Biomedical subjects

Thomas P Burghardt

Publications and source records attributed to Thomas P Burghardt.

13 recordsLinked to original sources

PubMed2006

Solution NMR of acetylcholine binding protein reveals agonist-mediated conformational change of the C-loop.

Previous X-ray crystallography, molecular dynamics simulation, fluorescence spectroscopy, and deuterium-hydrogen exchange of acetylcholine binding protein (AChBP) suggest that after binding of the agonist, the C-loop at the periphery of the binding site draws inward to cap the site and envelop the agonist. In this study, we use high-resolution solution NMR to monitor changes in the chemical environment of the C-loop without and with acetylcholine (ACh) bound. Substitution of [15N]cysteine for the native cysteines 123, 136, 187, and 188 provided intrinsic monitors of the chemical environments of the Cys- and C-loops, respectively. Two-dimensional transverse relaxation-optimized spectroscopy 15N-1H HSQC spectroscopy of apo-AChBP revealed seven well resolved cross-peaks for the group of cysteines. The spectrum of AChBP with Ser substituted for Cys 187 and 188 shows only two main cross-peaks, corresponding to Cys 123 and 136 from the Cys-loop, enabling resonance assignments. After binding of ACh, the five cross-peaks associated with cysteines from the C-loop condense into two predominant cross-peaks not observed in the spectrum from the apo protein, indicating a restricted range of conformations and change in chemical environment of the C-loop. The results show that isotopic cysteine can be incorporated into specified positions of AChBP expressed from a eukaryotic source, that the C-loop assumes multiple conformations without ACh, but that its conformation becomes restricted with ACh bound. The collective findings suggest a structural mechanism for agonist recognition in AChBP and related Cys-loop receptors.

Acetylcholine↗

PubMed2006

In situ single-molecule imaging with attoliter detection using objective total internal reflection confocal microscopy.

Confocal microscopy is widely used for acquiring high spatial resolution tissue sample images of interesting fluorescent molecules inside cells. The fluorescent molecules are often tagged proteins participating in a biological function. The high spatial resolution of confocal microscopy compared to wide field imaging comes from an ability to optically isolate and image exceedingly small volume elements made up of the lateral (focal plane) and depth dimensions. Confocal microscopy at the optical diffraction limit images volumes on the order of approximately 0.5 femtoliter (10(-15) L). Further resolution enhancement can be achieved with total internal reflection microscopy (TIRM). With TIRM, an exponentially decaying electromagnetic field (near-field) established on the surface of the sample defines a subdiffraction limit dimension that, when combined with conventional confocal microscopy, permits image formation from <7 attoL (10(-18) L) volumes [Borejdo et al. (2006) Biochim. Biophys. Acta, in press]. Demonstrated here is a new variation of TIRM, focused TIRM (fTIRM) that decreases the volume element to approximately 3 attoL. These estimates were verified experimentally by measuring characteristic times for Brownian motion of fluorescent nanospheres through the volume elements. A novel application for TIRM is in situ single-molecule fluorescence spectroscopy. Single-molecule studies of protein structure and function are well-known to avoid the ambiguities introduced by ensemble averaging. In situ, proteins are subjected to the native forces of the crowded environment in the cell that are not present in vitro. The attoL fluorescence detection volume of TIRM permits isolation of single proteins in situ. Muscle tissue contains myosin at a approximately 120 microM concentration. Evidence is provided that >75% of the bleachable fluorescence detected with fTIRM is emitted by five chromophore-labeled myosins in a muscle fiber.

Animals↗

PubMed2006

In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy.

Fluorescence detection of single molecules provides a means to investigate protein dynamics minus ambiguities introduced by ensemble averages of unsynchronized protein movement or of protein movement mimicking a local symmetry. For proteins in a biological assembly, taking advantage of the single molecule approach could require single protein isolation from within a high protein concentration milieu. Myosin cross-bridges in a muscle fiber are proteins attaining concentrations of approximately 120 muM, implying single myosin detection volume for this biological assembly is approximately 1 attoL (10(-18) L) provided that just 2% of the cross-bridges are fluorescently labeled. With total internal reflection microscopy (TIRM) an exponentially decaying electromagnetic field established on the surface of a glass-substrate/aqueous-sample interface defines a subdiffraction limit penetration depth into the sample that, when combined with confocal microscopy, permits image formation from approximately 3 attoL volumes. Demonstrated here is a variation of TIRM incorporating a nanometer scale metal film into the substrate/glass interface. Comparison of TIRM images from rhodamine-labeled cross-bridges in muscle fibers contacting simultaneously the bare glass and metal-coated interface show the metal film noticeably reduces both background fluorescence and the depth into the sample from which fluorescence is detected. High contrast metal film-enhanced TIRM images allow secondary label visualization in the muscle fibers, facilitating elucidation of Z-disk structure. Reduction of both background fluorescence and detection depth will enhance TIRM's usefulness for single molecule isolation within biological assemblies.

