Biomedical subjects

P D Vogel

Publications and source records attributed to P D Vogel.

12 recordsLinked to original sources

PubMed2001

Electron spin resonance and fluorescence studies of the bound-state conformation of a model protein substrate to the chaperone SecB.

SecB is a homotetrameric, cytosolic chaperone that forms part of the protein translocation machinery in Escherichia coli. We have investigated the bound-state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58-residue protein and contains three disulfide groups between residues 5 and 55, 14 and 38, as well as 30 and 51. Mutants of BPTI that contained only a single disulfide were reduced, and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide. The relative proximity of the labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy. The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB. Binding occurs at multiple sites on the substrate, and the binding site on each SecB monomer accommodates less than 21 substrate residues. In addition, we have labeled four solvent-accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The electron spin resonance data suggest that these cysteine residues are in close proximity (15 A) when no substrate protein is bound but move away to a distance of greater than 20 A when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein.

Alanine↗

PubMed2000

Site-directed spin-labeling of the catalytic sites yields insight into structural changes within the F0F1-ATP synthase of Escherichia coli.

Electron spin resonance (ESR) spectroscopy using site-specific cysteine spin-labeling of the catalytic nucleotide binding sites of F(1)-ATPase was employed to investigate conformational changes within the nucleotide binding sites of the enzyme. Mutant Escherichia coli F(1) that had been modified at position beta-Y331C with a spin label showed almost normal catalytic activity and enabled us to study the effects of binding of different nucleotides and of the F(o) subunit b on the conformation of the catalytic binding sites. The ESR spectra of the spin-labeled, nucleotide-depleted F(1) indicate asymmetry within the sites as is expected from the structural models of the enzyme. Nucleotide binding to the enzyme clearly affects the conformation of the sites; the most pronounced feature upon nucleotide binding is the formation of catalytic site(s) in a very open conformation. Using the same beta-331 spin-labeled F(1) and a truncated form of F(o) subunit b, b(24)(-)(156), we found that binding of b(24)(-)(156) to spin-labeled F(1) significantly changes the conformation of the catalytic sites. In this paper we present data that for the first time directly show that a conformational binding change takes place upon binding of nucleotides to the nucleotide binding sites and that also show that binding of b(24)(-)(156) strongly affects the conformation of the catalytic sites, most likely by increasing the population of binding sites that are in the open conformation.

Adenosine Diphosphate↗

PubMed2000

Insights into ATP synthase structure and function using affinity and site-specific spin labeling.

A variety of different approaches has been used during the last couple of decades to investigate structure and function relationships within the catalytic portion of the F0F1-ATP synthase and of its interactions with the proton-translocator F0. In our group, we employ ESR spectroscopy with the use of stable organic radicals, so-called spin labels, as reporter groups. The radicals are either attached to substrates/ligands or specifically inserted into the protein structure by "site-specific spin labeling." Both approaches bear intrinsic advantages for their special uses and result in the specific information that is available through ESR, e.g., structural changes due to binding of effector molecules (e.g., Mg2+ ions), conformational transitions during catalytic turnover, distance information on radicals bound at 20 A or less, and information on the binding characteristics of labeled substrates. This review summarizes the results of a variety of different approaches we have used during the last years to study, with the help of ESR spectroscopy, the structure of the nucleotide binding sites of F1-ATPases of different origins as well as interactions with F0 subunits.

Affinity Labels↗

PubMed1999

Photoaffinity labeling of wild-type and mutant forms of the yeast V-ATPase A subunit by 2-azido-[(32)P]ADP.

Molecular modeling studies have previously suggested the possible presence of four aromatic residues (Phe(452), Tyr(532), Tyr(535), and Phe(538)) near the adenine binding pocket of the catalytic site on the yeast V-ATPase A subunit (MacLeod, K. J., Vasilyeva, E., Baleja, J. D., and Forgac, M. (1998) J. Biol. Chem. 273, 150-156). To test the proximity of these aromatic residues to the adenine ring, the yeast V-ATPase containing wild-type and mutant forms of the A subunit was reacted with 2-azido-[(32)P]ADP, a photoaffinity analog that stably modifies tyrosine but not phenylalanine residues. Mutant forms of the A subunit were constructed in which the two endogenous tyrosine residues were replaced with phenylalanine and in which a single tyrosine was introduced at each of the four positions. Strong ATP-protectable labeling of the A subunit was observed for the wild-type and the mutant containing tyrosine at 532, significant ATP-protectable labeling was observed for the mutants containing tyrosine at positions 452 and 538, and only very weak labeling was observed for the mutants containing tyrosine at 535 or in which all four residues were phenylalanine. These results suggest that Tyr(532) and possibly Phe(452) and Tyr(538) are in close proximity to the adenine ring of ATP bound to the A subunit. In addition, the effects of mutations at Phe(452), Tyr(532), Tyr(535), and Glu(286) on dissociation of the peripheral V(1) and integral V(0) domains both in vivo and in vitro were examined. The results suggest that in vivo dissociation requires catalytic activity while in vitro dissociation requires nucleotide binding to the catalytic site.

