Impact of Polysorbate 80 Grade on the Interfacial Properties and Interfacial Stress Induced Subvisible Particle Formation in Monoclonal Antibodies.

Polysorbate 80 is a nonionic surfactant that is added to therapeutic protein formulations to mitigate protein particle formation when subjected to various mechanical stresses. Variations in the PS80 grade has recently sparked questions surrounding the effect of oleic acid content (OAC) on surfactant's ability to mitigate interface-induced protein particle formation when stressed. In this work, a Langmuir trough was used to apply interfacial dilatational stress to two IgG molecules (mAb1 and mAb2) in formulations containing Chinese pharmacopeia (CP) and multicompendial (MC) grades of PS80. The interfacial properties of these mAb formulations, with and without interfacial dilatational stresses, were correlated with subvisible particle count and particle size/morphology distributions as measured by Micro-flow imaging (MFI). Overall, differences in interfacial properties correlated well with protein particle formation for both molecules in the two PS80 formulations. Further, the impact of grade of PS80 on the interfacial properties and interfacial stress-induced protein particle formation depends on the adsorption kinetics of the IgG molecules as well as the concentration of the surfactant used. This study demonstrates that measuring the interfacial properties of mAb formulations can be a useful tool to predict interfacial stress induced protein particle formation in the presence of different excipients of varying quality.

Cross-linking of poly (vinyl alcohol) films under acidic and thermal stress.

Poly (vinyl alcohol), PVA, a commonly used excipient to coat tablets, forms insoluble films in the presence of acids and thermal stress. This may lead to drug products failing to meet dissolution specifications over time. Studies were conducted to understand the effect of acid strength, processing conditions, and storage stress on the mechanism of insoluble film formation using PVA and OpadryⓇ II as model systems. Aqueous cast films, prepared by incorporating hydrochloric acid (HCl) into the coating solutions or exposing pre-cast "as is" films to HCl vapors, were used as surrogates to develop analytical methods. To understand effect of acid and processing on coatings, acidified OpadryⓇ II was spray coated onto inert cores under "wet" or "dry" conditions. Samples stored at 50-60 °C were analyzed for film disintegration to understand physical/chemical changes in the polymer. Rate and extent of insoluble films formation was dependent on the acid concentration and thermal stress. Analysis of the films indicated significant de-acetylation and ether bond formation in insoluble aqueous cast films. In contrast, acidified coated films showed only ether bond formation, which increased on stress, forming insoluble films. The reduction in the time to form insoluble films for "wet" versus "dry" coated films was rationalized by considering effect of coating, drying, and storage on the microstructure of acidified PVA and ether bond propagation. The results highlight the need to develop an in-depth understanding of the design space for PVA coated products and storage conditions in presence of acids.

Bridging size and charge variants of a therapeutic monoclonal antibody by two-dimensional liquid chromatography.

Monoclonal antibodies are heterogeneous in nature and may contain numerous variants with differences in size, charge, and hydrophobicity, which may impact clinical efficacy, immunogenicity, and safety. Characterization of antibody variants is necessary to build structure-function correlation and establish a proper control strategy. Isolation and enrichment of variants by conventional chromatographic peak fractionation is labor-intensive and time-consuming. The instability of fractions during isolation and subsequent characterization may also be a concern. Hence, it is desirable to analyze antibody variants in an online and real-time manner. Here we demonstrate a 2D-LC methodology - multiple heart-cutting IEC-SEC- as an investigational tool to facilitate a charge variant characterization study. Both IEC modes - anion exchange (AEX) and cation exchange (CEX) chromatography are discussed. Using this approach, direct bridging of size and charge variants of an antibody molecule was achieved without offline peak fractionation. It was observed that antibody aggregates elute late on both the AEX and CEX columns, presumably due to secondary hydrophobic interactions. Additionally, we overcame the solvent mismatch issue and developed a 2D SEC-IEC method to confirm the bridging results. This is the first reported SEC-IEC 2D-LC application for the characterization of antibody size and charge variants.

Assessing the risk of drug crystallization in vivo.

