ABSTRACT
Whispering gallery mode (WGM) resonators are promising optical structures for microfluidic label-free biosensors mainly due to their high sensitivity, but from a practical point of view they present numerous constraints that make their use in real laboratory diagnosis application difficult. Herein we report on a monolithic lab on a chip fabricated by a hybrid femtosecond laser micromachining approach, for label-free biosensing. It consists of a polymer WGM microresonator sensor integrated inside a glass microfluidic chip, presenting a refractive index change sensitivity of 61 nm per RIU. The biosensing capabilities of the device have been demonstrated by exploiting the biotin-streptavidin binding affinity, obtaining a measurable minimum surface density increase of 67 × 103 molecules per µm2.
Subject(s)
Biosensing Techniques/instrumentation , Lab-On-A-Chip Devices , Optical Devices , Writing , Equipment Design , Glucose/analysisABSTRACT
Hydrodynamic synchronization is a fundamental physical phenomenon by which self-sustained oscillators communicate through perturbations in the surrounding fluid and converge to a stable synchronized state. This is an important factor for the emergence of regular and coordinated patterns in the motions of cilia and flagella. When dealing with biological systems, however, it is always hard to disentangle internal signaling mechanisms from external purely physical couplings. We have used the combination of two-photon polymerization and holographic optical trapping to build a mesoscale model composed of chiral propellers rotated by radiation pressure. The two microrotors can be synchronized by hydrodynamic interactions alone although the relative torques have to be finely tuned. Dealing with a micron sized system we treat synchronization as a stochastic phenomenon and show that the phase lag between the two microrotors is distributed according to a stationary Fokker-Planck equation for an overdamped particle over a tilted periodic potential. Synchronized states correspond to minima in this potential whose locations are shown to depend critically on the detailed geometry of the propellers.
Subject(s)
Models, Theoretical , Oscillometry/methods , Cilia/physiology , Flagella/physiology , Hydrodynamics , Models, BiologicalABSTRACT
This work primarily aims to fabricate and use two photon polymerization (2PP) microstructures capable of being optically manipulated into any arbitrary orientation. We have integrated optical waveguides into the structures and therefore have freestanding waveguides, which can be positioned anywhere in the sample at any orientation using optical traps. One of the key aspects to the work is the change in direction of the incident plane wave, and the marked increase in the numerical aperture demonstrated. Hence, the optically steered waveguide can tap from a relatively broader beam and then generate a more tightly confined light at its tip. The paper contains both simulation, related to the propagation of light through the waveguide, and experimental demonstrations using our BioPhotonics Workstation. In a broader context, this work shows that optically trapped microfabricated structures can potentially help bridge the diffraction barrier. This structure-mediated paradigm may be carried forward to open new possibilities for exploiting beams from far-field optics down to the subwavelength domain.
Subject(s)
Computer-Aided Design , Models, Theoretical , Molecular Imprinting/instrumentation , Refractometry/instrumentation , Surface Plasmon Resonance/instrumentation , Computer Simulation , Equipment Design , Equipment Failure Analysis , Light , Scattering, RadiationABSTRACT
We present a microfluidic cell sorter that is able to count, characterize and sort micrometer sized particles and cells. In addition to optical counting and characterization, also sorting is performed by optical forces. The device is optimized for simplicity. The microfluidic channels and optical waveguides that carry the illuminating, detecting and sorting light form a single integrated structure, all built from the same material in a single photopolymerization step.
Subject(s)
Microfluidics/instrumentation , Optics and Photonics , Flow Cytometry , Photochemistry , Polymers/chemistryABSTRACT
The structural changes in bacteriorhodopsin during the photocycle are investigated. Time resolved polarized infrared spectroscopy in combination with photoselection is used to determine the orientation and motion of certain structural units of the molecule: Asp-85, Asp-96, Asp-115, the Schiff base, and several amide I vibrations. The results are compared with recently published x-ray diffraction data with atomic resolution about conformational motions during the photocycle. The orientation of the measured vibrations are also calculated from the structure data, and based on the comparison of the values from the two techniques new information is obtained: several amide I bands in the infrared spectrum are assigned, and we can also identify the position of the proton in the protonated Asp residues.