Aluminum↗

PubMed2004

Agonist-mediated conformational changes in acetylcholine-binding protein revealed by simulation and intrinsic tryptophan fluorescence.

We delineated acetylcholine (ACh)-dependent conformational changes in a prototype of the nicotinic receptor ligand binding domain by molecular dynamics simulation and changes in intrinsic tryptophan (Trp) fluorescence. Prolonged molecular dynamics simulation of ACh-binding protein showed that binding of ACh establishes close register of Trps from adjacent subunits, Trp(143) and Trp(53), and draws the peripheral C-loop inward to occlude the entrance to the binding cavity. Close register of Trp(143) and Trp(53) was demonstrated by ACh-mediated quenching of intrinsic Trp fluorescence, elimination of quenching by mutation of one or both Trps to Phe, and decreased lifetime of Trp fluorescence by bound ACh. Occlusion of the binding cavity by the C-loop was demonstrated by restricted access of an extrinsic quencher of binding site Trp fluorescence by ACh. The collective findings showed that ACh initially establishes close register of conserved Trps from adjacent subunits and then draws the C-loop inward to occlude the entrance to the binding cavity.

Acetylcholine↗

PubMed2004

The myosin cardiac loop participates functionally in the actomyosin interaction.

The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on beta-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in beta-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using site-directed mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.

Actomyosin↗

PubMed2003

Cosolvent-induced aggregation inhibits myosin ATPase activity by stabilizing the predominant transition intermediate.

High concentration of the cosolvent poly(ethylene glycol) (PEG) induces reversible aggregation of skeletal myosin subfragment 1 (S1) and inhibition of its Mg-ATPase activity [Highsmith et al. (1998) Biophys. J. 74, 1465-1472]. In the present work the effect of aggregation on the various steps of the ATPase cycle was studied. The isomerization and hydrolysis steps of the cycle were not affected by S1 aggregation since the formation of the "trapped" S1.MgADP.phosphate analogue complexes, which mimic the prehydrolysis M*ATP and posthydrolysis M**ADP.P(i) transition states, proceeded without any hindrance. Similar conclusions could be reached from the chemical modification of Lys-83 and Cys-707 in the presence of MgATP and MgATPgammaS, which indicated that the most populated intermediate of the cycle in solubilized and aggregated S1 is M**ADP.P(i). The dissociation of the trapped S1.MgADP.phosphate analogue complexes resembling the M**ADP.P(i) state was strongly inhibited by PEG-6000, showing that the transition from this intermediate is prevented by the aggregation. This step is presumably inhibited because the coupled swinging of the lever arm from the closed to the open position is constrained by the close packing of aggregated S1.

Animals↗

PubMed2003

A novel approach to the characterization of substrate specificity in short/branched chain Acyl-CoA dehydrogenase.

Rat and human short/branched chain acyl-CoA dehydrogenases exhibit key differences in substrate specificity despite an overall amino acid identity of 85% between them. Rat short/branched chain acyl-CoA dehydrogenases (SBCAD) are more active toward substrates with longer carbon side chains than human SBCAD, whereas the human enzyme utilizes substrates with longer primary carbon chains. The mechanism underlying this difference in substrate specificity was investigated with a novel surface plasmon resonance assay combined with absorbance and circular dichroism spectroscopy, and kinetics analysis of wild type SBCADs and mutants with altered amino acid residues in the substrate binding pocket. Results show that a relatively few amino acid residues are critical for determining the difference in substrate specificity seen between the human and rat enzymes and that alteration of these residues influences different portions of the enzyme mechanism. Molecular modeling of the SBCAD structure suggests that position 104 at the bottom of the substrate binding pocket is important in determining the length of the primary carbon chain that can be accommodated. Conformational changes caused by alteration of residues at positions 105 and 177 directly affect the rate of electron transfer in the dehydrogenation reactions, and are likely transmitted from the bottom of the substrate binding pocket to beta-sheet 3. Differences between the rat and human enzyme at positions 383, 222, and 220 alter substrate specificity without affecting substrate binding. Modeling predicts that these residues combine to determine the distance between the flavin ring of FAD and the catalytic base, without changing the opening of the substrate binding pocket.

