Protein conformational changes and protonation dynamics probed by a single shot using quantum-cascade-laser-based IR spectroscopy.

Mid-IR spectroscopy is a powerful and label-free technique to investigate protein reactions. In this study, we use quantum-cascade-laser-based dual-comb spectroscopy to probe protein conformational changes and protonation events by a single-shot experiment. By using a well-characterized membrane protein, bacteriorhodopsin, we provide a comparison between dual-comb spectroscopy and our homebuilt tunable quantum cascade laser (QCL)-based scanning spectrometer as tools to monitor irreversible reactions with high time resolution. In conclusion, QCL-based infrared spectroscopy is demonstrated to be feasible for tracing functionally relevant protein structural changes and proton translocations by single-shot experiments. Thus, we envisage a bright future for applications of this technology for monitoring the kinetics of irreversible reactions as in (bio-)chemical transformations.

Dynamics and mechanism of a light-driven chloride pump.

Chloride transport by microbial rhodopsins is an essential process for which molecular details such as the mechanisms that convert light energy to drive ion pumping and ensure the unidirectionality of the transport have remained elusive. We combined time-resolved serial crystallography with time-resolved spectroscopy and multiscale simulations to elucidate the molecular mechanism of a chloride-pumping rhodopsin and the structural dynamics throughout the transport cycle. We traced transient anion-binding sites, obtained evidence for how light energy is used in the pumping mechanism, and identified steric and electrostatic molecular gates ensuring unidirectional transport. An interaction with the π-electron system of the retinal supports transient chloride ion binding across a major bottleneck in the transport pathway. These results allow us to propose key mechanistic features enabling finely controlled chloride transport across the cell membrane in this light-powered chloride ion pump.

Real-Time Tracking of Proton Transfer from the Reactive Cysteine to the Flavin Chromophore of a Photosensing Light Oxygen Voltage Protein.

LOV (light oxygen voltage) proteins are photosensors ubiquitous to all domains of life. A variant of the short LOV protein from Dinoroseobacter shibae (DsLOV) exhibits an exceptionally fast photocycle. We performed time-resolved molecular spectroscopy on DsLOV-M49S and characterized the formation of the thio-adduct state with a covalent bond between the reactive cysteine (C72) and C4a of the FMN. By use of a tunable quantum cascade laser, the weak absorption change of the vibrational band of S-H stretching vibration of C57 was resolved with a time resolution of 10 ns. Deprotonation of C72 proceeded with a time constant of 12 µs which tallies the rise of the thio-adduct state. These results provide valuable information for the mechanistic interpretation of light-induced structural changes in LOV domains, which involves the choreographed sequence of proton transfers, changes in electron density distributions, spin alterations of the latter, and transient bond formation and breakage. Such molecular insight will help develop new optogenetic tools based on flavin photoreceptors.

Electron inventory of the iron-sulfur scaffold complex HypCD essential in [NiFe]-hydrogenase cofactor assembly.

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN- and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN- and CO ligands on the scaffold complex HypCD.

Femtosecond-to-millisecond structural changes in a light-driven sodium pump.

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins.

The Two-Photon Reversible Reaction of the Bistable Jumping Spider Rhodopsin-1.

Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore-attached to the protein through a protonated Schiff base-and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography.

Effect of Cast Modification on Denture Base Adaptation Following Maxillary Complete Denture Processing.

PURPOSE: To investigate the effect of cast modifications on denture base adaptation in coronal and sagittal projections following maxillary complete denture processing. MATERIALS AND METHODS: A total of 60 edentulous maxillary casts (n = 10) were distributed among six groups. Group 1 was the control group with no modification, groups 2 through 6 included a butterfly postdam preparation, groups 3 and 4 also included a 10-mm wide/4-mm deep box with addition of four round holes in group 4, and groups 5 and 6 also included a 20-mm wide/4-mm deep box with addition of four round holes in group 6. The boxes were prepared at the mid-heel area of the casts. Two layers of baseplate wax (1 mm each) were used to standardize denture base thickness across the groups. A standard technique was used to replicate the denture tooth set-up, and standardized processing was done for all the groups. Following deflasking, casts with the dentures were sectioned in the coronal and sagittal directions. Microscopic pictures were taken at preselected points. Data were organized in tables, and statistical analyses were performed using repeated measure ANOVA, Tukey post hoc tests, and post hoc comparison tests set at 5% level of significance. RESULTS: Maximum gaps were measured at the mid-palatal area followed by nearby areas and the areas near ridge crests in both coronal and sagittal projections. The analyses revealed significant differences between the groups in coronal projection (1/2, 3/4, 5/6) and sagittal projection (1, 2, 3/4, 5/6) without significant differences within the pairs. The groups were ranked from the highest group 1 to the lowest group 6 relative to the gap means. Post hoc comparisons showed that points 1C and 2A had the highest gap means across the study groups. CONCLUSIONS: Within the limitations of this study, it can be extrapolated that the denture base adaptation can be effectively increased with the box preparation at the mid-heel aspect of the casts. Significant reduction of gaps was seen when the box size increased from 10 to 20 mm in coronal and sagittal projections; however, the addition of four holes had no significant effect on gap size alterations.

