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J M Beechem

Publications and source records attributed to J M Beechem.

At least 37 records · Page 2Linked to original sources
PubMed1997

Real-time measurement of multiple intramolecular distances during protein folding reactions: a multisite stopped-flow fluorescence energy-transfer study of yeast phosphoglycerate kinase.

Understanding the set of rules which dictate how the primary amino acid sequence determines tertiary structure is an unsolved problem in biophysics. If it were possible to simultaneously measure all of the intramolecular distances in a protein (in real time) during a folding reaction, the "second" genetic code problem would be solved. Regrettably, no such technique currently exists. As a first step toward this goal, an optical distance assay system has been developed for a two-domain protein, yeast phosphoglycerate kinase (PGK), using Förster resonance energy transfer [Lillo, M. P., et al. (1997) Biochemistry 36, 11261-11272]. In this study, real-time stopped-flow distance changes are measured using six unique pairs of donor/acceptor fluorescent labels strategically placed throughout the tertiary structure of PGK. These multiple donor/acceptor sites were genetically engineered into PGK by cysteine substitution mutagenesis followed by extrinsic labeling with fluorescent probes, 5-[[[(2-iodoacetyl)amino]ethyl]amino]naphthalenesulfonic acid (as a donor) and 5-iodoacetamidofluorescein (acceptor). The unfolding of PGK is found to be a sequential multistep process (native --> I1 --> I2 --> unfolded) with rate constants of 0.30, 0.16, and 0.052 s-1, respectively (from native to unfolded). Unique to this unfolding study, six intramolecular distance vectors have been resolved for both the I1 and I2 states. With this distance information, it is shown that the transition from the native to I1 state can be modeled as a large hinge-bending motion, in which both domains "swing away" from each other by about 15 A. As the domains move apart, the carboxyl-terminal domain rotates almost 90 degrees about the hinge region connecting the two domains. It is also shown that the amino-terminal domain remains intact during the native --> I1 transition, consistent with our previous site-specific tryptophan fluorescence anisotropy stopped-flow study [Beechem, J. M., et al. (1995) Biochemistry 34, 13943-13948]. Future experiments are proposed which will attempt to resolve in detail the unfolding/refolding transitions in this protein with a resolution of approximately 5-10 A.

Energy Transfer↗
PubMed1997

Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL.

The chaperonin GroEL is a double-ring structure with a central cavity in each ring that provides an environment for the efficient folding of proteins when capped by the co-chaperone GroES in the presence of adenine nucleotides. Productive folding of the substrate rhodanese has been observed in cis ternary complexes, where GroES and polypeptide are bound to the same ring, formed with either ATP, ADP or non-hydrolysable ATP analogues, suggesting that the specific requirement for ATP is confined to an action in the trans ring that evicts GroES and polypeptide from the cis side. We show here, however, that for the folding of malate dehydrogenase and Rubisco there is also an absolute requirement for ATP in the cis ring, as ADP and AMP-PNP are unable to promote folding. We investigated the specific roles of binding and hydrolysis of ATP in the cis and trans rings using mutant forms of GroEL that bind ATP but are defective in its hydrolysis. Binding of ATP and GroES in cis initiated productive folding inside a highly stable GroEL-ATP-GroES complex. To discharge GroES and polypeptide, ATP hydrolysis in the cis ring was required to form a GroEL-ADP-GroES complex with decreased stability, priming the cis complex for release by ATP binding (without hydrolysis) in the trans ring. These observations offer an explanation of why GroEL functions as a double-ring complex.

Adenosine Triphosphate↗
PubMed1997

Structure-function analysis of TAF130: identification and characterization of a high-affinity TATA-binding protein interaction domain in the N terminus of yeast TAF(II)130.

