scholarly journals Prolonged Residence Time of a Noncovalent Molecular Adapter, β-Cyclodextrin, within the Lumen of Mutant α-Hemolysin Pores

2001 ◽  
Vol 118 (5) ◽  
pp. 481-494 ◽  
Author(s):  
Li-Qun Gu ◽  
Stephen Cheley ◽  
Hagan Bayley
Keyword(s):  
Residence Time ◽  
Organic Molecules ◽  
Tight Binding ◽  
Major Effect ◽  
Site Directed Mutagenesis ◽  
Channel Blockers ◽  
Wild Type ◽  
Sensor Element ◽  
Kd Values ◽  
In The Wild

Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the α-hemolysin (αHL) pore, thereby offering a basis for the detection of a variety of organic molecules with αHL as a sensor element. β-Cyclodextrin (βCD) resides in the wild-type αHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric αHL pores that are capable of accommodating βCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane β barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind βCD ∼104-fold more avidly than the remaining αHL pores, including WT-αHL. The lower Kd values of these mutants are dominated by reduced values of koff. The major effect of the mutations is most likely a remodeling of the binding site for βCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral βCD molecule by electroosmotic flow. The mutant pores for which the dwell time of βCD is prolonged can serve as improved components for stochastic sensors.

scholarly journals Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

1994 ◽  
Vol 302 (2) ◽  
pp. 355-361 ◽  
Author(s):  
K Inukai ◽  
T Asano ◽  
H Katagiri ◽  
M Anai ◽  
M Funaki ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
In The Wild ◽  
Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

Development of surface pattern during division in Paramecium. II. Defective spatial control in the mutant kin241

Development ◽  
1992 ◽  
Vol 115 (1) ◽  
pp. 319-335 ◽  
Author(s):  
M. Jerka-Dziadosz ◽  
N. Garreau de Loubresse ◽  
J. Beisson
Keyword(s):  
Basal Body ◽  
Major Effect ◽  
The Body ◽  
Cortical Reorganization ◽  
Surface Pattern ◽  
Recessive Mutation ◽  
Wild Type ◽  
Cortical Organization ◽  
Nuclear Reorganization ◽  
In The Wild

kin241 is a monogenic nuclear recessive mutation producing highly pleiotropic effects on cell size and shape, generation time, thermosensitivity, nuclear reorganization and cortical organization. We have analyzed the nature of the cortical disorders and their development during division, using various specific antibodies labelling either one of the cortical cytoskeleton components, as was previously done for analysis of cortical pattern formation in the wild type. Several abnormalities in basal body properties were consistently observed, although with a variable frequency: extra microtubules in either the triplets or in the lumen; nucleation of a second kinetodesmal fiber; abnormal orientation of the newly formed basal body with respect to the mother one. The latter effect seems to account for the major observed cortical disorders (reversal, intercalation of supplementary ciliary rows). The second major effect of the mutation concerns the spatiotemporal map of cortical reorganization during division. Excess basal body proliferation occurs and is correlated with modified boundaries of some of the cortical domains identified in the wild type on the basis of their basal body duplication pattern. This is the first mutant described in a ciliate in which both the structure and duplication of basal bodies and the body plan are affected. The data support the conclusion that the mutation does not alter the nature of the morphogenetic signal(s) which pervade the dividing cell, nor the competence of cytoskeletal structures to respond to signalling, but affects the local interpretation of the signals.

scholarly journals A 13C-NMR study of the role of Asn-155 in stabilizing the oxyanion of a subtilisin tetrahedral adduct

1997 ◽  
Vol 326 (3) ◽  
pp. 861-866 ◽  
Author(s):  
Timothy P. O'CONNELL ◽  
Regina M. DAY ◽  
Ekaterina V. TORCHILIN ◽  
William W. BACHOVCHIN ◽  
J. Paul G. MALTHOUSE
Keyword(s):  
13C Nmr ◽  
Chemical Shifts ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Imidazolium Cation ◽  
Nmr Study ◽  
In The Wild ◽  
Main Factor

By removing one of the hydrogen-bond donors in the oxyanion hole of subtilisin BPN, we have been able to determine how it affects the catalytic efficiency of the enzyme and the pKa of the oxyanion formed in a choloromethane inhibitor derivative. Variant 8397 of subtilisin BPN contains five mutations which enhance its stability. Site-directed mutagenesis was used to prepare the N155A mutant of this variant. The catalytic efficiencies of wild-type and variant 8397 are similar, but replacing Asn-155 with alanine reduces catalytic efficiency approx. 300-fold. All three forms of subtilisin were alkylated using benzyloxycarbonylglycylglycyl[2-13C]phenylalanylchloromethane and examined by 13C-NMR. A single signal due to the 13C-enriched carbon was detected in all the derivatives and it was assigned to the hemiketal carbon of a tetrahedral adduct formed between the hydroxy group of Ser-221 and the inhibitor. This signal had chemical shifts in the range 98.3–103.6 p.p.m., depending on the pH. The titration shift of 4.7–4.8 p.p.m. was assigned to oxyanion formation. The oxyanion pKa values in the wild-type and 8397 variants were 6.92 and 7.00 respectively. In the N155A mutant of the 8397 variant the oxyanion pKa increased to 8.09. We explain why such a small increase is observed and we conclude that it is the interaction between the oxyanion and the imidazolium cation of the active-site histidine that is the main factor responsible for lowering the oxyanion pKa.

