Complementation of one RecA protein point mutation by another. Evidence for trans catalysis of ATP hydrolysis

The RecA residues Lys248 and Glu96 are closely opposed across the RecA subunit-subunit interface in some recent models of the RecA nucleoprotein filament. The K248R and E96D single mutant proteins of the Escherichia coli RecA protein each bind to DNA and form nucleoprotein filaments but do not hydrolyze ATP or dATP. A mixture of K248R and E96D single mutant proteins restores dATP hydrolysis to 25% of the wild type rate, with maximum restoration seen when the proteins are present in a 1:1 ratio. The K248R/E96D double mutant RecA protein also hydrolyzes ATP and dATP at rates up to 10-fold higher than either single mutant, although at a reduced rate compared with the wild type protein. Thus, the K248R mutation partially complements the inactive E96D mutation and vice versa. The complementation is not sufficient to allow DNA strand exchange. The K248R and E96D mutations originate from opposite sides of the subunit-subunit interface. The functional complementation suggests that Lys248 plays a significant role in ATP hydrolysis in trans across the subunit-subunit interface in the RecA nucleoprotein filament. This could be part of a mechanism for the long range coordination of hydrolytic cycles between subunits within the RecA filament.     

On evolutionary conservation of thermodynamic coupling in proteins

The inherent complexity of thermodynamic coupling in proteins presents a major challenge in understanding and engineering protein function. Recent work has argued that the study of proteins can be simplified by the use of correlated mutations in the evolutionary record to locate a small subset of thermodynamically coupled residues that participate in functionally important, evolutionarily conserved energetic pathways. To test this hypothesis, we examined the predictions of correlated mutation algorithms for a number of proteins for which coupling between residues has been determined by analysis of double mutant cycles. We find that correlated mutation algorithms can find residue pairs that are physically close and that physically close residue pairs tend to be thermodynamically coupled. We find little evidence, however, for the hypothesis that thermodynamic coupling is limited to the subset of evolutionarily constrained residue positions.     

The Mathematics of Mosaic Analysis I: The Relationship between Sturts and Distance

In mosaic fate mapping the fraction of mosaics in which two structures are of different genotype is calculated. This frequency of separation has been called a "distance" and the units of this distance are called "sturts". The fundamental assumption of fate mapping is that the frequency of separation increases continuously with the actual distance between the anlage for these structures on the blastoderm. This paper shows that the frequency of separation does not increase beyond a certain value.—For the current theory to work as proposed, each mosaic animal must be half mutant and half normal. This is rarely the case in collections of mosaics. It has been thought that if some flies are less than half mutant and others more than half, these two types would introduce compensating errors in mapping distance. We show that this is not true and describe the nature of the errors introduced. It is probable that these errors are the main reason that mapping distances reported from different sets of mosaics have not been reproducible. This paper presents methods for the proper handling of data from mosaics with different amounts of mutant tissue.—We prove here that for mosaics with an arbitrary fraction of mutant tissue (m), the largest frequency of separation that can occur is 2m. We prove that sturts underestimate actual distance on the blastoderm by a factor of r/m, where r is the radius of the mutant patch, and that sturts give no information on distances greater than 2r. This, and not double crossing over, is the reason for the nonadditivity of sturts and the shrinking of large distances in sturt measures. Sturtoids overestimate distances by a factor of 1/(2r) and also give no information on distances over 2r. This paper gives formulae for correctly estimating distance when using a collection of mosaics with varying amounts of mutant tissue. We also describe the nature of the errors introduced by convoluted or elongate mosaic boundaries and by multiple mosaic patches.     

Role of the electrostatic loop charged residues in Cu,Zn superoxide dismutase.