Subject(s)
Bacteriorhodopsins/chemistry , Light , Spectroscopy, Fourier Transform Infrared/methods , Aspartic Acid/chemistry , Halobacterium/metabolism , Normal Distribution , Photochemistry , Protein Conformation , Spectrophotometry, Infrared , Time Factors , X-Ray DiffractionABSTRACT
The light-driven proton pump bacteriorhodopsin (bR) was functionally expressed in Xenopus laevis oocytes and in HEK-293 cells. The latter expression system allowed high time resolution of light-induced current signals. A detailed voltage clamp and patch clamp study was performed to investigate the DeltapH versus Deltapsi dependence of the pump current. The following results were obtained. The current voltage behavior of bR is linear in the measurable range between -160 mV and +60 mV. The pH dependence is less than expected from thermodynamic principles, i.e., one DeltapH unit produces a shift of the apparent reversal potential of 34 mV (and not 58 mV). The M(2)-BR decay shows a significant voltage dependence with time constants changing from 20 ms at +60 mV to 80 ms at -160 mV. The linear I-V curve can be reconstructed by this behavior. However, the slope of the decay rate shows a weaker voltage dependence than the stationary photocurrent, indicating that an additional process must be involved in the voltage dependence of the pump. A slowly decaying M intermediate (decay time > 100 ms) could already be detected at zero voltage by electrical and spectroscopic means. In effect, bR shows optoelectric behavior. The long-lived M can be transferred into the active photocycle by depolarizing voltage pulses. This is experimentally demonstrated by a distinct charge displacement. From the results we conclude that the transport cycle of bR branches via a long-lived M(1)* in a voltage-dependent manner into a nontransporting cycle, where the proton release and uptake occur on the extracellular side.
Subject(s)
Bacteriorhodopsins/metabolism , Energy Transfer/physiology , Oocytes/metabolism , Proton Pumps/physiology , Animals , Cell Membrane/physiology , Cells, Cultured/metabolism , Cytoplasm/physiology , Electrophysiology , Female , Halobacterium , Humans , Hydrogen-Ion Concentration , Kinetics , Light , Membrane Potentials , Patch-Clamp Techniques/methods , Xenopus , Xenopus laevisABSTRACT
The transport of protons across membranes is an important process in cellular bioenergetics. The light-driven proton pump bacteriorhodopsin is the best-characterized protein providing this function. Photon energy is absorbed by the chromophore retinal, covalently bound to Lys 216 via a protonated Schiff base. The light-induced all-trans to 13-cis isomerization of the retinal results in deprotonation of the Schiff base followed by alterations in protonatable groups within bacteriorhodopsin. The changed force field induces changes, even in the tertiary structure, which are necessary for proton pumping. The recent report of a high-resolution X-ray crystal structure for the late M intermediate of a mutant bacteriorhopsin (with Asp 96-->Asn) displays the structure of a proton pathway highly disturbed by the mutation. To observe an unperturbed proton pathway, we determined the structure of the late M intermediate of wild-type bacteriorhodopsin (2.25 A resolution). The cytoplasmic side of our M2 structure shows a water net that allows proton transfer from the proton donor group Asp 96 towards the Schiff base. An enlarged cavity system above Asp 96 is observed, which facilitates the de- and reprotonation of this group by fluctuating water molecules in the last part of the cycle.
Subject(s)
Bacteriorhodopsins/chemistry , Proton Pumps/chemistry , Bacteriorhodopsins/genetics , Bacteriorhodopsins/metabolism , Biological Transport , Crystallography, X-Ray , Cytoplasm/metabolism , Models, Molecular , Point Mutation , Protein Conformation , Protein Structure, Tertiary , Proton Pumps/metabolism , ProtonsABSTRACT
Bacteriorhodopsin (bR) and halorhodopsin (hR) are light-induced ion pumps in the cell membrane of Halobacterium salinarium. Under normal conditions bR is an outward proton transporter, whereas hR is an inward Cl- transporter. There is strong evidence that at very low pH and in the presence of Cl-, bR transports Cl- ions into the cell, similarly to hR. The chloride pumping activity of bR is connected to the so-called acid purple state. To account for the observed effects in bR a tentative complex counterion was suggested for the protonated Schiff base of the retinal chromophore. It would consist of three charged residues: Asp-85, Asp-212, and Arg-82. This quadruplet (including the Schiff base) would also serve as a Cl- binding site at low pH. We used Fourier transform infrared difference spectroscopy to study the structural changes during the transitions between the normal, acid blue, and acid purple states. Asp-85 and Asp-212 were shown to participate in the transitions. During the normal-to-acid blue transition, Asp-85 protonates. When the pH is further lowered in the presence of Cl-, Cl- binds and Asp-212 also protonates. The binding of Cl- and the protonation of Asp-212 occur simultaneously, but take place only when Asp-85 is already protonated. It is suggested that HCl is taken up in undissociated form in exchange for a neutral water molecule.