We report structure-function analyses of TAF130, the single-copy essential yeast gene encoding the 130,000-Mr yeast TATA-binding protein (TBP)-associated factor TAF(II)130 (yTAF(II)130). A systematic family of TAF130 mutants was generated, and these mutant TAF130 alleles were introduced into yeast in both single and multiple copies to test for their ability to complement a taf130delta null allele and support cell growth. All mutant proteins were stably expressed in vivo. The complementation tests indicated that a large portion (amino acids 208 to 303 as well as amino acids 367 to 1037) of yTAF(II)130 is required to support cell growth. Direct protein blotting and coimmunoprecipitation analyses showed that two N-terminal deletions which remove portions of yTAF(II)130 amino acids 2 to 115 dramatically decrease the ability of these mutant yTAF(II)130 proteins to bind TBP. Cells bearing either of these two TAF130 mutant alleles also exhibit a slow-growth phenotype. Consistent with these observations, overexpression of TBP can correct this growth deficiency as well as increase the amount of TBP interacting with yTAF(II)130 in vivo. Our results provide the first combined genetic and biochemical evidence that yTAF(II)130 binds to yeast TBP in vivo through yTAF(II)130 N-terminal sequences and that this binding is physiologically significant. By using fluorescence anisotropy spectroscopic binding measurements, the affinity of the interaction of TBP for the N-terminal TBP-binding domain of yTAF(II)130 was measured, and the Kd was found to be about 1 nM. Moreover, we found that the N-terminal domain of yTAF(II)130 actively dissociated TBP from TATA box-containing DNA.

Amino Acid Sequence↗
PubMed1996

Real-time fluorescence assay system for gene transcription: simultaneous observation of protein/DNA binding, localized DNA melting, and mRNA production.

This article describes the development of an in vitro multicolor fluorescence assay system for studying protein/DNA complex formation, transcription bubble formation, and mRNA production. These studies were accomplished using three different fluorescent spectroscopic probes: rhodamine-labeled DNA (at the 5' position) to monitor protein/DNA complex formation, DNA internally labeled with the base analog 2-aminopurine in place of adenine to monitor transcription bubble formation, and gamma-fluorophore-labeled UTP nucleotide to measure mRNA transcription rates. Combining these three assay systems allows the simultaneous determination of protein/DNA binding, localized DNA melting transitions, and mRNA production at physiological concentrations of reagents (pM-nM) and millisecond timing resolution. The application of this multicolor fluorescence assay to Escherichia coli RNA polymerase reactions (binding, open complex formation, and mRNA production) demonstrates the importance of kinetically coupled events in gene transcription.

2-Aminopurine↗
PubMed1996

Dynamics of loading the beta sliding clamp of DNA polymerase III onto DNA.

A "minimal" DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports processive DNA synthesis by DNA polymerase III core in the presence of the beta sliding clamp, gamma complex clamp loader, and single-stranded binding protein from Escherichia coli. This primer-template system was used to measure the loading of the beta sliding clamp by the gamma complex in an ATP-dependent reaction. Bound protein-DNA complexes were detected by monitoring fluorescence depolarization of DNA. Steady state and time-resolved anisotropies were measured, and stopped-flow pre-steady state fluorescence measurements allowed visualization of the loading reactions in real time. The rate of loading beta onto DNA was 12 s-1, demonstrating that clamp assembly is rapid on the time scale required for lagging strand Okazaki fragment synthesis. The association rate appears to be limited by an intramolecular step occurring prior to the clamp-loading reaction, possibly the opening of the toroidal beta dimer.

Adenosine Triphosphate↗
PubMed1996

Unusual binding stoichiometries and cooperativity are observed during binary and ternary complex formation in the single active pore of R67 dihydrofolate reductase, a D2 symmetric protein.

R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that confers resistance to the antibacterial drug, trimethoprim. This DHFR variant is not homologous in either sequence or structure to chromosomal DHFRs. A recent crystal structure of the active tetrameric species describes a single active site pore that traverses the length of the protein (Narayana et al., 1995). Related sites (due to a 222 symmetry element at the center of the active site pore) are used for binding of ligands, i.e., each half-pore can accommodate either the substrate, dihydrofolate, or the cofactor, NADPH, although dihydrofolate and NADPH are bound differently. Ligand binding in R67 DHFR was evaluated using time-resolved fluorescence anisotropy and isothermal titration calorimetry techniques. Under binary complex conditions, two molecules of either NADPH, folate, dihydrofolate, or N10 propargyl-5,8-dideazafolate (CB3717) can be bound. Binding of NADPH displays negative cooperativity, binding of either folate or dihydrofolate shows positive cooperativity, and binding of CB3717 shows two identical sites. Any asymmetry introduced by binding of one ligand is proposed to induce the cooperativity associated with binding of the second ligand. Evaluation of ternary complex formation demonstrates that one molecule of folate binds to a 1:1 mixture of R67 DHFR+NADPH. These binding results indicate a maximum of two ligands bind in the pore. A mechanism describing catalysis is proposed that is consistent with the binding results.