We have expressed and characterized a mutant of Xenopus laevis Cu,Zn superoxide dismutase in which four highly conserved charged residues belonging to the electrostatic loop have been replaced by neutral side chains: Lys120 --> Leu, Asp130 --> Gln, Glu131 --> Gln, and Lys134 --> Thr. At low ionic strength, the mutant enzyme is one of the fastest superoxide dismutases ever assayed (k = 6.7 x 10(9) M(-1) s(-1), at pH 7 and mu = 0.02 M). Brownian dynamics simulations give rise to identical enzyme-substrate association rates for both wild-type and mutant enzymes, ruling out the possibility that enhancement of the activity is due to pure electrostatic factors. Comparative analysis of the experimental catalytic rate of the quadruple and single mutants reveals the nonadditivity of the mutation effects, indicating that the hyperefficiency of the mutant is due to a decrease of the energy barrier and/or to an alternative pathway for the diffusion of superoxide within the active site channel. At physiological ionic strength the catalytic rate of the mutant at neutral pH is similar to that of the wild-type enzyme as it is to the catalytic rate pH dependence. Moreover, mutation effects are additive. These results show that, at physiological salt conditions, electrostatic loop charged residues do not influence the diffusion pathway of the substrate and, if concomitantly neutralized, are not essential for high catalytic efficiency of the enzyme, pointing out the role of the metal cluster and of the invariant Arg141 in determining the local electrostatic forces facilitating the diffusion of the substrate towards the active site.     

Complete replacement of basic amino acid residues with cysteines in Rickettsia prowazekii ATP/ADP translocase

The ATP/ADP translocase (Tlc) of Rickettsia prowazekii is a basic protein with isoelectric point (pI)=9.84. It is conceivable, therefore, that basic residues in this protein are involved in electrostatic interactions with negatively charged substrates. We tested this hypothesis by individually mutating all basic residues in Tlc to Cys. Unexpectedly, mutations of only 20 out of 51 basic residues resulted in greater than 80% inhibition of transport activity. Moreover, 12 of 51Cys-substitution mutants exhibited higher than wild-type (WT) activity. At least in one case this up-effect was additive and the double mutant Lys422Cys Lys427Cys transported ATP five-fold better than WT protein. Since in these two single mutants and in the corresponding double mutant K(m)'s were similar to that of WT protein, we conclude that Tlc may have evolved a mechanism that limits the transporter's exchange rate and that at least these two basic residues play a key role in that mechanism. Based on the alignment of 16 Tlc homologs, the loss of activity in the mutants poorly correlates with charge conservation within the Tlc family. Also, despite the presence of three positively charged and one negatively charged intramembrane residues, we have failed to identify potential charge pairs (salt bridges) by either charge reversal or charge neutralization approaches.     

Evaluation of direct and cooperative contributions towards the strength of buried hydrogen bonds and salt bridges

An experimental approach to evaluate the net binding free energy of buried hydrogen bonds and salt bridges is presented. The approach, which involves a modified multiple-mutant cycle protocol, was applied to selected interactions between TEM-1-beta-lactamase and its protein inhibitor, BLIP. The selected interactions (two salt bridges and two hydrogen bonds) all involving BLIP-D49, define a distinct binding unit. The penta mutant, where all side-chains constructing the binding unit were mutated to Ala, was used as a reference state to which combinations of side-chains were introduced. At first, pairs of interacting residues were added allowing the determination of interaction energies in the absence of neighbors, using double mutant cycles. Addition of neighboring residues allowed the evaluation of their cooperative effects on the interaction. The two isolated salt bridges were either neutral or repulsive whereas the two hydrogen bonds contribute 0.3 kcal mol(-1 )each. Conversely, a double mutant cycle analysis of these interactions in their native environment showed that they all stabilize the complex by 1-1.5 kcal mol(-1). Examination of the effects of neighboring residues on each of the interactions revealed that the formation of a salt bridge triad, which involves two connected salt bridges, had a strong cooperative effect on stabilizing the complex independent of the presence or absence of additional neighbors. These results demonstrate the importance of forming net-works of buried salt bridges. We present theoretical electrostatic calculations which predict the observed mode of cooperativity, and suggest that the cooperative networking effect results from the favorable contribution of the protein to the interaction. Furthermore, a good correlation between calculated and experimentally determined interaction energies for the two salt bridges, and to a lesser extent for the two hydrogen bonds, is shown. The data analysis was performed on values of DeltaDeltaG(double dagger)K(d) which reflect the strength of short range interactions, while DeltaDeltaG(o)K(D) values which include the effects of long range electrostatic forces that alter specifically DeltaDeltaG(double dagger)k(a) were treated separately.     

Importance of long-range interactions in protein folding

Long-range interactions play an active role in the stability of protein molecules. In this work, we have analyzed the importance of long-range interactions in different structural classes of globular proteins in terms of residue distances. We found that 85% of residues are involved in long-range contacts. The residues occurring in the range of 4-10 residues apart contribute more towards long-range contacts in all-alpha proteins while the range is 11-20 in all-beta proteins. The hydrophobic residues Cys, Ile and Val prefer the 11-20 range and all other residues prefer the 4-10 range. The residues in all-beta proteins have an average of 3-8 long-range contacts whereas the residues in other classes have 1-4 long-range contracts. Furthermore, the preference of residue pairs to the folding and stability will be discussed.     