Acyl Coenzyme A↗

PubMed2003

Energy transduction optical sensor in skeletal myosin.

The skeletal myosin cross-bridge in dynamic association with actin is the unitary energy transducer in muscle, converting free energy from ATP hydrolysis into contractile force. Myosin's conserved ATP-sensitive tryptophan (AST) is an energy transduction optical sensor signaling transduction-related transient conformation change by modulating its fluorescence intensity amplitude and relaxation rate. Recently introduced techniques have provided the means of observing the time-resolved intensity decay from this single residue in the native protein to elucidate the mechanism of its ATP sensitivity. AST signal characteristics could be derived from local protein structure by a scenario involving interactions with excited-state tryptophan. This investigation suggests the very different possibility that hypochromism induced in the tryptophan absorption band, a ground-state effect, is a significant structural effector of optical transduction sensing. This possibility makes feasible the interpretation of the transient AST optical signal in terms of dynamical protein structure, thereby raising the empirical signal to the level of a structural determinant. Using the crystallographically based geometry from several myosin structures, the maximum calculated AST hypochromism is <10% to be compared with the value of approximately 30% observed here experimentally. Rationalizing the discrepancy invites further investigation of S1 dynamical structure local to the AST during transduction.

Actins↗

PubMed2003

Mass spectral determination of skeletal/cardiac actin isoform ratios in cardiac muscle.

Skeletal and cardiac muscle contains actin isoforms that vary by two juxtaposed amino acids and two amino acid substitutions (Met299Leu and Ser358Thr). This close sequence homology does not allow cardiac and skeletal actin isoforms to be resolved in traditional SDS-PAGE analysis as the molecular weights (Deltamass = 32 Da) are not significantly different and the pIs are identical (5.2). Although cardiac actin is the predominant form in cardiac muscle, there appears to be a specific skeletal/cardiac actin ratio in a normal heart that may vary in a compromised or diseased heart. In an effort to ascertain the validity of this hypothesis we developed a mass spectrometric technique to measure the ratio of skeletal to cardiac actin. The technique involves purification of muscle actin and subsequent liquid chromatography coupled with electrospray ionization Fourier transform ion cylcotron resonance (LC/FTICR-MS) mass spectrometry. A 7 Tesla FTICR mass spectrometer was utilized to compare skeletal/cardiac actin isoform ratios. Additionally, a new dual electrospray ionization source was employed to determine accurate masses of the alpha-skeletal and alpha-cardiac actins.

Actins↗

PubMed2003

Chemical decoupling of ATPase activation and force production from the contractile cycle in myosin by steric hindrance of lever-arm movement.

The myosin motor protein generates force in muscle by hydrolyzing Adenosine 5'-triphosphate (ATP) while interacting transiently with actin. Structural evidence suggests the myosin globular head (subfragment 1 or S1) is articulated with semi-rigid catalytic and lever-arm domains joined by a flexible converter domain. According to the prevailing hypothesis for energy transduction, ATP binding and hydrolysis in the catalytic domain drives the relative movement of the lever arm. Actin binding and reversal of the lever-arm movement (power stroke) applies force to actin. These domains interface at the reactive lysine, Lys84, where trinitrophenylation (TNP-Lys84-S1) was observed in this work to block actin activation of myosin ATPase and in vitro sliding of actin over myosin. TNP-Lys84-S1's properties and interactions with actin were examined to determine how trinitrophenylation causes these effects. Weak and strong actin binding, the rate of mantADP release from actomyosin, and actomyosin dissociation by ATP were equivalent in TNP-Lys84-S1 and native S1. Molecular dynamics calculations indicate that lever-arm movement inhibition during ATP hydrolysis and the power stroke is caused by steric clashes between TNP and the converter or lever-arm domains. Together these findings suggest that TNP uncouples actin activation of myosin ATPase and the power stroke from other steps in the contraction cycle by inhibiting the converter and lever-arm domain movements.

Actins↗

PubMed2002

Purification and characterization of two polymorphic variants of short chain acyl-CoA dehydrogenase reveal reduction of catalytic activity and stability of the Gly185Ser enzyme.