Retentive strength and marginal discrepancies of a ceramic-reinforced calcium phosphate luting agent: An in vitro pilot study.

STATEMENT OF PROBLEM: Information on the properties of a relatively new luting agent with a unique formulation (ceramic-reinforced calcium phosphate) is limited. PURPOSE: The purpose of this in vitro study was to compare the retentive strengths and marginal discrepancies of a ceramic-reinforced calcium phosphate luting agent (CM) with a self-adhesive resin luting agent (RX) and to determine and compare the mode of failure of dislodged cemented copings. MATERIAL AND METHODS: Forty extracted human molar teeth were prepared to receive zirconia copings. After cementation, the specimens were divided into 4 subgroups (n=10): CM A (axial loading), CM OA (off-axis loading), RX A (axial loading), and RX OA (off-axis loading). For each subgroup, 9 of the specimens received experimental treatment (thermocycling and dynamic loading), and the tenth received no experimental treatment. Eight copings were pulled off in a universal testing machine (MTS Insight; MTS). The ninth specimen was treated experimentally but was not pull tested. The marginal discrepancy and the dentin interface of the specimens that were not pull tested were analyzed with scanning electron microscopy and energy dispersion spectroscopy. The mode of failure of the dislodged copings was also subjectively evaluated. RESULTS: The mean retentive strengths were 5.92 MPa for CM A, 5.81 MPa for CM OA, 5.75 MPa for RX A, and 5.69 MPa for RX OA. The marginal discrepancy recorded for both CM and RX ranged from 30 to 45 µm, (mean, 36 ±4.6 µm). Energy dispersion spectroscopy analysis showed the presence of calcium, phosphorus, silicon, and aluminum for the CM marginal discrepancy and the presence of aluminum in the dentinal tubules adjacent to the CM. Calcium and phosphorus were detected in lesser amounts adjacent to the RX marginal discrepancy. The mode of failure for CM was primarily adhesive to the tooth preparation, and, for RX, the failure mode was predominantly adhesive to the coping. CONCLUSIONS: CM had statistically significantly higher mean retentive strength compared with RX. Subgroups loaded axially had statistically significantly higher retentive strengths compared with those loaded off axis.

Vibronic Dynamics of the Ultrafast all-trans to 13-cis Photoisomerization of Retinal in Channelrhodopsin-1.

Channelrhodopsins are light-gated ion channels with extensive applications in optogenetics. Channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) exhibits a red-shifted absorption spectrum as compared to Channelrhodopsin-2, which is highly beneficial for optogenetic application. The primary event in the photocycle of CaChR1 involves an isomerization of the protein-bound retinal chromophore. Here, we apply highly time-resolved vibronic spectroscopy to reveal the electronic and structural dynamics associated with the first step of the photocycle of CaChR1. We observe vibrationally coherent formation of the P1 intermediate exhibiting a twisted 13-cis retinal with a 110 ± 7 fs time constant. Comparison with low-temperature resonance Raman spectroscopy of the corresponding trapped photoproduct demonstrates that this rapidly formed P1 intermediate is stable for several hundreds of nanoseconds.

Peri-Implant Strain in an In Vitro Model.

An in vitro experimental model was designed and tested to determine the influence that peri-implant strain may have on the overall crestal bone. Strain gages were attached to polymethylmethacrylate (PMMA) models containing a screw-type root form implant at sites 1 mm from the resin-implant interface. Three different types of crown superstructures (cemented, 1-screw [UCLA] and 2-screw abutment types) were tested. Loading (1 Hz, 200 N load) was performed using a MTS Mechanical Test System. The strain gage data were stored and organized in a computer for statistical treatment. Strains for all abutment types did not exceed the physiological range for modeling and remodeling of cancellous bone, 200-2500 µÎµ (microstrain). For approximately one-quarter of the trials, the strain values were less than 200 µÎµ the zone for bone atrophy. The mean microstrain obtained was 517.7 µÎµ. In conclusion, the peri-implant strain in this in vitro model did not exceed the physiologic range of bone remodeling under axial occlusal loading.