scholarly journals An Unusual Response Regulator Influences Sporulation at Early and Late Stages in Streptomyces coelicolor

2007 ◽  
Vol 189 (7) ◽  
pp. 2873-2885 ◽  
Author(s):  
Yuqing Tian ◽  
Kay Fowler ◽  
Kim Findlay ◽  
Huarong Tan ◽  
Keith F. Chater
Keyword(s):  
Streptomyces Coelicolor ◽  
Point Mutations ◽  
Aerial Mycelium ◽  
Site Directed Mutagenesis ◽  
Null Mutant ◽  
Wild Type ◽  
New Variant ◽  
In The Wild ◽  
Pcr Targeting ◽  
Spore Maturation

ABSTRACT WhiI, a regulator required for efficient sporulation septation in the aerial mycelium of Streptomyces coelicolor, resembles response regulators of bacterial two-component systems but lacks some conserved features of typical phosphorylation pockets. Four amino acids of the abnormal “phosphorylation pocket” were changed by site-directed mutagenesis. Unlike whiI null mutations, these point mutations did not interfere with sporulation septation but had various effects on spore maturation. Transcriptome analysis was used to compare gene expression in the wild-type strain, a D27A mutant (pale gray spores), a D69E mutant (wild-type spores), and a null mutant (white aerial mycelium, no spores) (a new variant of PCR targeting was used to introduce the point mutations into the chromosomal copy of whiI). The results revealed 45 genes that were affected by the deletion of whiI. Many of these showed increased expression in the wild type at the time when aerial growth and development were taking place. About half of them showed reduced expression in the null mutant, and about half showed increased expression. Some, but not all, of these 45 genes were also affected by the D27A mutation, and a few were affected by the D69E mutation. The results were consistent with a model in which WhiI acts differently at sequential stages of development. Consideration of the functions of whiI-influenced genes provides some insights into the physiology of aerial hyphae. Mutation of seven whiI-influenced genes revealed that three of them play roles in spore maturation.

scholarly journals Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c

1994 ◽  
Vol 302 (1) ◽  
pp. 95-101 ◽  
Author(s):  
A Schejter ◽  
T I Koshy ◽  
T L Luntz ◽  
R Sanishvili ◽  
I Vig ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
General Increase ◽  
Reduction Potentials ◽  
Cytochromes C ◽  
In The Wild ◽  
Order Of Magnitude ◽  
The Stability ◽  
Haem Iron

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

scholarly journals The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function

1995 ◽  
Vol 312 (1) ◽  
pp. 273-280 ◽  
Author(s):  
M Haraguchi ◽  
S Yamashiro ◽  
K Furukawa ◽  
K Takamiya ◽  
H Shiku ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Kinetics Analysis ◽  
Glycosylation Sites ◽  
In The Wild ◽  
Polymerase Chain ◽  
The Stability

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

scholarly journals Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei

2007 ◽  
Vol 405 (3) ◽  
pp. 445-454 ◽  
Author(s):  
Tanja Schlecker ◽  
Marcelo A. Comini ◽  
Johannes Melchers ◽  
Thomas Ruppert ◽  
R. Luise Krauth-Siegel
Keyword(s):  
Glutathione Peroxidase ◽  
Trypanosoma Brucei ◽  
Catalytic Mechanism ◽  
Disulfide Bridge ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Glutathione Peroxidases ◽  
Thiolate Anion ◽  
In The Wild

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical genes for cysteine-homologues of the selenocysteine-containing glutathione peroxidases. The enzymes, which are essential for the parasites, lack glutathione peroxidase activity but catalyse the trypanothione/Tpx (tryparedoxin)-dependent reduction of hydroperoxides. Cys47, Gln82 and Trp137 correspond to the selenocysteine, glutamine and tryptophan catalytic triad of the mammalian selenoenzymes. Site-directed mutagenesis revealed that Cys47 and Gln82 are essential. A glycine mutant of Trp137 had 13% of wild-type activity, which suggests that the aromatic residue may play a structural role but is not directly involved in catalysis. Cys95, which is conserved in related yeast and plant proteins but not in the mammalian selenoenzymes, proved to be essential as well. In contrast, replacement of the highly conserved Cys76 by a serine residue resulted in a fully active enzyme species and its role remains unknown. Thr50, proposed to stabilize the thiolate anion at Cys47, is also not essential for catalysis. Treatment of the C76S/C95S but not of the C47S/C76S double mutant with H2O2 induced formation of a sulfinic acid and covalent homodimers in accordance with Cys47 being the peroxidative active site thiol. In the wild-type peroxidase, these oxidations are prevented by formation of an intramolecular disulfide bridge between Cys47 and Cys95. As shown by MS, regeneration of the reduced enzyme by Tpx involves a transient mixed disulfide between Cys95 of the peroxidase and Cys40 of Tpx. The catalytic mechanism of the Tpx peroxidase resembles that of atypical 2-Cys-peroxiredoxins but is distinct from that of the selenoenzymes.