Short chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric flavoenzyme that catalyzes the first intramitochondrial step in the beta-oxidation of fatty acids. Two polymorphisms in the coding region of the SCAD gene, 511C>T (R147W) and 625G>A (G185S), have been shown to be associated with an increased level of ethylmalonic acid excretion in urine, a clinical characteristic of SCAD deficiency. To characterize the biochemical consequences of these variations, in vitro site-directed mutagenesis and prokaryotic expression were used to produce the corresponding SCAD variant proteins. Both variant proteins were unstable when produced in Escherichia coli, but could be rescued and subsequently purified by coexpressing them with the bacterial chaperonin GroEL/ES. The k(cat)/K(m) values of the green wild-type, R147W, and G185S SCAD enzymes coexpressed with GroEL/ES were 33, 30, and 10 microM(-)(1) s(-)(1), respectively. There were minimal differences in the kinetic parameters measured for the green, degreened, and wild-type enzymes coexpressed with GroEL/ES, and the R147W variant when butyryl-CoA was used as a substrate. The catalytic efficiency of the G185S variant enzyme, however, was reduced compared to that of the wild-type enzyme. The thermal and guanidine HCl stability of the purified enzymes as determined by fluorescence, far-UV CD spectroscopy, and incubation-induced rest activity showed the following order of relative stability: wild-type enzyme > R147W > G185S. Near-UV CD spectroscopy indicated that these impairments are caused by decreased flexibility in the tertiary conformation of the two mutant enzymes. The common SCAD polymorphisms may lead to clinically relevant alterations in enzyme function.

Acyl-CoA Dehydrogenase↗

PubMed2002

Cation-pi interaction in a folded polypeptide.

Cationic and aromatic side chains from protein residues interact to stabilize tertiary structure. The stabilization energy originates in part from electrostatic attraction between the cation, and regions of high electron density in pi-orbitals of the aromatic group, leading to the name cation-pi interaction. The lysine and tyrosine containing peptide, N-acetyl-Pro-Pro-Lys-Tyr-Asp-Lys-NH(2), has near uv CD characteristic of tyrosine in a structured environment. Nuclear Overhauser effect (NOE), coupling constant, and ring current chemical shift constraints obtained with (1)H NMR confirm that the peptide (t6p) folds. Simulated annealing consistent with all NMR constraints produces a 40-structure ensemble for t6p with potential energies within one standard deviation of the lowest value observed. Calculated binding energies indicate that cation-pi and cation-phenolic OH interactions exists between the Lys3 and Tyr4 side chains in most of the structures. The t6p peptide in solution is a model for these interactions in a protein. A perturbing electric field from the cationic ground state charge intermingles the excited states of the aromatic group. This intermingling effect may provide a cation-pi signature effect in the tyrosine spectroscopy. The absorption and CD for the lowest energy electronic transitions of the tyrosine phenol were computed for the ensemble. Red-shifted peak energy and hypochromicity in the absorbance band, and decreasing rotational strength, correlates with increasing binding energy of the complex indicating the cation-pi spectroscopic signature. The ensemble average spectroscopic signature effects in t6p are small and in agreement with observation.

Amino Acid Sequence↗

PubMed2002

Tyrosine mediated tryptophan ATP sensitivity in skeletal myosin.

Myosin is the molecular motor in muscle that generates torque and transiently reacts with actin. The mechanical work performed by the motor occurs by successive decrements in the free energy of the myosin-nucleotide system. The seat of these transitions is the globular "head" domain of the myosin molecule (subfragment 1 or S1). A very useful (hitherto empirical) signal of these transitions has been optical, namely, detection of state-dependent changes in absorbance or fluorescence of S1. This effect has now been found to arise in a particular myosin residue (Trp510 in rabbit skeletal muscle), enabling the study of its intimate mechanism. In this work, based on measuring time-dependent signals, we find that the signal change upon nucleotide binding is adequately explained by assuming that nucleotide binding to a remote site causes a transition from a situation in which Trp510 is strongly statically quenched to a situation in which it is weakly statically quenched. The Trp510-static quencher interaction is also responsible, in part, for the changing tryptophan optical density in S1 upon nucleotide binding. Using crystallographically based geometry, calculation of the Trp510 electronic wave function indicates that Tyr503 is the static quencher.

Adenosine Diphosphate↗