INTRODUCTION: Low intrinsic solubility leading to poor oral bioavailability is a common challenge in drug discovery that can often be overcome by formulation strategies, however, it remains a potential limitation that can pose challenges for early risk assessment and represent a significant obstacle to drug development. We identified a selective inhibitor (BMS-986126) of the IL-1 receptor-associated kinase 4 (IRAK4) with favorable properties as a lead candidate, but with unusually low intrinsic solubility of <1 µg/mL. METHODS: Conventional histopathology identified the issue of crystal formation in vivo. Subsequent investigative work included confocal Raman micro-spectroscopy, MALDI-MS, polarized light microscopy of fresh wet-mount tissue scrapings and transmission electron microscopy. RESULTS: BMS-986126 was advanced into a 2-week toxicology study in rats. The main finding in this study was minimal granulomatous inflammation in the duodenum, associated with the presence of birefringent crystals at the highest dosage of 100 mg/kg/day. Considering the safety margin, and the single location of the lesion, BMS-986126 was further progressed into IND-enabling toxicology studies where tolerability deteriorated with increasing dosing duration. Birefringent crystals and granulomatous inflammation were detected in multiple organs at dosages ≥20 mg/kg/day. Raman spectroscopy confirmed the identity of the crystals as BMS-986126. Therefore, follow up investigations were conducted to further characterize drug crystallization and to evaluate detection methods for their potential to reliably detect in vivo crystallization early. DISCUSSION: The purpose of our efforts was to identify critical factors influencing in vivo drug crystallization and to provide a preliminary assessment (based on one compound) which method would be best suited for identifying crystals. Results indicated a combination of methods was required to provide a complete assessment of drug crystallization and that a simple technique, scraping of freshly collected tissue followed by evaluation under polarizing light was suitable for detecting crystals. However, dosing for 2 weeks was required for crystals to grow to a clearly detectable size.

Characterization of a Liquid Crystal System for Sustained Release of a Peptide BMS-686117.

Liquid crystal lipid-based formulations are an effective approach to prolong pharmacokinetics and reduce burst release of a drug on subcutaneous delivery. The objective of this paper was to investigate the influence of phase structures of a lipid-based liquid crystal delivery system and its associated mechanical properties on the release profile of a peptide. It was hypothesized that release of drug molecules are closely related to the mechanical properties that are controlled by phase structures. Experimentally, the relationship between phase structures of lipid liquid crystal system-soy phosphatidyl choline (SPC) and glycerol dioleate (GDO) in water were characterized by polarized light microscopy and small angle X-ray diffraction. Their rheological properties were evaluated with a rheometer and the in vitro release of the peptide as a measure drug release from the LC-depot injection. Three phases: disordered phase, lamellar phase, mixtures of cubic, lamellar, and hexagonal phases were detected by varying formulation compositions. A significant difference in rheological behavior was observed. The disordered phase displayed some attributes of typical Newtonian fluid with lowest viscosity while the lamellar phase showed a shear thinning behavior. Regarding the mechanical strength, the lamellar phase presents the highest storage modulus due to its layer structure followed by mixed phases. Comparing release profiles, the lamellar phase produced a fast release followed by the mixture of phases. In conclusion, this study demonstrates the ability to characterize LC phase structures with microscopy, small angle X-ray diffraction, and rheological measurements and their link to modulating a peptide release profile.

Understanding Protein-Interface Interactions of a Fusion Protein at Silicone Oil-Water Interface Probed by Sum Frequency Generation Vibrational Spectroscopy.

Protein adsorbed at the silicone oil-water interface can undergo a conformational change that has the potential to induce protein aggregation on storage. Characterization of the protein structures at interface is therefore critical for understanding the protein-interface interactions. In this article, we have applied sum frequency generation (SFG) spectroscopy for studying the secondary structures of a fusion protein at interface and the surfactant effect on protein adsorption to silicone oil-water interface. SFG and chiral SFG spectra from adsorbed protein in the amide I region were analyzed. The presence of beta-sheet vibrational band at 1635 cm-1 implies the protein secondary structure was likely perturbed when protein adsorbed at silicone oil interface. The time-dependent SFG study showed a significant reduction in the SFG signal of preadsorbed protein when polysorbate 20 was introduced, suggesting surfactant has stronger interaction with the interface leading to desorption of protein from the interface. In the preadsorbed surfactant and a mixture of protein/polysorbate 20, SFG analysis confirmed that surfactant can dramatically prevent the protein adsorption to silicone oil surface. This study has demonstrated the potential of SFG for providing the detailed molecular level understanding of protein conformation at interface and assessing the influence of surfactant on protein adsorption behavior.