The effects of loading on the preload and dimensions of the abutment screw for a 3-unit cantilever-fixed prosthesis design.

OBJECTIVE: The purpose of this study was to use an in vitro model system to compare the effects on the screw torque and screw dimensions within 2 commercially available implant systems from occlusal loading on a cantilevered-fixed partial denture. MATERIALS AND METHODS: Cantilevered implant-supported 3-unit prostheses with 2 premolar abutments and 1 premolar pontic (7.3 mm in length) were made on resin casts containing 2 implant analogs for 2 implant systems: BioLok Silhouette Tapered Implant System (Birmingham, AL) and Zimmer Tapered Screw-Vent Implant System (Carlsbad, CA) with 10 samples in each group. Each sample was loaded with either of 2 protocols: (1) a load of 50 N on the cantilevered pontic unit and (2) a loading of 150 N on all 3 units. The outcome measures were (1) changes in residual torque of the abutment screws and (2) changes in screw dimension. RESULTS: The BioLok Silhouette Tapered Implant group demonstrated slight but statistically significant torque loss 18.8% to 28.5% in both abutment screws for both protocols, P ≤ 0.05, without any changes in screw dimension. In the Zimmer Tapered Screw-Vent Implant group, there was a significant elongation of the abutment screws and a markedly significant 44.4%, (P ≤ 0.01) loss in torque in the mesial screw and a 28.5%, (P ≤ 0.05) loss in torque in the distal screw when the cantilever alone was loaded. CONCLUSIONS: Differences in screw design influence the maintenance of preload and distortion of the shank. The influence of the interface design, namely an internal hex of 1 mm versus an external hex did not influence the preload. Cantilevered prostheses can cause loss of torque and dimensional changes in abutment screws.

Nickel-oxido structure of a water-oxidizing catalyst film.

The atomic structure of an electrodeposited Ni catalyst film is dominated by extensive di-µ-oxido bridging between Ni(III/IV) ions, as revealed by X-ray absorption spectroscopy. The structure is surprisingly similar to that of an analogous Co-based film and colloidal Mn-based catalysts. Structural requirements for water oxidation are discussed.

The effects of laser microtextured collars upon crestal bone levels of dental implants.

PURPOSE: The purpose of this study was to examine the crestal bone, connective tissue, and epithelial cell response to a laser microtextured collar compared with a machined collar, in the dog model. MATERIALS: Six mongrel dogs had mandibular premolars and first molars extracted and after healing replaced with BioLok implants 4 x 8 mm. Each dog had 3 control implants placed on one side of the mandible and 3 experimental, laser microtextured, implants placed contralaterally. After 3 months, 1 dog was killed. Bridges were placed on the implants of 4 of the dogs. The sixth dog served as a negative control for the duration of the experiment. Two of the dogs were killed 3 months after loading, of the dogs were killed 6 months after loading as was the negative (unloaded) control. Histology, electron microscopy, and histomorpho-metric analysis was done on histologic sections obtained from block sections of the mandible containing the implants. RESULTS: Initially the experimental implants showed greater bone attachment along the collar. With time the bone heights along the control and experimental collars were equivalent. However, the controls had more soft tissue downgrowth, greater osteoclastic activity, and increased saucerization compared with sites adjacent to experimental implants. There was closer adaptation of the bone to the laser microtextured collars. CONCLUSION: Use of tissue-engineered collars with microgrooving seems to promote bone and soft tissue attachment along the collar and facilitate development of a biological width.

Effects of load and indicator type upon occlusal contact markings.