Adenosine Diphosphate↗

PubMed1997

Nucleotide hydrolysis-dependent conformational changes in p21(ras) as studied using ESR spectroscopy.

We have employed ESR spectroscopy using guanine nucleotides that contain a spin label at the 2',3'-position of the ribose to investigate structural changes in the proto-oncogene product p21(ras) that are dependent on nucleotide hydrolysis. The three nucleotide analogs used were 2',3'-(2,2,5, 5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid ester (SL) GTP, SL-GDP, and the non-hydrolyzable analog SL-guanylylimidodiphosphate. SL-GTP was hydrolyzed by p21 with rates similar to those for GTP hydrolysis and appears to be an excellent substrate analog. The ESR spectra of SL-GTP and SL-GDP in complex with p21 differ significantly when acquired at 0 degrees C or 5 degrees C indicating different environments (conformations) of the protein-bound radicals depending on the phosphorylation state of the bound nucleotide. We calculated the rate constant for the conformational change as deduced from the changes in the corresponding ESR spectra upon incubation of the p21.SL-GTP complex at 25 degrees C and compared it to the rate constant of hydrolysis of SL-GTP at the same temperature. The rate constant deduced from the ESR method was similar to that determined by a high performance liquid chromatography technique. The data are in agreement with the idea that a conformational change during GTP hydrolysis by p21 occurs simultaneously with the actual hydrolysis step.

Binding Sites↗

PubMed1997

ATP-binding properties of human Hsp90.

Hsp90 is one of the most abundant proteins in the cytosol of eukaryotic cells. Under physiological conditions Hsp90 has been shown to play a major role in several specific signaling pathways, including maturation of various kinases and maintenance of steroid receptors in an activable state. It is well established that the level of Hsp90 increases severalfold under stress conditions, and it has been shown that the chaperone function of Hsp90 is ATP-independent. Although yeast Hsp90 does not bind ATP, as determined by a number of methods monitoring tight binding, ATP-dependent functions of Hsp90 in the presence of co-factors and elevated temperatures are still under discussion. Here, we have reinvestigated ATP-binding properties and ATPase activity of human Hsp90 under various conditions. We show that human Hsp90 does not bind ATP tightly and does not exhibit detectable ATPase activity. However, using electron spin resonance spectroscopy, weak binding of spin-labeled ATP analogues with half-maximal binding at 400 microM ATP was detected. The functional significance of this weak interaction remains enigmatic.

Adenosine Triphosphatases↗

PubMed1997

Asymmetry of catalytic but not of noncatalytic sites on Escherichia coli F1-ATPase in solution as observed using electron spin resonance spectroscopy.

We have employed electron spin resonance (ESR) spectroscopy using different spin-labeled nucleotides to probe the environment of nucleotides bound at catalytic and noncatalytic nucleotide binding sites of the Escherichia coli F1-ATPase. We found that nucleotides bound in the noncatalytic binding sites were strongly immobilized and resulted in ESR spectra with one single corresponding spectral component. Nucleotide bound at the catalytic binding sites gave rise to two different signals in the ESR spectra indicative of two distinct conformations of the catalytic sites of the protein. One conformation of the catalytic sites is very tight, resulting in signals identical to those of the noncatalytic sites, while the second type of catalytic sites permitted an unusually high mobility of the bound spin-labeled nucleotide. The findings are compared to the requirements of the binding change mechanism and to the features of the nucleotide binding sites as elucidated from the X-ray structural model of the beef heart mitochondrial enzyme.

Electron Spin Resonance Spectroscopy↗

PubMed1996

Nucleotide binding to the heat-shock protein DnaK as studied by ESR spectroscopy.