The Impact of Disintegrant Type, Surfactant, and API Properties on the Processability and Performance of Roller Compacted Formulations of Acetaminophen and Aspirin.

In formulation development, certain excipients, even though used in small quantities, can have a significant impact on the processability and performance of the dosage form. In this study, three common disintegrants, croscarmellose sodium (CCS), crospovidone (xPVP), and sodium starch glycolate (SSG) as well as the surfactant sodium lauryl sulfate (SLS) were evaluated for their impact on the processability and performance of a typical dry granulation formulation. Two model compounds, the mechanically brittle and chemically inert acetaminophen and the mechanically ductile carboxylic acid aspirin, were used for the evaluation. It was found that the disintegrants were generally identical in their impact on the processability and little difference was observed in the granulation and compression processes. The exception is that when xPVP was used in the formulation of the brittle acetaminophen, lower compression forces were needed to reach the same tablet hardness, suggesting a binding effect of xPVP for such systems. In general, CCS and xPVP tend to provide slightly better disintegration than SSG. However, in the case of aspirin, a strong hydrogen bonding interaction between the carboxylic acid group of aspirin and the carbonyl group of xPVP was observed, resulting in slower release of the drug after fast disintegration. SLS was found to have a significant impact on the processability due to its lubricating effect, resulting in higher compression forces needed to achieve the target tablet hardness. Due to the higher degree of compression, the disintegration and dissolution of both drugs became slower despite the wetting effect of SLS.

Characterization of Phase Separation Propensity for Amorphous Spray Dried Dispersions.

A generalized screening approach, applying isothermal calorimetry at 37 °C 100% RH, to formulations of spray dried dispersions (SDDs) for two active pharmaceutical ingredients (APIs) (BMS-903452 and BMS-986034) is demonstrated. APIs 452 and 034, with similar chemotypes, were synthesized and promoted during development for oral dosing. Both APIs were formulated as SDDs for animal exposure studies using the polymer hydroxypropylmethlycellulose acetyl succinate M grade (HPMCAS-M). 452 formulated at 30% (wt/wt %) was an extremely robust SDD that was able to withstand 40 °C 75% RH open storage conditions for 6 months with no physical evidence of crystallization or loss of dissolution performance. Though 034 was a chemical analogue with similar physical chemical properties to 452, a physically stable SDD of 034 could not be formulated in HPMCAS-M at any of the drug loads attempted. This study was used to develop experience with specific physical characterization laboratory techniques to evaluate the physical stability of SDDs and to characterize the propensity of SDDs to phase separate and possibly crystallize. The screening strategy adopted was to stress the formulated SDDs with a temperature humidity screen, within the calorimeter, and to apply orthogonal analytical techniques to gain a more informed understanding of why these SDDs formulated with HPMCAS-M demonstrated such different physical stability. Isothermal calorimetry (thermal activity monitor, TAM) was employed as a primary stress screen wherein the SDD formulations were monitored for 3 days at 37 °C 100% RH for signs of phase separation and possible crystallization of API. Powder X-ray diffraction (pXRD), modulated differential scanning calorimetry (mDSC), Fourier transform infrared spectroscopy (FTIR), and solid state nuclear magnetic resonance (ssNMR) were all used to examine formulated SDDs and neat amorphous drug. 452 SDDs formulated at 30% (wt/wt %) or less did not show phase separation behavior upon exposure to 37 °C 100% RH for 3 days. 034 SDD formulations from 10 through 50% (wt/wt %) all demonstrated thermal traces consistent with exothermic phase separation events over 3 days at 37 °C 100% RH in the TAM. However, only the 15, 30, and 50% containing 034 samples showed pXRD patterns consistent with crystalline material in post-TAM samples. Isothermal calorimetry is a useful screening tool to probe robust SDD physical performance and help investigate the level of drug polymer miscibility under a humid stress. Orthogonal analytical techniques such as pXRD, ssNMR, and FTIR were key in this SDD formulation screening to gain physical understanding and confirm or refute whether physical changes occur during the observed thermal events characterized by the calorimetric screening experiments.