scholarly journals Trapping of Organic Blockers by Closing of Voltage-dependent K+ Channels

1997 ◽  
Vol 109 (5) ◽  
pp. 527-535 ◽  
Author(s):  
Miguel Holmgren ◽  
Paula L. Smith ◽  
Gary Yellen
Keyword(s):  
Quaternary Ammonium ◽  
Organic Molecules ◽  
Slow Phase ◽  
Channel Blockers ◽  
Wild Type ◽  
Squid Axon ◽  
Small Organic Molecules ◽  
K Channels ◽  
Voltage Dependent ◽  
Ammonium Compounds

Small organic molecules, like quaternary ammonium compounds, have long been used to probe both the permeation and gating of voltage-dependent K+ channels. For most K+ channels, intracellularly applied quaternary ammonium (QA) compounds such as tetraethylammonium (TEA) and decyltriethylammonium (C10) behave primarily as open channel blockers: they can enter the channel only when it is open, and they must dissociate before the channel can close. In some cases, it is possible to force the channel to close with a QA blocker still bound, with the result that the blocker is “trapped.” Armstrong (J. Gen. Physiol. 58:413–437) found that at very negative voltages, squid axon K+ channels exhibited a slow phase of recovery from QA blockade consistent with such trapping. In our studies on the cloned Shaker channel, we find that wild-type channels can trap neither TEA nor C10, but channels with a point mutation in S6 can trap either compound very efficiently. The trapping occurs with very little change in the energetics of channel gating, suggesting that in these channels the gate may function as a trap door or hinged lid that occludes access from the intracellular solution to the blocker site and to the narrow ion-selective pore.

scholarly journals Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1

2000 ◽  
Vol 350 (3) ◽  
pp. 677-683 ◽  
Author(s):  
Ana M. PAJOR ◽  
Esther S. KAHN ◽  
Rama GANGULA
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Conserved Residues ◽  
In The Wild ◽  
Cationic Amino Acids

The role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased Km for succinate of 1.8mM, compared with 0.3mM in the wild-type NaDC-1. The R349A mutant had Na+ and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding.

Pressure-jump Relaxation Kinetics of Unfolding and Refolding Transitions of Staphylococcal Nuclease and Proline Isomerization Mutants

1996 ◽  
Author(s):  
Gedlminas J. A. Vidugiris ◽  
Raj Thomas
Keyword(s):  
Globular Proteins ◽  
Staphylococcal Nuclease ◽  
Site Directed Mutagenesis ◽  
Pressure Jump ◽  
Wild Type ◽  
Relaxation Kinetics ◽  
Signal Activity ◽  
In The Wild ◽  
Pressure Jump Relaxation ◽  
Kinetics Of

We present here the first report of the pressure dependence of pressure-jump relaxation kinetics for protein folding transitions. We have studied the relaxation kinetics for the unfolding/refolding of wild-type Staphylococcal nuclease and have found that the relaxation kinetics observed at high pressure are much slower than those observed by pH or denaturant jumps at atmospheric pressure. This indicates that these processes have large, positive values for the activation volumes, most likely stemming from exclusion of solvent from a transition state that is less well packed than the native state. We examined the pressure-jump relaxation kinetics of three single-site mutations in nuclease that lead to alterations in the interactions between the two domains of the protein and changes in the equilibrium constant for isomerization of the lysine-116 to proline 117 peptide bond away from the cis form that predominates in the wild-type enzyme. At comparable pressures, the relaxation times for these mutants were significantly shorter than those observed for the wild type, indicating lower values of the activation volumes. We propose that these mutations cause a decrease in the cooperativity of the unfolding of the two domains, leading to a decrease in the degree of solvent exclusion at the rate-limiting step. The mechanism by which a particular amino acid sequence determines the fold and stability of globular proteins remains one of the most interesting and important unresolved issues in biophysical chemistry. The approaches to increasing our understanding of this phenomenon typically have involved perturbation of the proteins by chemical means or by temperature extremes. The equilibrium or time-dependent responses to these perturbations are then monitored (using a spectroscopic signal, activity, or some other observable) to extract the energetic or kinetic aspects of the unfolding or refolding transitions. Another means of perturbing the system is to modify the protein itself, either chemically or by site-directed mutagenesis, and to assess the effects of modification on the equilibrium or kinetic folding or refolding profiles. This approach has generated a great deal of information about small globular proteins that denature reversibly.

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