Subject(s)
Bacteriorhodopsins/metabolism , Chlorides/metabolism , Arginine/chemistry , Aspartic Acid/chemistry , Bacteriorhodopsins/chemistry , Binding Sites , Biophysical Phenomena , Biophysics , Chlorides/chemistry , Halobacterium salinarum/metabolism , Hydrogen-Ion Concentration , Ion Transport , Schiff Bases , Spectroscopy, Fourier Transform InfraredABSTRACT
We have recently introduced a method, made possible by an improved orienting technique using a combination of electric and magnetic fields, that allows the three-dimensional detection of the intramolecular charge displacements during the photocycle of bacteriorhodopsin. This method generates electric asymmetry, a prerequisite for the detection of electric signal on the macroscopic sample, in all three spatial dimensions. Purple membrane fragments containing bacteriorhodopsin were oriented so that their permanent electric dipole moment vectors were perpendicular to the membrane plane and pointed in the same direction. The resulting cylindrical symmetry was broken by photoselection, i. e., by flash excitation with low intensity linearly polarized light. From the measured electric signals, the three-dimensional motion of the electric charge center in the bacteriorhodopsin molecules was calculated for the first 400 microseconds. Simultaneous absorption kinetic recording provided the time-dependent concentrations of the intermediates. Combining the two sets of data, we determined the discrete dipole moments of intermediates up to M. When compared with the results of current molecular dynamics calculations, the data provided a decisive experimental test for selecting the optimal theoretical model for the proton transport and should eventually lead to a full description of the mechanism of the bacteriorhodopsin proton pump.
Subject(s)
Bacteriorhodopsins/chemistry , Electrochemistry/methods , Photosynthesis , Models, Biological , Proton PumpsABSTRACT
Using temperature-derivative spectroscopy in the temperature range below 100 K, we have studied the dependence of the Soret band on the recombination barrier in sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation at 12 K. The spectra were separated into contributions from the photodissociated species, Mb*CO, and CO-bound myoglobin. The line shapes of the Soret bands of both photolyzed and liganded myoglobin were analyzed with a model that takes into account the homogeneous bandwidth, coupling of the electronic transition to vibrational modes, and static conformational heterogeneity. The analysis yields correlations between the activation enthalpy for rebinding and the model parameters that characterize the homogeneous subensembles within the conformationally heterogeneous ensemble. Such couplings between spectral and functional parameters arise when they both originate from a common structural coordinate. This effect is frequently denoted as "kinetic hole burning." The study of these correlations gives direct insights into the structure-function relationship in proteins. On the basis of earlier work that assigned spectral parameters to geometric properties of the heme, the connections with the heme geometry are discussed. We show that two separate structural coordinates influence the Soret line shape, but only one of the two is coupled to the enthalpy barrier for rebinding. We give evidence that this coordinate, contrary to widespread belief, is not the iron displacement from the mean heme plane.
Subject(s)
Metmyoglobin/chemistry , Protein Conformation , Animals , Binding Sites , Ligands , Male , Protein Binding , Spectrum Analysis , Spermatozoa/metabolism , WhalesABSTRACT
We have measured the Soret band of the photoproduct obtained by complete photolysis of sperm whale carbonmonoxymyoglobin at 10 K. The experimental spectrum has been modeled with an analytical expression that takes into account the homogeneous bandwidth, the coupling of the electronic transition with both high and low frequency vibrational modes, and the effects of static conformational heterogeneity. The comparison with deoxymyoglobin at low temperature reveals three main differences. In the photoproduct, the Soret band is shifted to red. The band is less asymmetric, and an enhanced coupling to the heme vibrational mode at 674 cm-1 is observed. These differences reflect incomplete relaxation of the active site after ligand dissociation. The smaller band asymmetry of the photoproduct can be explained by a smaller displacement of the iron atom from the mean porphyrin plane, in quantitative agreement with the X-ray structure analysis. The enhanced vibrational coupling is attributed to a subtle heme distortion from the planar geometry that is barely detectable in the X-ray structure.