Use of a disulfide cross-linking strategy to study muscarinic receptor structure and mechanisms of activation.

To gain insight into the molecular architecture of the cytoplasmic surface of G protein-coupled receptors, we have developed a disulfide cross-linking strategy using the m3 muscarinic receptor as a model system. To facilitate the interpretation of disulfide cross-linking data, we initially generated a mutant m3 muscarinic receptor (referred to as m3'(3C)-Xa) in which most native Cys residues had been deleted or substituted with Ala or Ser (remaining Cys residues Cys-140, Cys-220, and Cys-532) and in which the central portion of the third intracellular loop had been replaced with a factor Xa cleavage site. Radioligand binding and second messenger assays showed that the m3'(3C)-Xa mutant receptor was fully functional. In the next step, pairs of Cys residues were reintroduced into the m3'(3C)-Xa construct, thus generating 10 double Cys mutant receptors. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the second intracellular loop and a second Cys within the C-terminal portion of the third intracellular loop, at positions 484-493. Radioligand binding studies and phosphatidylinositol assays indicated that all double Cys mutant receptors were properly folded. Membrane lysates prepared from COS-7 cells transfected with the different mutant receptor constructs were incubated with factor Xa protease and the oxidizing agent Cu(II)-(1,10-phenanthroline)3, and the formation of intramolecular disulfide bonds between juxtaposed Cys residues was monitored by using a combined immunoprecipitation/immunoblotting strategy. To our surprise, efficient disulfide cross-linking was observed with 8 of the 10 double Cys mutant receptors studied (Cys-169/Cys-484 to Cys-491), suggesting that the intracellular m3 receptor surface is characterized by pronounced backbone fluctuations. Moreover, [35S]guanosine 5'-3-O-(thio)triphosphate binding assays indicated that the formation of intramolecular disulfide cross-links prevented or strongly inhibited receptor-mediated G protein activation, suggesting that the highly dynamic character of the cytoplasmic receptor surface is a prerequisite for efficient receptor-G protein interactions. This is the first study using a disulfide mapping strategy to examine the three-dimensional structure of a hormone-activated G protein-coupled receptor.     

Fast folding of cytochrome c.

Native iso-2 cytochrome c contains two residues (His 18, Met 80) coordinated to the covalently attached heme. On unfolding of iso-2, the His 18 ligand remains coordinated to the heme iron, whereas Met 80 is displaced by a non-native heme ligand, His 33 or His 39. To test whether non-native His-heme ligation slows folding, we have constructed a double mutant protein in which the non-native ligands are replaced by asparagine and lysine, respectively (H33N,H39K iso-2). The double mutant protein, which cannot form non-native histidine-heme coordinate bonds, folds significantly faster than normal iso-2 cytochrome c: gamma = 14-26 ms for H33N,H39K iso-2 versus gamma = 200-1,100 ms for iso-2. These results with iso-2 cytochrome c strongly support the hypothesis that non-native His-heme ligation results in a kinetic barrier to fast folding of cytochrome c. Assuming that the maximum rate of a conformational search is about 10(11) s-1, the results imply that the direct folding pathway of iso-2 involves passage through on the order of 10(9) or fewer partially folded conformers.     

The evolution of nonhuman primate loud calls: Acoustic adaptation for long-distance transmission

Many nonhuman primates produce species-typical loud calls used to communicate between and within groups over long distances. Given their observed spacing functions, primate loud calls are likely to show acoustic adaptations to increase their propagation over distance. Here we evaluate the hypothesis that primates emit loud calls at relatively low sound frequencies to minimize their attenuation. We tested this hypothesis within and between species. First, we compared the frequencies of loud calls produced by each species with those of other calls from their vocal repertoires. Second, we investigated the relationship between loud call frequency and home range size across a sample of primate species. Comparisons indicated that primates produce loud calls at lower frequencies than other calls within their vocal repertoires. In addition, a significant negative relationship exists between loud call frequency and home range size among species. The relationship between call frequency and range size holds after controlling for the potentially confounding effects of body size and phylogeny. These results are consistent with the hypothesis that nonhuman primates produce loud calls at relatively low frequencies to facilitate their transmission over long distances.     

