Biomedical subjects

Katalin Ajtai

Publications and source records attributed to Katalin Ajtai.

9 recordsLinked to original sources

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

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

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

Myosin: a noncovalent stabilizer of fibrin in the process of clot dissolution.

Myosin modulates the fibrinolytic process as a cofactor of the tissue plasminogen activator and as a substrate of plasmin. We report now that myosin is present in arterial thrombi and it forms reversible noncovalent complexes with fibrinogen and fibrin with equilibrium dissociation constants in the micromolar range (1.70 and 0.94 microM, respectively). Competition studies using a peptide inhibitor of fibrin polymerization (glycl-prolyl-arginyl-proline [GPRP]) indicate that myosin interacts with domains common in fibrinogen and fibrin and this interaction is independent of the GPRP-binding polymerization site in the fibrinogen molecule. An association rate constant of 1.81 x 10(2) M(-1) x s(-1) and a dissociation rate constant of 3.07 x 10(-4) s(-1) are determined for the fibrinogen-myosin interaction. Surface plasmon resonance studies indicate that fibrin serves as a matrix core for myosin aggregation. The fibrin clots equilibrated with myosin are stabilized against dissolution initiated by plasminogen and tissue-type plasminogen activator (tPA) or urokinase (at fibrin monomer-myosin molar ratio as high as 30) and by plasmin under static and flow conditions (at fibrin monomer-myosin molar ratio lower than 15). Myosin exerts similar effects on the tPA-induced dissolution of blood plasma clots. Covalent modification involving factor XIIIa does not contribute to this stabilizing effect; myosin is not covalently attached to the clot by the time of complete cross-linking of fibrin. Thus, our in vitro data suggest that myosin detected in arterial thrombi binds to the polymerized fibrin, in the bound form its tPA-cofactor properties are masked, and the myosin fibrin clot is relatively resistant to plasmin.

Binding Sites↗

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

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↗