The effect of polymeric excipients on the physical properties and performance of amorphous dispersions: Part I, free volume and glass transition.

PURPOSE: To investigate the structural effect of polymeric excipients on the behavior of free volume of drug-polymer dispersions in relation to glass transition. METHODS: Two drugs (indomethacin and ketoconazole) were selected to prepare amorphous dispersions with PVP, PVPVA, HPC, and HPMCAS through spray drying. The physical attributes of the dispersions were characterized using SEM and PXRD. The free volume (hole-size) of the dispersions along with drugs and polymers was measured using positron annihilation lifetime spectroscopy (PALS). Their glass transition temperatures (Tgs) were determined using DSC and DMA. FTIR spectra were recorded to identify hydrogen bonding in the dispersions. RESULTS: The chain structural difference-flexible (PVP and PVPVA) vs. inflexible (HPC and HPMCAS)-significantly impacts the free volume and Tgs of the dispersions as well as their deviation from ideality. Relative to Tg, free volume seems to be a better measure of hydrogen bonding interaction for the dispersions of PVP, HPC, and HPMCAS. The free volume of polymers and their dispersions in general appears to be related to their conformations in solution. CONCLUSIONS: Both the backbone chain rigidity of polymers as well as drug-polymer interaction can impact the free volume and glass transition behaviors of the dispersions.

Low level drug product API form analysis - Avalide tablet NIR quantitative method development and robustness challenges.

Avalide(@), a medication used for the treatment of hypertension, is a combination of Irbesartan, and Hydrochlorothiazide. Irbesartan, one of the active pharmaceutical ingredients (API) in Avalide products, exists in two neat crystalline forms: Form A and Form B. Irbesartan Form A is the API form used in a wet granulation for the preparation of Avalide tablets. The presence of the less soluble Irbesartan Form B in Avalide tablets may result in the slower dissolution. In this paper, we have presented our work on the method development, verification and challenges of quantitatively detecting, via NIR and ssNMR, very small amounts of Irbesartan Form B in Avalide tablets. As part of the NIR method development and qualification, limit of detection, linearity and accuracy were examined. In addition, a limited study of the robustness of the method was conducted and a bias in the level of Form B was correlated to the ambient humidity. ssNMR, a primary method for the determination of polymorphic composition, was successfully used as an orthogonal technique to verify the accuracy of the NIR method and added to the confidence in the NIR method. The speed and efficiency of the NIR method make it a suitable and convenient tool for routine analysis of Avalide tablets for Form B in a QC environment.

Anharmonic vibrational calculations modeling the raman spectra of intermediates in the photoactive yellow protein (PYP) photocycle.

The role of anharmonic effects in the vibrational spectroscopy of the dark state and two major chromophore intermediates of the photoactive yellow protein (PYP) photocycle is examined via ab initio vibrational self-consistent field (VSCF) calculations and time-resolved resonance Raman spectroscopy. For the first time, anharmonicity is considered explicitly in calculating the vibrational spectra of an ensemble consisting of the PYP chromophore surrounded by model compounds used as mimics of the important active-site residues. Predictions of vibrational frequencies on an ab initio corrected semiempirical potential energy surface show remarkable agreement with experimental frequencies for all three states, thus shedding light on the potential along the reaction path. For example, calculated frequencies for vibrational modes of the red-shifted intermediate, PYPL, exhibit an overall average error of 0.82% from experiment. Upon analysis of anharmonicity patterns in the PYP modes we observe a decrease in anharmonicity in the C8-C9 stretching mode nu29 (trans-cis isomerization marker mode) with the onset of the cis configuration in PYPL. This can be attributed to the loss of the hydrogen-bonding character of the adjacent C9-O2 to the methylamine (Cys69 backbone). For several of the modes, the anharmonicity is mostly due to mode-mode coupling, while for others it is mostly intrinsic. This study shows the importance of the inclusion of anharmonicity in theoretical spectroscopic calculations, and the sensitivity of experiments to anharmonicity. The characterization of protein active-site residues by small molecular mimics provides an acceptable chemical structural representation for biomolecular spectroscopy calculations.