Subject(s)
Heme/chemistry , Myoglobin/chemistry , Animals , Cold Temperature , Photochemistry , Spectrum Analysis , WhalesABSTRACT
Electric signals associated with the photocycle of bacteriorhodopsin carry valuable information about the proton transport process. Photocurrents measured by different experimental methods are interpreted in terms of intramolecular charge displacements. Permanent electrical asymmetry of the sample is considered to be a prerequisite for the detection of electric signals. The various photoelectric measuring techniques can be distinguished by the way of achievement of this asymmetry. A common feature of the available methods, however, is that the samples are cylindrically symmetric. Consequently, intramembraneous charge displacements can normally be monitored only along the axis of the membrane normal. We developed a novel method that allows also the detection of the in-plane components of the charge displacements. Samples containing oriented purple membrane fragments were used in the experiments, and the rotational symmetry was transiently broken via anisotropic excitation of the bR molecules by linearly polarized light. Kinetics of the normal and in-plane components were measured and interpreted as a result of spatial charge displacements associated with the proton transport process in bacteriorhodopsin.
ABSTRACT
Previous C13-NMR studies showed that two of the four internal aspartic acid residues (Asp-96 and Asp-115) of bacteriorhodopsin (bR) are protonated up to pH = 10, but no accurate pKa of these residues has been determined. In this work, infrared spectroscopy with the attenuated total reflection technique was used to characterize pH-dependent structural changes of ground-state, dark-adapted wild-type bacteriorhodopsin and its mutant (D96N) with aspartic acid-96 replaced by asparagine. Data indicated deprotonation of Asp-96 at high pH (pKa = 11.4 +/- 0.1), but no Asp-115 titration was observed. The analysis of the whole spectral region characteristic to complex conformational changes in the protein showed a more complicated titration with an additional pKa value (pKa1 = 9.3 +/- 0.3 and pKa2 = 11.5 +/- 0.2). Comparison of results obtained for bR and the D96N mutant of bR shows that the pKa approximately 11.5 characterizes not a direct titration of Asp-96 but a protein conformational change that makes Asp-96 accessible to the external medium.
Subject(s)
Bacteriorhodopsins/chemistry , Aspartic Acid , Bacteriorhodopsins/isolation & purification , Bacteriorhodopsins/metabolism , Carbon Isotopes , Halobacterium salinarum/metabolism , Hydrogen-Ion Concentration , Kinetics , Magnetic Resonance Spectroscopy , Point Mutation , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectroscopy, Fourier Transform Infrared/instrumentation , Spectroscopy, Fourier Transform Infrared/methods , Structure-Activity RelationshipABSTRACT
Fouier-transform infrared (FTIR) difference spectra of several His-E7 and Val-E11 mutants of sperm whale carbonmonoxymyoglobin were obtained by photodissociation at cryogenic temperatures. The IR absorption of the CO ligand shows characteristic features for each of the mutants, both in the ligand-bound (A) state and in the photodissociated (B) state. For most of the mutants, a single A substate band is observed, which points to the crucial role of the His-E7 residue in determining the A substrate spectrum of the bound CO in the native structure. The fact that some of the mutants show more than one stretch band of the bound CO indicates that the appearance of multiple A substates is not exclusively connected to the presence of His-E7. In all but one mutant, multiple stretch bands of the CO in the photodissociated state are observed; these B substates are thought to arise from discrete positions and/or orientations of the photodissociated ligand in the heme pocket. The red shifts of the B bands with respect to the free-gas frequency indicate weak binding in the heme pocket. The observation of similar red shifts in microperoxidase (MP-8), where there is no residue on the distal side, suggests that the photodissociated ligand is still associated with the heme iron. Photoselection experiments were performed to determine the orientation of the bound ligand with respect to the heme normal by photolyzing small fractions of the sample with linearly polarized light at 540 nm. The resulting linear dichroism in the CO stretch spectrum yielded angles alpha > 20 degrees between the CO molecular axis and the heme normal for all of the mutants. We conclude that the off-axis position of the CO ligand in the native structure does not arise from steric constraints imposed by the distal histidine. There is no clear correlation between the size of the distal residue and the alpha of the CO ligand.
