＣＡ和精氨酸的密码子ＣＧＴ,得到了４个ａｒｇｓ的突变体ａｒｇｓ３７８ＫＡ,ａｒｇｓ３７８ＫＲ,ａｒｇｓ３８１ＫＡ和ａｒｇｓ３８１ＫＲ,将它们分别连接到ｐＵＣ１８上,转入大肠杆菌ＴＧ１,在ＴＧ１转化子中,ＡｒｇＲＳ及其变种的表达量约为ＴＧ１中的１２０倍以上。结果表明Ｌｙｓ３７８为Ａｒｇ和Ａｌａ取代分别使活力下降０％和１０％;Ｌｙｓ３８１变为Ａｌａ和Ａｒｇ后,活力分别下降３３％和１０％左右。Ｌｙｓ３７８不为酶活力必需。Ｌｙｓ３８１部位的正电荷对酶活力是重要的。     

The conserved charged residues of the C-terminal region of FliG, a rotor component of the Na+-driven flagellar motor

FliG is an essential component of the flagellar motor and functions in flagellar assembly, torque generation and regulation of the direction of flagellar rotation. The five charged residues important for the rotation of the flagellar motor were identified in Escherichiacoli FliG (FliG(E)). These residues are clustered in the C terminus and are all conserved in FliG(V) of the Na(+)-driven motor of Vibrioalginolyticus (Lys284, Arg301, Asp308, Asp309 and Arg317). To investigate the roles of these charged residues in the Na(+)-driven motor, we cloned the VibriofliG gene and introduced single or multiple substitutions into the corresponding positions in FliG(V). FliG(V) with double Ala replacements in all possible combinations at these five conserved positions still retained significant motile ability, although some of the mutations completely eliminated the function of FliG(E). All of the triple mutants constructed in this study also remained motile. These results suggest that the important charged residues may be located in different places and the conserved charged residues are not so important for the Na(+)-driven flagellar motor of Vibrio. The chimeric FliG protein (FliG(VE)), composed of the N-terminal domain from V.alginolyticus and the C-terminal domain from E.coli, functions in Vibrio cells. The mutations of the charge residues of the C-terminal region in FliG(VE) affected swarming ability as in E.coli. Both the FliG(V) and the FliG(VE) proteins with the triple mutation were more susceptible to proteolysis than proteins without the mutation, suggesting that their conformations were altered.     

Direct identification of lysine-33 as the principal cationic center of the omega-amino acid binding site of the recombinant kringle 2 domain of tissue-type plasminogen activator.

We have generated site-specific mutants of the kringle 2 domain of tissue-type plasminogen activator [( K2tPA]) in order to identify directly the cationic center of the protein that is responsible for its interaction with the carboxyl group of important omega-amino acid effector molecules, such as epsilon-amino caproic acid (EACA). Molecular modeling of [K2tPA], docked with EACA, based on crystal structures of the kringle 2 region of prothrombin and the kringle 4 domain of human plasminogen, clearly shows that Lys33 is the only positively charged amino acid in [K2tPA] that is sufficiently proximal to the carboxyl group of the ligand to stabilize this interaction. In order to examine directly the importance of this particular amino acid residue in this interaction, we have constructed, expressed, and purified three recombinant (r) mutants of [K2tPA], viz., Lys33Thr, Lys33Leu, and Lys33Arg, and found that only the last variant retained significant ability to interact with EACA and several of its structural analogues at neutral pH. In addition, another mutated r-[K2tPA], i.e., Lys33His, interacts very weakly with omega-amino acids at neutral pH and much more strongly at lower pH values where His33 would be expected to undergo protonation. This demonstrates that any positively charged amino acid at position 33 satisfies the requirement for mediation of significant bindings to this class of molecules. Since, in other kringles, positively charged residues at amino acid sequence positions homologous to Lys68, Arg70, and Arg71 of [K2tPA] have been found to participate in kringle interactions with EACA-like compounds, we have also examined the binding of EACA, and some of its analogues, to three additional r-[K2tPA] variants, i.e., Lys68Ala, Arg70Ala, and Arg71Ala. In each case, binding of these omega-amino acids to the variant kringles was observed, with only the Lys68Ala variant showing a slightly diminished capacity for this interaction. These investigations provide clear and direct evidence that Lys33 is the principal cationic site in wild-type r-[K2tPA] that directly interacts with the carboxyl group of omega-amino acid effector molecules.     

Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha subunit

It has recently been shown that replacement of the border residues (Gln-111 and Asn-122) of the H1-H2 extracellular domain of the sheep Na,K-ATPase alpha subunit with the charged amino acids Arg and Asp generates a ouabain-resistant enzyme (Price, E. M. and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to further study structure-function relationships in Na,K-ATPase, six additional mutations have been made at these border positions. Two of these mutants were single amino acid substitutions (Gln-111 to Arg or Asn-122 to Asp). These mutations change one or the other H1-H2 border residue to a charged amino acid. The remaining substitutions were double mutants in which both of the H1-H2 border residues were simultaneously changed to charged amino acids. Changes were made which introduced either positively charged amino acids (Lys at positions 111 and 122), negatively charged amino acids (Glu at positions 111 and 122) or oppositely charged amino acids (Lys at position 111 and Glu at 122; Asp at position 111 and Arg at 122) at the borders of the H1-H2 extracellular domain. HeLa cells transfected with any of these sheep Na,K-ATPase alpha subunit mutants were able to grow in concentrations of ouabain that were toxic to untransfected cells or cells transfected with the wild type sheep alpha subunit. Crude membranes isolated from the transfectants were analyzed for ouabain inhibitable Na,K-ATPase activity. All of the transfectants contained a relatively ouabain-resistant component of enzyme activity, with the ouabain I50 values ranging from 4 x 10(-3) M to 1 x 10(-6) M. The most resistant enzyme was the double mutant that contained Asp at position 111 and Arg at 122, whereas the least resistant were the enzymes containing the single amino acid substitutions. There was no correlation between the type of charged amino acid present at the border position and the degree of ouabain resistance. These data demonstrate the functional importance, in terms of ouabain binding, of the border positions of the H1-H2 extracellular domain of the Na,K-ATPase alpha subunit.