Combined spectroscopic and topographic characterization of nanoscale domains and their distributions of a redox protein on bacterial cell surfaces.

Redox protein nanoscale domains on the cell surface of a bacterium, Shewanella oneidensis MR1, grown in the absence and presence of electron acceptors, is topographically characterized using combined atomic force microscopy (AFM) and confocal surface enhanced Raman scattering (SERS) spectroscopy. The protruding nanoscale domains on the outer membrane of S. oneidensis were observed, as was their disappearance upon exposure to electron acceptors such as oxygen, nitrate, fumarate, and iron nitrilotriacetate (FeNTA). Using SERS spectroscopy, a redox heme protein was identified as a major component of the cell surface domains. This conclusion was further confirmed by the disappearance of Raman vibrational frequencies, characteristic of heme proteins, upon exposure of the cells to electron acceptors. Our experimental results from our AFM imaging and SERS spectroscopy, consistent with the literature, suggest the protruding nanoscale surface domains as heme-containing secretions. Our results on the distributions of redox proteins on microbial cell surfaces will be helpful for a mechanistic understanding of the behaviors of surface proteins and their interactions with redox environments.

Nitric oxide binding to prokaryotic homologs of the soluble guanylate cyclase beta1 H-NOX domain.

The heme cofactor in soluble guanylate cyclase (sGC) is a selective receptor for NO, an important signaling molecule in eukaryotes. The sGC heme domain has been localized to the N-terminal 194 amino acids of the beta1 subunit of sGC and is a member of a family of conserved hemoproteins, called the H-NOX family (Heme-Nitric Oxide and/or OXygen-binding domain). Three new members of this family have now been cloned and characterized, two proteins from Legionella pneumophila (L1 H-NOX and L2 H-NOX) and one from Nostoc punctiforme (Np H-NOX). Like sGC, L1 H-NOX forms a 5-coordinate Fe(II)-NO complex. However, both L2 H-NOX and Np H-NOX form temperature-dependent mixtures of 5- and 6-coordinate Fe(II)-NO complexes; at low temperature, they are primarily 6-coordinate, and at high temperature, the equilibrium is shifted toward a 5-coordinate geometry. This equilibrium is fully reversible with temperature in the absence of free NO. This process is analyzed in terms of a thermally labile proximal Fe(II)-His bond and suggests that in both the 5- and 6-coordinate Fe(II)-NO complexes of L2 H-NOX and Np H-NOX, NO is bound in the distal heme pocket of the H-NOX fold. NO dissociation kinetics for L1 H-NOX and L2 H-NOX have been determined and support a model in which NO dissociates from the distal side of the heme in both 5- and 6-coordinate complexes.

Characterization of functional heme domains from soluble guanylate cyclase.

Soluble guanylate cyclase (sGC) is a heterodimeric, nitric oxide (NO)-sensing hemoprotein composed of two subunits, alpha1 and beta1. NO binds to the heme cofactor in the beta1 subunit, forming a five-coordinate NO complex that activates the enzyme several hundred-fold. In this paper, the heme domain has been localized to the N-terminal 194 residues of the beta1 subunit. This fragment represents the smallest construct of the beta1 subunit that retains the ligand-binding characteristics of the native enzyme, namely, tight affinity for NO and no observable binding of O(2). A functional heme domain from the rat beta2 subunit has been localized to the first 217 amino acids beta2(1-217). These proteins are approximately 40% identical to the rat beta1 heme domain and form five-coordinate, low-spin NO complexes and six-coordinate, low-spin CO complexes. Similar to sGC, these constructs have a weak Fe-His stretch [208 and 207 cm(-)(1) for beta1(1-194) and beta2(1-217), respectively]. beta2(1-217) forms a CO complex that is very similar to sGC and has a high nu(CO) stretching frequency at 1994 cm(-)(1). The autoxidation rate of beta1(1-194) was 0.073/min, while the beta2(1-217) was substantially more stable in the ferrous form with an autoxidation rate of 0.003/min at 37 degrees C. This paper has identified and characterized the minimum functional ligand-binding heme domain derived from sGC, providing key details toward a comprehensive characterization.