Subject(s)
Hemeproteins/chemistry , Histidine , Myoglobin/chemistry , Valine , Amino Acid Sequence , Animals , Ligands , Mutagenesis, Site-Directed , Point Mutation , Protein Binding , Recombinant Proteins/chemistry , Spectroscopy, Fourier Transform Infrared/methods , WhalesABSTRACT
Phenomena occurring in the heme pocket after photolysis of carbonmonoxymyoglobin (MbCO) below about 100 K are investigated using temperature-derivative spectroscopy of the infrared absorption bands of CO. MbCO exists in three conformations (A substrates) that are distinguished by the stretch bands of the bound CO. We establish connections among the A substates and the substates of the photoproduct (B substates) using Fourier-transform infrared spectroscopy together with kinetic experiments on MbCO solution samples at different pH and on orthorhombic crystals. There is no one-to-one mapping between the A and B substates; in some cases, more than one B substate corresponds to a particular A substate. Rebinding is not simply a reversal of dissociation; transitions between B substates occur before rebinding. We measure the nonequilibrium populations of the B substates after photolysis below 25 K and determine the kinetics of B substate transitions leading to equilibrium. Transitions between B substates occur even at 4 K, whereas those between A substates have only been observed above about 160 K. The transitions between the B substates are nonexponential in time, providing evidence for a distribution of substates. The temperature dependence of the B substate transitions implies that they occur mainly by quantum-mechanical tunneling below 10 K. Taken together, the observations suggest that the transitions between the B substates within the same A substate reflect motions of the CO in the heme pocket and not conformational changes. Geminate rebinding of CO to Mb, monitored in the Soret band, depends on pH. Observation of geminate rebinding to the A substates in the infrared indicates that the pH dependence results from a population shift among the substates and not from a change of the rebinding to an individual A substate.
Subject(s)
Heme/chemistry , Myoglobin/chemistry , Animals , Binding Sites , Biophysical Phenomena , Biophysics , Ligands , Photochemistry , Protein Binding , Protein Conformation , Spectrophotometry , Temperature , Thermodynamics , WhalesABSTRACT
Infrared spectroscopy is used to characterize the transitions in the photocycle of bR involving the M intermediate. It has been shown previously that in this part of the photocycle a large protein conformational change takes place that is important for proton pumping. In this work we separate the spectra of the L, M, and N intermediates in order to better describe the timing of the molecular changes. We use the photoreaction of the M intermediate to separate its spectrum from those of L and N. At temperatures between 220 and 270 K a mixture of M and L or N is produced by illumination with green light. Subsequent blue illumination selectively drives M back into the ground state and the difference between the spectra before and after blue excitation yields the spectrum of M. Below about 250 K and L/M mixture is separated; at higher temperatures an M/N mixture is seen. We find that the spectrum of M is identical in the two temperature regions. The large protein conformational change is seen to occur during the M to N transition. Our results confirm that Asp-96 is transiently deprotonated in the L state. The only aspartic protonation changes between M and bR are the protonation of Asp-85 and Asp-212 that occur simultaneously during the L to M transition. Blue-light excitation of M results in deprotonation of both. The results suggest a quadrupolelike interaction of the Schiff base, Asp-85, Asp-212, and an additional positive charge in bR.
Subject(s)
Bacteriorhodopsins/chemistry , Halobacterium salinarum/chemistry , Spectrophotometry, Infrared , PhotochemistryABSTRACT
The photoinduced electric response of oriented purple membranes associated with processes before the K-intermediate decay of bacteriorhodopsin was measured in the 180-300 K temperature range. These response signals consist of two kinetically distinct components (both temperature dependent). The experimental data show a correlation between the time constants of the rise of the signal and solution resistance. A model is proposed to assign these components to two diffusion-limited processes of charge displacement in the solution. The displacement is caused by the electric field of the photoinduced transient dipole which is formed in the primary act of the bacteriorhodopsin photocycle. The two processes are assigned as: (a) the conduction of electrical current through H-bonds (time resolved only in the temperature range 180-200 K) and (b) the diffusion of charges through the interfacial layer.