Allosteric Regulation↗
PubMed1996

Characterization of the active intermediate of a GroEL-GroES-mediated protein folding reaction.

Recent studies of GroE-mediated protein folding indicate that substrate proteins are productively released from a cis ternary complex in which the nonnative substrate is sequestered within the GroEL channel underneath GroES. Here, we examine whether protein folding can occur in this space. Stopped-flow fluorescence anisotropy of a pyrene-rhodanese-GroEl complex indicates that addition of GroES and ATP (but not ADP) leads to a rapid change in substrate flexibility at GroEL. Strikingly, when GroES release is blocked by the use of either a nonhydrolyzable ATP analog or a single-ring GroEL mutant, substrates complete folding while remaining associated with chaperonin. We conclude that the cis ternary complex, in the presence of ATP, is the active state intermediate in the GroE-mediated folding reaction: folding is initiated in this state and for some substrates may be completed prior to the timed release of GroES triggered by ATP hydrolysis.

Adenosine Triphosphate↗
PubMed1996

Large-amplitude picosecond anisotropy decay of the intrinsic fluorescence of double-stranded DNA.

The conformational flexibility of the DNA double helix is of great interest because of its potential role in protein recognition, packaging into chromosomes, formation of photodefects, and interaction with drugs. Theory finds that DNA is very flexible; however, there is a scarcity of experimental results that examine intrinsic properties of the DNA bases for the inherent flexibility in solution. We have studied the dynamics of poly(dA).poly(dT) and (dA)20.(dT)20 in a 50 mM cacodylate, 0.1 M NaCl, pH 7 buffer by using the time-correlated picosecond fluorescence anisotropy of thymine selectively excited at 293 nm. For both nucleic acids, a large-amplitude biphasic decrease in the anisotropy is observed that has a very fast, large-amplitude component on the picosecond time scale and a slower, smaller-amplitude component on the nanosecond time scale. These modes are sensitive to sucrose concentration, and are greatly attenuated at 77% sucrose by volume. This observation suggests that motions of the bases make a significant contribution to the observed fluorescence depolarization (in the absence of sucrose). Measurements on the single-stranded systems poly(dT) and (dT)20 reveal a much smaller amplitude of the very fast depolarization mode. These observations are consistent with a mechanism that involves concerted motions in the interior of the double-stranded systems.

Biophysical Phenomena↗
PubMed1995

The interaction of epidermal growth factor with its receptor in A431 cell membranes: a stopped-flow fluorescence anisotropy study.

We describe a quantitative examination of the interaction of epidermal growth factor (EGF) with the EGF receptor using A431 cell membrane vesicles as a receptor source. Using T-format steady-state fluorescence anisotropy detection coupled with stopped-flow mixing, we measured the association and EGF-induced dissociation kinetics of fluorescein 5-isothiocyanate-labeled mEGF (FITC-EGF) with the EGF receptor over a wide range of FITC-EGF concentrations, membrane dilutions, and time scales (milliseconds to minutes). Fluorescence anisotropy-based equilibrium binding titrations were also performed. All studies utilized the same receptor preparation, ligand preparation, and detection system. The entire data surface (approximately 78,000 data points) was simultaneously analyzed using global analysis techniques with a variety of kinetic models. Our analysis identified with a high level of confidence receptor populations with two association rate constants (k(on) = 1.2 x 10(6) M-1 s-1, 7.2 x 10(6) M-1 s-1) and three dissociation rate constants (k(off0 = 0.95 x 10(-2) s-1, 0.13 x 10(-2) s-1, 0.32 x 10(-3) s-1), which reflect the presence of at least two distinct receptor populations in A431 cell membranes. Analysis of the kinetic data was found to be much more sensitive to the presence of multiple receptor populations than was the analysis of the equilibrium binding data.

Animals↗
PubMed1995

Probing intradomain and interdomain conformational changes during equilibrium unfolding of phosphoglycerate kinase: fluorescence and circular dichroism study of tryptophan mutants.