Alanine-Scanning Mutagenesis of Protein Phosphatase Type 1 in the Yeast Saccharomyces Cerevisiae

Protein phosphatase type 1, encoded by GLC7 in Saccharomyces cerevisiae, is an essential serine/threonine phosphatase implicated in the regulation of a diverse array of physiological functions. We constructed and examined 20 mutant alleles of GLC7 in which codons encoding clusters of charged residues were changed to alanine codons. Three of 20 mutant alleles alter residues in the active site of the phosphatase and are unable to rescue the lethality of a glc7::LEU2 disruption. The 17 alleles that support growth confer a range of mutant traits including cell cycle arrest, 2-deoxyglucose resistance, altered levels of glycogen, sensitivity to high salt, and sporulation defects. For some traits, such as 2-deoxyglucose resistance and cell cycle arrest, the mutated residues map to specific regions of the protein whereas the mutated residues in glycogen-deficient mutants and sporulation-defective mutants are more widely distributed over the protein surface. Many mutants have complex phenotypes, each displaying a diverse range of defects. The wide range of phenotypes identified from the collection of mutant alleles is consistent with the hypothesis that Glc7p-binding proteins, which are thought to regulate the specificity of Glc7p, have overlapping binding sites on the surface of Glc7p. This could account for the high level of sequence conservation found among type 1 protein phosphatases from different species.     

ENDOR structural characterization of a catalytically competent acylenzyme reaction intermediate of wild-type TEM-1 beta-lactamase confirms glutamate-166 as the base catalyst

The catalytically competent active-site structure of a true acylenzyme reaction intermediate of TEM-1 beta-lactamase formed with the kinetically specific spin-labeled substrate 6-N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carboxyl)-penicillanic acid isolated under cryoenzymologic conditions has been determined by angle-selected electron nuclear double resonance (ENDOR) spectroscopy. Cryoenzymologic experiments with use of the chromophoric substrate 6-N-[3-(2-furanyl)-propen-2-oyl]-penicillanic acid showed that the acylenzyme reaction intermediate could be stabilized in the -35 to -75 degrees C range with a half-life suitably long to allow freeze-quenching of the reaction species for ENDOR studies while a noncovalent Michaelis complex could be optically identified at temperatures only below -70 degrees C. The wild-type, Glu166Asn, Glu240Cys, and Met272Cys mutant forms of the mature enzyme were overexpressed in perdeuterated minimal medium to allow detection and assignment of proton resonances specific for the substrate and chemically modified amino acid residues in the active site. From analysis of the dependence of the ENDOR spectra on the setting of the static laboratory magnetic field H0, the dipolar contributions to the principal hyperfine coupling components were estimated to calculate the separations between the unpaired electron of the nitroxyl group and isotopically identified nuclei. These electron-nucleus distances were applied as constraints to assign the conformation of the substrate in the active site and of amino acid side chains by molecular modeling. Of special interest was that the ENDOR spectra revealed a water molecule sequestered in the active site of the acylenzyme of the wild-type protein that was not detected in the deacylation impaired Glu166Asn mutant. On the basis of the X-ray structure of the enzyme, the ENDOR distance constraints placed this water molecule within hydrogen-bonding distance to the carboxylate side chain of glutamate-166 as if it were poised for nucleophilic attack of the scissile ester bond. The ENDOR results provide experimental evidence of glutamate-166 in its functional role as the general base catalyst in the wild-type enzyme for hydrolytic breakdown of the acylenzyme reaction intermediate of TEM-1 beta-lactamase.     

Contributions of engineered surface salt bridges to the stability of T4 lysozyme determined by directed mutagenesis.