We employed ESR spectroscopy using spin-labeled adenine nucleotides to investigate nucleotide binding to the 70-kDa heat shock protein, DnaK, from Escherichia coli. Binding stoichiometries of 1 mol/ mol for both ATP and ADP to previously nucleotide-depleted protein in the presence of Mg2+ were determined directly and under equilibrium binding conditions. Of the spin-labeled adenine nucleotides available to us, only the derivatives with the spin label attached to the C8 position of the adenine moiety, 8-SL-AdoP3 and 8-SL-AdoP2 [8-(2,2,6,6-tetramethyl-piperidin-4-yl -1-oxyl-)amino-adenosine-5'-triphosphate or diphosphate], were bound sufficiently tightly by the heat-shock protein, resulting in ESR spectra typical for immobilized radicals. In the absence of Mg2+, only approximately 0.5 mol were bound. Subsequent addition of Mg2+, however, led to the previously observed maximum binding of 1 mol/mol. Both 8-SL-AdoP3 and 8-SL-AdoP2 were fully exchangeable upon addition of excess ATP or ADP suggesting that the analogs bound directly to the nucleotide binding sites within the protein. 8-SL-AdoP2 release was also observed in the presence of the co-chaperone GrpE, indicating that the spin-labeled analogs of adenine nucleotides function like the natural nucleotide-substrates of the heat-shock protein. Small differences in the ESR spectra of 8-SL-AdoP3 and 8-SL-AdoP2 in complex with DnaK were observed.

Adenine Nucleotides↗

PubMed1994

Effects of magnesium ions on the relative conformation of nucleotide binding sites of F1-ATPases as studied by electron spin resonance spectroscopy.

Cations like Mg2+ play an important role in the catalytic mechanism of F1-ATPases. In this study we applied ESR spectroscopy and used the ATP analog 2-azido-2',3'-(2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid ester)ATP (2-N3-SL-ATP) to investigate the effects of Mg2+ ions on the structure of the nucleotide binding sites of F1-ATPases from beef heart mitochondria (MF1) and from the thermophilic bacterium PS3 (TF1). The results demonstrated that Mg2+ ions not only influenced the binding of the nucleotide analogs to F1 but also altered the structure and geometry of the nucleotide binding sites. We observed that the dipolar interactions that are indicative of the close proximity of enzyme-bound 2-N3-SL-ANP (Vogel, P.D., Nett, J.H., Sauer, H.E., Schmadel, K., Cross, R.L., and Trommer, W.E. (1992) J. Biol. Chem. 267, 11982-11986) were only detectable in MF1-ATPase when the enzyme was preincubated with Mg2+ ions. In the absence of Mg2+, the enzyme exhibited ESR spectra indicative of spin label bound in at least two different environments (binding sites) with no dipolar interactions visible. TF1-ATPase did not exhibit clear dipolar interactions in the presence or absence of Mg2+. The ESR spectra of TF1 in the absence of Mg2+ indicated two different environments of the spin labels. Subsequent addition of Mg2+, however, led to exactly the same spectra as if the enzyme was incubated with the ions, indicating a rearrangement of the nucleotide binding sites. In summary, clear differences in the structures of the nucleotide binding sites of MF1 and TF1 in the presence or absence of Mg2+ were observed. Conformational differences between F1-bound spin-labeled nucleotides were also observed between TF1- and MF1-ATPases.

Adenosine Triphosphate↗

PubMed1993

Supported arm exercise vs unsupported arm exercise in the rehabilitation of patients with severe chronic airflow obstruction.

OBJECTIVE: Compare unsupported (UAEx) vs supported (SAEx) arm exercise in training of patients with severe chronic airflow obstruction (CAO). DESIGN: Randomized trial of UAEx vs SAEx training added to a 10-week outpatient program of lower extremity (LE) exercise training, respiratory muscle training, breathing retraining, psychological support, and teaching. SETTING: The Lahey Clinic Medical Center, a tertiary referral center. PATIENTS: Forty patients with CAO entered the rehabilitation program with 32 completing training and testing. INTERVENTIONS: All underwent progressive bicycle ergometer and treadmill training and respiratory muscle training using a threshold inspiratory pressure trainer. Patients were randomized to progressive SAEx training (arm cycle ergometer, n = 17) or UAEx training (raising weighted dowel, n = 18). MAIN OUTCOME MEASURES AND RESULTS: There was no significant difference in disease severity or exercise capacity between the two groups. Twelve-min walk test, bicycle ergometer power output, and respiratory muscle function improved with no significant difference in improvement between the two groups. Both groups showed similar improvements in arm ergometer testing while those trained with UAEx showed greater improvement in dowel testing (UAEx > SAEx, p = 0.002). In 17 patients VO2isotime (time at which patient performed pre-training and post-training tests) was measured during dowel testing. Only those trained with UAEx showed decreases in VO2isotime (UAEx trained, p = 0.02; SAEx, p = 0.18). VO2 during the last minute of a 2-min period of simple arm elevation was also measured in 17 patients. Only those trained with UAEx showed decreases in VO2 (UAEx, p = 0.02; SAEx, p = 0.20). CONCLUSION: We confirm that a pulmonary rehabilitation program incorporating exercise training improves LE and respiratory muscle function. Arm exercise training improved arm activity with greater increases in unsupported arm activity seen in those trained with unsupported arm training. Metabolic cost of UAEx decreased only in those trained with UAEx. As UAEx is typical of activities of daily living in patients with CAO, the changes seen with UAEx training may be of greater clinical significance. Arm training should be incorporated in exercise training and a simple program of UAEx appears the optimal format.