Phosphoglycerate kinase is a monomeric protein composed of two globular domains of the alpha/beta type. Extensive domain-domain interactions involve three segments of the polypeptide chain that are distant from one another in the primary sequence: the N-terminus, the C-terminus, and a centrally located alpha-helix. In order to monitor spectroscopically the conformational changes that occur in the individual domains and at the interdomain interface during the unfolding process, we have constructed a series of single-tryptophan mutants. In addition to two previously described mutants, each with single tryptophans in the C-terminal domain (W308 and W333) [Szpikowska, B. K., Beechem, J. M., Sherman, M. A., & Mas, M. T. (1994) Biochemistry 33, 2217-2225], four new single-tryptophan mutants have been constructed: two with tryptophans located in the interdomain region (W194 and W399) and two with tryptophans in the N-terminal domain (W48 and W122). The equilibrium unfolding transitions induced by guanidine hydrochloride were monitored using far-UV CD, near-UV CD, steady-state, and time-resolved fluorescence. These studies reveal two unfolding transitions and suggest a sequential unfolding process for the mutants described in this paper. During the first transition (Cm approximately 0.5 M) the interdomain region and C-terminal domain unfold; the N-terminal domain remains relatively compact but lacks much of the tertiary structure that characterizes the native state. A hyperfluorescent intermediate is detected during this transition by tryptophan probes placed within the N-terminal domain. Complete unfolding of the N-terminal domain occurs during the second transition (Cm approximately 0.9 M).

Circular Dichroism↗
PubMed1995

Sequential domain unfolding in phosphoglycerate kinase: fluorescence intensity and anisotropy stopped-flow kinetics of several tryptophan mutants.

Stopped-flow total intensity and anisotropy experiments on single tryptophan containing mutants of yeast phosphoglycerate kinase (PGK) located in either the carboxy-terminal domain (W308 and W333), amino-terminal domain (W48 and W122), or "hinge" region (W194 and W399) were performed. The results obtained for single tryptophans in individual domains suggest that the unfolding of PGK by guanidinium hydrochloride is a sequential process in which unfolding of the carboxy-terminal domain is followed by the unfolding of the amino-terminal domain. A kinetic intermediate has been detected which consists of an unfolded carboxy-terminal domain and an altered amino-terminal domain, identical in hydrodynamic properties with the native state, but hyperfluorescent. In contrast to the C-terminal tryptophans, which exhibit concurrent total intensity and anisotropy changes in the entire denaturant concentration range (0-->2 M), the N-terminal tryptophans experience a large increase in fluorescence intensity and a constant anisotropic environment at low concentrations of denaturant, corresponding to the first transition region of the equilibrium unfolding profile. Anisotropy changes for the N-terminal probes are observed above 1 M Gdn-HCl, the region corresponding to the second equilibrium unfolding transition. Stopped-flow experiments performed on PGK mutants with two tryptophans (i.e., with a single tryptophan in each domain) confirm that each domain unfolds independently, and that the individual site-specific mutations do not significantly alter the unfolding pathway. Unfolding kinetics experiments with tryptophans situated in the hinge reveal that the region sensed by W399 unfolds before the carboxy-terminal domain, whereas W194 senses unfolding of both domains.

Fluorescence Polarization↗
PubMed1995

Yeast TATA binding protein interaction with DNA: fluorescence determination of oligomeric state, equilibrium binding, on-rate, and dissociation kinetics.