Six designed mutants of T4 lysozyme were created in an attempt to create putative salt bridges on the surface of the protein. The first three of the mutants, T115E (Thr 115 to Glu), Q123E, and N144E, were designed to introduce a new charged side chain close to one or more existing charged groups of the opposite sign on the surface of the protein. In each of these cases the putative electrostatic interactions introduced by the mutation include possible salt bridges between residues within consecutive turns of an alpha-helix. Effects of the mutations ranged from no change in stability to a 1.5 degrees C (0.5 kcal/mol) increase in melting temperature. In two cases, secondary (double) mutants were constructed as controls in which the charge partner was removed from the primary mutant structure. These controls proteins indicate that the contributions to stability from each of the engineered salt bridges is very small (about 0.1-0.25 kcal/mol in 0.15 M KCl). The structures of the three primary mutants were determined by X-ray crystallography and shown to be essentially the same as the wild-type structure except at the site of the mutation. Although the introduced charges in the T115E and Q123E structures are within 3-5 A of their intended partner, the introduced side chains and their intended partners were observed to be quite mobile. It has been shown that the salt bridge between His 31 and Asp 70 in T4 lysozyme stabilizes the protein by 3-5 kcal/mol [Anderson, D. E., Becktel, W. J., & Dahlquist, F. W. (1990) Biochemistry 29, 2403-2408]. To test the effectiveness of His...Asp interactions in general, three additional double mutants, K60H/L13D, K83H/A112D, and S90H/Q122D, were created in order to introduce histidine-aspartate charge pairs on the surface of the protein. Each of these mutants destabilizes the protein by 1-3 kcal/mol in 0.15 M KCl at pH values from 2 to 6.5. The X-ray crystallographic structure of the mutant K83H/A112D has been determined and shows that there are backbone conformational changes of 0.3-0.6 A extending over several residues. The introduction of the histidine and aspartate presumably introduces strain into the folded protein that destabilizes this variant. It is concluded that pairs of oppositely charged residues that are on the surface of a protein and have freedom to adopt different conformations do not tend to come together to form structurally localized salt bridges. Rather, such residues tend to remain mobile, interact weakly if at all, and do not contribute significantly to protein stability. It is argued that the entropic cost of localizing a pair of solvent-exposed charged groups on the surface of a protein largely offsets the interaction energy expected from the formation of a defined salt bridge. There are examples of strong salt bridges in proteins, but such interactions require that the folding of the protein provides the requisite driving energy to hold the interacting partners in the correct rigid alignment.     

Interaction between yeast sgs1 helicase and DNA topoisomerase III

The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases that includes the human Bloom's syndrome and Werner's syndrome proteins. In this work, we report studies on the interaction between Sgs1 and DNA topoisomerase III in vitro and in vivo. Affinity chromatography experiments with various fragments of Sgs1, a 1447-amino acid polypeptide, suggested that its N-terminal one-fifth was sufficient for interaction with DNA topoisomerase III. Gel electrophoretic mobility shift assays also indicated that a fragment Sgs1(1-283), containing residues 1-283, inhibited the binding of DNA topoisomerase III to single-stranded DNA. A shorter protein fragment containing residues 1-107 also showed partial inhibition in these assays. Studies of a sgs1 top1 double mutant lacking both Sgs1 and DNA topoisomerase I showed that the slow growth phenotype of this double mutant is suppressed by expressing full-length Sgs1, but not Sgs1 without the N-terminal 107 amino acid residues. In sgs1 top3 cells devoid of DNA topoisomerase III, however, expression of full-length Sgs1 or Sgs1 lacking the N-terminal 107 amino acid residues has the same effect of reducing the growth rate of the double mutant. These in vitro and in vivo data indicate that Sgs1 and DNA topoisomerase III physically interact and that this interaction is physiologically significant.     

Contribution of individual histidines to the global stability of human prolactin

A member of the family of hematopoietic cytokines human prolactin (hPRL) is a 23k kDa polypeptide hormone, which displays pH dependence in its structural and functional properties. The binding affinity of hPRL for the extracellular domain of its receptor decreases 500‐fold over the relatively narrow, physiologic pH range from 8 to 6; whereas, the affinity of human growth hormone (hGH), its closest evolutionary cousin, does not. Similarly, the structural stability of hPRL decreases from 7.6 to 5.6 kcal/mol from pH 8 to 6, respectively, whereas the stability of hGH is slightly increased over this same pH range. hPRL contains nine histidines, compared with hGH's three, and they are likely responsible for hPRL's pH‐dependent behavior. We have systematically mutated each of hPRL's histidines to alanine and measured the effect on pH‐dependent global stability. Surprisingly, a vast majority of these mutations stabilize the native protein, by as much as 2–3 kcal/mol. Changes in the overall pH dependence to hPRL global stability can be rationalized according to the predominant structural interactions of individual histidines in the hPRL tertiary structure. Using double mutant cycles, we detect large interaction free energies within a cluster of nearby histidines, which are both stabilizing and destabilizing to the native state. Finally, by comparing the structural locations of hPRL's nine histidines with their homologous residues in hGH, we speculate on the evolutionary role of replacing structurally stabilizing residues with histidine to introduce pH dependence to cytokine function.     