ABSTRACT
Infrared spectral changes in bacteriorhodopsin (bR) were followed during the slow decay of the M intermediate in the temperature region 240-260 K. The decay of the M form is characterized by the disappearance of the ethylenic bands and the bands indicating the reprotonation of the Schiff base. The route of Schiff-base reprotonation completely changes between 240 K and 260 K. At 240 K reprotonation occurs from Asp-85, the group to which the proton was released during M formation, and there is no pumping. At 260 K Schiff-base reprotonation takes place through Asp-96 from the cytoplasmic side, in the normal sequence assumed for proton pumping. The dramatic change in the route of Schiff-base reprotonation is coupled to a protein conformational change characterized by the change of the ratio of the two amide I bands at 1658 cm-1 and 1669 cm-1. This conformational change is interpreted as the conformational switch crucial for proton pumping: a protein relaxation following M formation results in a local rearrangement of the group, in the vicinity of the Schiff base. The rearrangement changes the accessibility of the Schiff base and provides that its deprotonation and reprotonation occur on different sides. The conformational change has characteristics typical for relaxations in proteins. In addition, it is shown that at 260 K an equilibrium exists between the M and N forms.
Subject(s)
Bacteriorhodopsins/metabolism , Halobacterium/metabolism , Kinetics , Models, Structural , Protein Conformation , Protons , Schiff Bases , Spectrophotometry, Infrared/methods , ThermodynamicsABSTRACT
Myoglobin, a simppe dioxygen-storage protein, is a good laboratory for the investigation of the connection between protein structure, dynamics, and function. Fourier-transform infrared spectroscopy on carbon-monoxymyoglobin (MbCO) shows three major CO bands. These bands are excellent probes for the investigation of the structure-function relationship. They have different CO binding kinetics and their CO dipoles form different angles with respect to the heme normal, implying that MbCO exists in three major conformational substates, A0, A1, and A3. The entropies and enthalpies of these substates depend on temperature above approximately 180 K and are influenced by pH, solvent, and pressure. These results suggest that even a protein as simple as Mb can assume a small number of clearly different structures that perform the same function, but with different rates. Moreover, protein structure and dynamics depend strongly on the interaction of the protein with its environment.
Subject(s)
Myoglobin/metabolism , Fourier Analysis , Kinetics , Models, Structural , Protein Conformation , Spectrophotometry, Infrared , ThermodynamicsABSTRACT
The rebinding kinetics of CO to myoglobin after flash photolysis is nonexponential in time below approximately 180 K; the kinetics is governed by a distribution of enthalpic barriers. This distribution results from inhomogeneities in the protein conformation, referred to as conformational substates. Hole-burning experiments on the Soret and IR CO-stretch bands test the assumption that an inhomogeneous distribution of conformational substates results in inhomogeneously broadened spectra. CO was slowly photolyzed at different wavelengths in the Soret band at 10 K. Both the Soret band and the CO-stretch band A1, centered at 1,945 cm-1, shift during photolysis, demonstrating that different wavelengths excite different parts of the distributed population. We have also done kinetic hole-burning experiments by measuring peak shifts in the Soret and A1 bands as the CO molecules rebind. The shifts indicate that the spectral and enthalpic distributions are correlated. In the A1 band, the spectral and enthalpic distributions are highly correlated while in the Soret the correlation is weak. From the peak shifts in the spectral and kinetic hole-burning experiments the inhomogeneous broadening is estimated to be approximately 15% of the total width in the Soret band and approximately 60% in A1. We have previously measured the tilt angle alpha between the bound CO and the heme normal (Ormos, P., D. Braunstein, H. Frauenfelder, M. K. Hong, S.-L. Lin, T. B. Sauke, and R. D. Young. 1988. Proc. Natl. Acad. Sci. USA. 85:8492-8496) and observed a wave number dependence of the tilt angles within the CO-stretch A bands. Thus the spectral and enthalpic distributions of the A bands are coupled to a heterogeneity of the structure.