A combination of steady-state, stopped-flow, and time-resolved fluorescence of intrinsic tryptophan and extrinsically labeled fluorescent DNA is utilized to examine the interaction of yeast TATA binding protein (TBP) with DNA. TBP is composed of two structural domains, the carboxy domain (residues 61-240), which is responsible for DNA binding and initiation of basal level transcription, and an amino terminal domain (residues 1-60), whose function is currently unknown. The steady-state fluorescence emission spectrum of the single tryptophan in the amino terminal domain of TBP undergoes a huge (30-40 nm) red-shift upon interaction with stoichiometric amounts of TATA box containing DNA. From time-resolved tryptophan fluorescence anisotropy studies, we demonstrate that, in the absence of DNA, the protein exists as a multimer in solution and it contains (at least) two primary conformations, one with the amino terminus associated tightly with the protein(s) in a hydrophobic environment and one with the amino terminus decoupled away from the rest of the protein and solvent-exposed. Upon binding DNA, the protein dissociates into a monomeric complex, upon which only the solvent-exposed amino terminus conformation remains. Kinetic and equilibrium binding studies were performed on TATA box containing DNA which was extrinsically labeled with a fluorescent probe Rhodamine-X at the 5'-end. This "fluorescent" DNA allowed for the collection of quantitative spectroscopic binding, kinetic on-rate, and kinetic off-rate data at physiological concentrations. Global analysis of equilibrium binding studies performed from 500 pM to 50 nM DNA reveals a single dissociation constant (Kd) of approximately 5 nM. Global analysis of stopped-flow anisotropy on-rate experiments, with millisecond timing resolution and TBP concentrations ranging from 20 to 600 nM (20 nM DNA), can be perfectly described by a single second-order rate constant of 1.66 x 10(5) M(-1) s(-1). These measurements represent the very first stopped-flow anisotropy study of a protein/DNA interaction. Stopped-flow anisotropy off-rate experiments reveal a single exponential k(off) of 4.3 x 10(-2) min-1 (1/k(off) = 23 min) From the ratio of on-rate to off-rate, a predicted Kd of 4.3 nM is obtained, revealing that the kinetic and equilibrium studies are internally consistent. Deletion of the amino terminal domain of TBP decreases the k(on) of TBP approximately 45-fold and eliminates classic second-order behavior.

Base Sequence↗
PubMed1995

Local and global dynamics during the folding of Escherichia coli dihydrofolate reductase by time-resolved fluorescence spectroscopy.

Time-resolved fluorescence techniques were utilized to monitor the kinetic refolding reaction of Escherichia coli dihydrofolate reductase (DHFR). Measurements of emission and anisotropy decay lifetimes of both the five intrinsic tryptophan residues and the fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) during the folding reaction were used to characterize the compactness and development of tertiary structure in transient intermediates formed during the folding of DHFR. Experiments monitoring bound ANS show that a rapidly-formed intermediate (< 20 ms) has a rotational time of approximately 10 ns and, therefore, a compactness similar to that for the native conformation. All of the tryptophan residues in this burst phase species rotate as freely as in the unfolded state. In the set of four intermediates which then appear over the next few hundred milliseconds, the apparent rotational time measured by ANS fluorescence increases to a maximum rotational time of approximately 20 ns. An increase in the average tryptophan lifetime for these intermediates suggests these side chains become excluded from solvent and associated dynamic quenching mechanisms. As the folding reaction proceeds to a set of four native conformers the bound ANS rotational time then decreases to approach that for the native protein, 10.5 ns, and the average tryptophan rotational time increases to the same value. During these rate-limiting, final steps in folding, the static quenching effects which reflect the formation of specific tertiary contacts involving tryptophans also appear.

Adenine↗
PubMed1994

Pre-steady-state kinetic analysis of sequence-dependent nucleotide excision by the 3'-exonuclease activity of bacteriophage T4 DNA polymerase.

The effects of local DNA sequence on the proofreading efficiency of wild-type T4 DNA polymerase were examined by measuring the kinetics of removal of the fluorescent nucleotide analog 2-aminopurine deoxynucleoside monophosphate (dAPMP) from primer/templates of defined sequences. The effects of (1) interactions with the 5'-neighboring bases, (2) base pair stability, and (3) G.C content of the surrounding sequences on the pre-steady-state kinetics of dAPMP excision were measured. Rates of excision dAPMP from a primer 3'-terminus located opposite a template T (AP.T base pair) increased, over a 3-fold range, with the 5'-neighbor to AP in the order C < G < T < A. Rates of removal of dAPMP from AP.X base pairs located in the same surrounding sequence increased as AP.T < AP.A < AP.C < AP.G, which correlates with the decrease in the stabilities of these base pairs predicted by Tm measurements. A key finding was that AP was excised at a slower rate when mispaired opposite C located next to four G.C base pairs than when correctly paired opposite T next to four A.T base pairs, suggesting that exonuclease mismatch removal specificities may be enhanced to a much greater extent by instabilities of local primer termini than by specific recognition of incorrect base pairs. In polymerase-initiated reactions, biphasic reaction kinetics were observed for the excision of AP within most but not all sequence contexts. Rates of the rapid phases (30-40 s-1) were relatively insensitive to sequence context. Rapid-phase rates reflect the rate constants for exonucleolytic excision of dAPMP from melted primer termini for both correct and incorrect base pairs and were roughly comparable to rates of removal of dAPMP from single-stranded DNA (65-80 s-1). Rates of the slow phases (3-13 s-1) were dependent on sequence context; the slow phase may reflect the rate of switching from the polymerase to the exonuclease active site, or perhaps the conversion of a primer/template terminus from an annealed to a melted state in the exonuclease active site. These data, using wild-type T4 DNA polymerase and two exonuclease-deficient T4 polymerases, support a model in which exonuclease excision occurs on melted primer 3'-termini for both mismatched and correctly matched primer termini, and where specificity favoring removal of terminally mismatched base pairs is determined by the much larger fraction of melted-out primer 3'-termini for mispairs compared to that for correct pairs.