Rational improvement of simvastatin synthase solubility in Escherichia coli leads to higher whole-cell biocatalytic activity

Simvastatin is the active pharmaceutical ingredient of the blockbuster cholesterol lowering drug Zocor. We have previously developed an Escherichia coli based whole-cell biocatalytic platform towards the synthesis of simvastatin sodium salt (SS) starting from the precursor monacolin J sodium salt (MJSS). The centerpiece of the biocatalytic approach is the simvastatin synthase LovD, which is highly prone to misfolding and aggregation when overexpressed from E. coli. Increasing the solubility of LovD without decreasing its catalytic activity can therefore elevate the performance of the whole-cell biocatalyst. Using a combination of homology structural prediction and site-directed mutagenesis, we identified two cysteine residues in LovD that are responsible for nonspecific intermolecular crosslinking, which leads to oligomer formation and protein aggregation. Replacement of Cys40 and Cys60 with alanine residues resulted in marked gain in both protein solubility and whole-cell biocatalytic activities. Further mutagenesis experiments converting these two residues to small or polar natural amino acids showed that C40A and C60N are the most beneficial, affording 27% and 26% increase in whole cell activities, respectively. The double mutant C40A/C60N combines the individual improvements and displayed approximately 50% increase in protein solubility and whole-cell activity. Optimized fed-batch high-cell-density fermentation of the double mutant in an E. coli strain engineered for simvastatin production quantitatively (>99%) converted 45 mM MJSS to SS within 18 h, which represents a significant improvement over the performance of wild-type LovD under identical conditions. The high efficiency of the improved whole-cell platform renders the biocatalytic synthesis of SS an attractive substitute over the existing semisynthetic routes.     

Molecular interactions of yeast frequenin (Frq1) with the phosphatidylinositol 4-kinase isoform,Pik1

Frq1, a 190-residue N-myristoylated calcium-binding protein, associates tightly with the N terminus of Pik1, a 1066-residue phosphatidylinositol 4-kinase. Deletion analysis of an Frq1-binding fragment, Pik1-(10-192), showed that residues within 80-192 are necessary and sufficient for Frq1 association in vitro. A synthetic peptide (residues 151-199) competed for binding of [(35)S]Pik1-(10-192) to bead-immobilized Frq1, whereas shorter peptides (164-199 and 174-199) did not. Correspondingly, a deletion mutant, Pik1(delta152-191), did not co-immunoprecipitate efficiently with Frq1 and did not support growth at elevated temperature. Site-directed mutagenesis of Pik1-(10-192) suggested that recognition determinants lie over an extended region. Titration calorimetry demonstrated that binding of an 83-residue fragment, Pik1-(110-192), or the 151-199 peptide to Frq1 shows high affinity (K(d) approximately 100 nm) and is largely entropic, consistent with hydrophobic interaction. Stoichiometry of Pik1-(110-192) binding to Frq1 was 1:1, as judged by titration calorimetry, by changes in NMR spectrum and intrinsic tryptophan fluorescence, and by light scattering. In cell extracts, Pik1 and Frq1 exist mainly in a heterodimeric complex, as shown by size exclusion chromatography. Cys-15 in Frq1 is not S-palmitoylated, as assessed by mass spectrometry; a Frq1(C15A) mutant and even a non-myristoylated Frq1(G2A,C15A) double mutant rescued the inviability of frq1Delta cells. This study defines the segment of Pik1 required for high affinity binding of Frq1.     

Structure of spin-labeled methylmethanethiolsulfonate in solution and bound to TEM-1 beta-lactamase determined by electron nuclear double resonance spectroscopy.