STATEMENT OF PROBLEM: Clinicians use occlusal indicators to identify tooth contacts. However, the reliability of these indicators has been questioned. At times occlusal contacts are not identified or false positive occlusal contacts are observed. PURPOSE OF STUDY: This study was designed to compare the number and size of occlusal indicator marks from both thick and thin occlusal indicator materials with different loads. MATERIALS AND METHODS: Ivorine casts were articulated and mounted on an Hana Mate nonadjustable articulator. Loads of 100N, 150N, and 200N were applied with Accufim (25 microm thick, Parkell, Farmingdale, NY) and Articulating Paper (60 microm thick, G.E. Rudischauer Dental Articulating Paper, Brooklyn, NY) as the occlusal indicators. A fresh piece of indicator was used for each trial. Comparisons were made of the number and size of the contacts for both the thick and thin occlusal indicators at the different loads. RESULTS: Observation of the marks recorded with the thicker occlusal indicator demonstrated both a greater number marks and a larger size to the marks when compared to the thinner Accufilm, p < or = 0.02-0.0001. However, there was no significant increase in the number or size of the marks with an increased load for either material.

Long-term effects of storage and thermal cycling on the marginal adaptation of provisional resin crowns: a pilot study.

STATEMENT OF PROBLEM: Provisional resin crowns may be used for an extended period while complex treatments are completed. The crowns function intraorally; therefore, moisture absorption and thermal cycling may affect the physical properties of acrylic resin, causing a change in marginal gap size. PURPOSE: The purpose of this pilot study was to examine the effect of long-term water absorption and thermal cycling on marginal gap size of polymethyl methacrylate copolymer and bis-acrylic composite resin crowns. MATERIAL AND METHODS: Specimens (n = 10) were fabricated from 2 acrylic resins: a polymethyl methacrylate (Alike) and a bis-acrylic composite resin (Provitec). Specimens were first fabricated on a metal master die. Custom die stems were fabricated for each specimen from a low-fusing alloy (Cerroblend) to eliminate the factor of polymerization shrinkage. Specimens were then fitted to assure a standardized, pre-experimental marginal gap range of < or = 25 microm. Specimens were stored in a humidor at 37 degrees C and 97% relative humidity for 1 year and subsequently thermal cycled (5 degrees C to 60 degrees C, 6-second dwell time, for 8000 cycles). Measurements in micrometers of the marginal gap were recorded using a microscope equipped with a digital video camera and image analysis software before and after treatment. A 2-way analysis of variance with a split design was performed for factors of materials and treatment (alpha = .05). RESULTS: For the factor of material, there was no significant difference; however, there was a significant difference between treatments, with a significantly greater increase in marginal gap size after thermal cycling (P < .002). CONCLUSION: Provisional crowns made from either a bis-acrylic resin composite or a polymethyl methacrylate copolymer demonstrated loss of marginal adaptation during a simulated long-term period of service.

Sensory responses from loading of implants: a pilot study.

PURPOSE: Osseointegrated implants lack a periodontal ligament. Nevertheless, masticatory function in subjects with implant-supported restorations appears similar to function in those with natural dentition. It is not clear how the neurophysiologic mechanisms that modulate jaw movement are associated with osseointegrated implants. This study examined the output from the inferior alveolar nerve during implant loading. MATERIALS AND METHODS: In 3 dogs, 3 premolars were extracted in the mandible and 2 endosseous titanium implants were placed, allowed to osseointegrate for 3 months, and loaded with vibration force at the threshold response for tooth vibration, at 2x threshold, and at 3x threshold. Neurophysiologic recordings were made from the inferior alveolar nerve during loading of both implants and the adjacent molar and canine. The response magnitude in action potentials in the 50- ms poststimulus period and latency of inferior alveolar afferents in milliseconds were compared following implant loading. RESULTS: Detectable inferior alveolar nerve responses were recorded following loading from both the implants and the teeth at 2x and 3x threshold. However, the response magnitude of teeth (canine, 2.38 +/- 0.18 at 2x, 2.78 +/- 0.2 at 3x; molar, 2.2 +/- 0.16 at 2x, 2.5 +/- 0.21 at 3x) was twice that of the implants (anterior, 1.3 +/- 0.12 at 2x, 1.68 +/- 0.13 at 3x; posterior, 0.8 +/- 0.1 at 2x, 1.53 +/- 0.15 at 3x). The differences in response magnitude between the teeth and implants were significant (P < .05). The latency of response was similar. DISCUSSION: Management of the occlusion for implant-supported restorations has been empirically developed. An underlying assumption has been that implant-guided jaw function lacks significant proprioception to modulate mastication and related jaw movements. This animal study provides preliminary evidence that force application to implants does elicit a proprioceptive response. CONCLUSION: Loading of implants does elicit a sensory response that can be observed in the inferior alveolar nerve. The implications are that during occlusal function, information from regions associated with the implant can provide knowledge that could potentially modulate jaw activity in a manner similar to natural teeth.