2-Aminopurine↗
PubMed1994

Equilibrium unfolding of yeast phosphoglycerate kinase and its mutants lacking one or both native tryptophans: a circular dichroism and steady-state and time-resolved fluorescence study.

Yeast 3-phosphoglycerate kinase contains two tryptophans, both situated in the carboxy-terminal domain, and seven tyrosines, five in the amino-terminal domain, one in the domain-domain interface, and one in the carboxy-terminal domain. Site-specific mutagenesis has been used to construct two single-tryptophan mutants and one no-tryptophan mutant by replacing one or both native tryptophans, W308 and W333, with phenylalanines. The mutations have been shown to have a relatively small effect on the overall structure and enzymatic properties of the mutants. Both tryptophans are quenched in the folded state. The steady-state emission spectra and tryptophan quantum yields are the same in the single-tryptophan mutants and in the wild-type protein. Large changes in the tryptophan emission maxima and steady-state emission intensities are observed upon unfolding. Far-UV circular dichroism and steady-state as well as time-resolved fluorescence spectroscopy have been used to monitor the equilibrium unfolding transitions of these mutants and wild-type PGK. For each protein, the transitions followed by CD and steady-state fluorescence are nearly coincident, suggesting that the structural changes monitored by local fluorescence probes and ellipticity changes, which are sensitive to the changes in the overall structure, report a single cooperative transition, consistent with a two-state unfolding mechanism. Both tryptophans have three lifetimes, which follow a similar pattern as a function of denaturant concentration. The amplitude terms associated with the two longer lifetimes increase with unfolding while the short lifetime amplitude decreases. It thus appears that these population amplitudes represent markers for the unfolded and folded states, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1994

Resolution of multiphasic reactions by the combination of fluorescence total-intensity and anisotropy stopped-flow kinetic experiments.

Multiphasic kinetics are often observed in stopped-flow investigations. To characterize further these kinetic phases, we have developed a methodology whereby fluorescence total intensity and anisotropy stopped-flow data can be combined in a single analysis. Fluorescence total intensity and anisotropy are highly interrelated and contain two very complementary forms of information. Total-intensity changes are useful in determining changes in populations with differing quantum yields, whereas anisotropy changes contain additional contributions caused by the rotational dynamics of the species. For cases in which the fluorescence quantum yield increases, the observed rate of anisotropy change will be more rapid than the total-intensity change, whereas in cases in which the total intensity decreases, the observed change in anisotropy will lag behind. In all cases, with quantum yield changes the stopped-flow anisotropy signals cannot be fit with models consisting of exponentials. Case studies examining these effects are described for the protein folding/refolding transitions of Staphylococcal nuclease and phosphoglycerate kinase. A multiphasic DNA exonuclease reaction using bacteriophage T4 DNA polymerase is also examined. In all of these cases, combined analysis of both data types revealed insights into reaction mechanism, which could not be obtained by either data type in isolation. Quantum yields and steady-state anisotropies associated with transiently populated intermediate species can be resolved. The data analysis methodologies described allow characterization of multiphasic reactions in terms of internally consistent kinetic rates, quantum yields, and steady-state anisotropies.

Bacteriophage T4↗