Site-directed spin-labeling of proteins whereby the spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)methanethiolsulfonate (SLMTS) is reacted with the -SH groups of cysteinyl residues incorporated into a protein by mutagenesis has been successfully applied to investigate secondary structure and conformational transitions of proteins. In these studies, it is expected that the spin-label moiety adopts different conformations dependent on its local environment. To determine the conformation of SLMTS in solution reacted with L-cysteine (SLMTCys) and bound in the active site of the Glu240Cys mutant of TEM-1 beta-lactamase, we have synthesized SLMTS both of natural abundance isotope composition and in site-specifically deuterated forms for electron nuclear double resonance (ENDOR) studies. ENDOR-determined electron-proton distances from the unpaired electron of the nitroxyl group of the spin-label to the methylene and methyl protons of SLMTS showed three conformations of the oxypyrrolinyl ring with respect to rotation around the S-S bond dependent on the solvent dielectric constant. For SLMTCys, two conformations of the molecule were compatible with the ENDOR-determined electron-nucleus distances to the side-chain methylene protons and to H(alpha) and H(beta1,2) of cysteine. To determine SLMTS conformation reacted with the Glu240Cys mutant of TEM-1 beta-lactamase, enzyme was overexpressed in both ordinary and perdeuterated minimal medium. Resonance features of H(alpha) and H(beta1,2) of the Cys240 residue of the mutant and of the side-chain methylene protons within the spin-label moiety yielded electron-proton distances that sterically accommodated the two conformations of free SLMTCys in solution.     

Diverse interactions between the individual mutations in a double mutant at the active site of staphylococcal nuclease.

In principle, the quantitative effect of a second mutation on a mutant enzyme may be antagonistic, absent, partially additive, additive, or synergistic with respect to the first mutation. Depending on the kinetic or thermodynamic parameter measured, the D21E and R87G mutations of staphylococcal nuclease exhibit four of these five categories of interaction in the double mutant. While Vmax of the R87G single mutant of staphylococcal nuclease is 10(4.8)-fold lower than that of the wild-type enzyme and the Vmax of the D21E single mutant is 10(3.0)-fold below that of wild type, the double mutant D21E + R87G was found to lose a factor of only 10(4.1) in Vmax relative to wild type, rather than the product of the two single mutations (10(7.8)). These results suggest antagonistic structural effects of the individual R87G and D21E mutations. An alternative explanation for the nonadditivity of effects, namely, the separate functioning of these residues in a stepwise mechanism involving the prior attack of water on phosphorus followed by protonation of the leaving group by Arg-87, is unlikely since no enzyme-bound phosphorane intermediate (less than 1% of [enzyme]) was found under steady-state conditions on the R87G mutant by 31P NMR at 242.9 MHz. Like the effects on Vmax, quantitatively similar antagonistic effects of the two mutations were detected on the binding of divalent cations in binary enzyme-Ca2+ and enzyme-Mn2+ complexes and in the ternary enzyme-Ca2(+)-5'-pdTdA complex, suggesting that the effects on Vmax result from antagonistic structural changes at the Ca2+ binding site. Simple additive weakening effects of the two mutations were found on the binding of the substrate 5'-pdTdA, in both the absence and the presence of the divalent cations, Mn2+ and Ca2+. However, synergistic effects of the two mutations were found on the binding of the substrate analogue 3',5'-pdTp, profoundly weakening its binding to the double mutant in both the absence and the presence of divalent cations. Such synergistic effects of the two mutations may result from negative cooperativity or strain in the binding of 3',5'-pdTp to the wild-type enzyme. It is concluded that the quantitative interactions of two active-site mutations of an enzyme can vary greatly depending on which parameter of the enzyme is measured. When the two mutations interact in the same way on several parameters, a common underlying mechanism is suggested.     

Fatty acylation of phospholipase D1 on cysteine residues 240 and 241 determines localization on intracellular membranes.

We have reported previously that phospholipase D1 (PLD1) is labeled specifically with [(3)H]palmitate following transient expression and immunoprecipitation and that this modification appeared important both for membrane localization and catalytic activity. In this work we identify by mutagenesis that the acylation sites on PLD1 are cysteine residues 240 and 241, with the cysteine at position 241 accounting for most but not all of the modification. Replacement of both cysteine residues with either serines or alanines resulted in a mutant protein that contained undetectable [(3)H]palmitate. In comparison with the wild type protein, the double mutant showed reduced catalytic activity in vivo, whereas its activity in vitro was unchanged. In addition, the localization of the double mutant was altered in comparison with the wild type protein, whereas wild type PLD1 is primarily on intracellular membranes and on punctate structures, the double mutant was on plasma membrane. Because cysteines 240 and 241 lie within a putative pleckstrin homology domain of PLD1, it is likely that fatty acylation on these residues modulates the function of the PLD1 pleckstrin homology domain.