Adult↗

PubMed1992

Nucleotide binding sites on mitochondrial F1-ATPase. Electron spin resonance spectroscopy and photolabeling by azido-spin-labeled adenine nucleotides support an adenylate kinase-like orientation.

A spin-labeled photoaffinity ATP analog, 2-N3-2',3'-SL-ATP (2-N3-SL-ATP) was specifically loaded at catalytic (exchangeable) or noncatalytic (nonexchangeable) nucleotide-binding sites on nucleotide-depleted mitochondrial F1-ATPase. Photolysis of the enzyme complexes resulted in the specific modification of beta-Tyr-345 when the catalytic sites were occupied and beta-Tyr-368 when noncatalytic sites were filled. These are the same amino acid assignments that were made previously using 2-N3ATP. The results demonstrate that the attachment of a spin label moiety to the ribose ring does not prevent proper binding of the analog at both types of nucleotide sites on F1-ATPase and suggest that the probe can be used for investigations of the nucleotide-binding sites using ESR spectroscopy. Enzyme that is in complex with the 2-N3-SL-ATP exhibits an ESR spectrum that is typical for highly immobilized nitroxyl radicals both in the dark or after photolysis. Additional peaks in the high- and low-field regions arise due to dipolar spin interactions most likely involving a pair of catalytic and noncatalytic sites. The two sites are calculated to be approximately 15 A apart. This distance, obtained through ESR spectroscopy, combined with the finding that the 2 labeled amino acids are only 23 residues apart from each other, further supports an adenylate kinase-like arrangement of nucleotide binding sites on F1-ATPase where catalytic and noncatalytic sites are in close proximity (Vogel, P. D., and Cross, R. L. (1991) J. Biol. Chem. 266, 6101-6105).

Adenine Nucleotides↗

PubMed1991

Adenine nucleotide-binding sites on mitochondrial F1-ATPase. Evidence for an adenylate kinase-like orientation of catalytic and noncatalytic sites.

Nucleotide-depleted mitochondrial F1-ATPase (F1[0,0]) is inhibited by the diadenosine oligophosphate compounds, AP4A, AP5A, and AP6A (where APxA stands for 5',5'-diadenosine oligophosphates having a chain of x phosphoryl groups linking the two adenosine moieties). When F1[0,0] is preincubated with these compounds and then assayed for ATP hydrolysis activity under conditions that normally allow turnover at all three catalytic sites, the maximal level of inhibition observed is 80%. However, when assayed at lower ATP concentrations under conditions that allow simultaneous turnover at only two of the three sites, no inhibition is observed. A decrease in the number of phosphoryl groups that links the adenosine moieties to less than 4 (AP3A, AP2A) converts the compound to an activator of ATP hydrolysis, similar in effect to that obtained when one mol of ADP or 2-azido-ADP binds at a catalytic site on F1[0,0]. Inhibition by the compounds requires the presence of at least one vacant noncatalytic site. Evidence is provided that the probes also interact with a catalytic site. The stoichiometry for maximal inhibition by AP4A is 0.94 mol/mol of F1. The data presented support a model for the structure of nucleotide-binding sites on F1 that places catalytic and noncatalytic sites in close proximity in an orientation analogous to the ATP and AMP binding sites on adenylate kinase. Inhibition of the enzyme by the dinucleotide compounds can be explained by the cross-bridging of one of the catalytic sites to a noncatalytic site in analogy to the inhibition of adenylate kinase by AP5A. The residual capacity for bi-site catalysis indicates that the second and third catalytic sites remain catalytically active.

Adenine↗