Probing inhomogeneous vibrational reorganization energy barriers of interfacial electron transfer.

An atomic force microscopy (AFM) and confocal Raman microscopy study of the interfacial electron transfer of a dye-sensitization system, i.e., alizarin adsorbed upon TiO(2) nanoparticles, has revealed the distribution of the mode-specific vibrational reorganization energies encompassing different local sites ( approximately 250-nm spatial resolution). Our experimental results suggest inhomogeneous vibrational reorganization energy barriers and different Franck-Condon coupling factors of the interfacial electron transfer. The total vibrational reorganization energy was inhomogeneous from site to site; specifically, mode-specific analyses indicated that energy distributions were inhomogeneous for bridging normal modes and less inhomogeneous or homogeneous for nonbridging normal modes, especially for modes far away from the alizarin-TiO(2) coupling hydroxyl modes. The results demonstrate a significant step forward in characterizing site-specific inhomogeneous interfacial charge-transfer dynamics.

Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis.

Soluble guanylate cyclase (sGC) is a nitric oxide- (NO-) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. Characterization of two of these proteins is reported here. The first is a 181 amino acid protein cloned from Vibrio cholerae (VCA0720) that is encoded in a histidine kinase-containing operon. The ferrous unligated form of VCA0720 is 5-coordinate, high-spin. The CO complex is low-spin, 6-coordinate, and the NO complex is high-spin and 5-coordinate. These ligand-binding properties are very similar to those of sGC. The second protein is the N-terminal 188 amino acids of Tar4 (TtTar4H), a predicted methyl-accepting chemotaxis protein (MCP) from the strict anaerobe Thermoanaerobacter tengcongensis. TtTar4H forms a low-spin, 6-coordinate ferrous-oxy complex, the first of this sGC-related family that binds O2. TtTar4H has ligand-binding properties similar to those of the heme-containing O2 sensors such as AxPDEA1. sGC does not bind O2 despite having a porphyrin with a histidyl ligand like the globins. The results reported here, with sequence-related proteins from prokaryotes but in the same family as the sGC heme domain, show that these proteins have evolved to discriminate between ligands such as NO and O2; hence, we term this family H-NOX domains (heme-nitric oxide/oxygen).

Time-resolved resonance raman structural studies of the pB' intermediate in the photocycle of photoactive yellow protein.

Time-resolved resonance Raman spectroscopy is used to obtain chromophore vibrational spectra of the pR, pB', and pB intermediates during the photocycle of photoactive yellow protein. In the pR spectrum, the C8-C9 stretching mode at 998 cm(-1) is approximately 60 cm(-1) lower than in the dark state, and the combination of C-O stretching and C7H=C8H bending at 1283 cm(-1) is insensitive to D2O substitution. These results indicate that pR has a deprotonated, cis chromophore structure and that the hydrogen bonding to the chromophore phenolate oxygen is preserved and strengthened in the early photoproduct. However, the intense C7H=C8H hydrogen out-of-plane (HOOP) mode at 979 cm(-1) suggests that the chromophore in pR is distorted at the vinyl and adjacent C8-C9 bonds. The formation of pB' involves chromophore protonation based on the protonation state marker at 1174 cm(-1) and on the sensitivity of the COH bending at 1148 cm(-1) as well as the combined C-OH stretching and C7H=C8H bending mode at 1252 cm(-1) to D2O substitution. The hydrogen out-of-plane Raman intensity at 985 cm(-1) significantly decreases in pB', suggesting that the pR-to-pB' transition is the stage where the stored photon energy is transferred from the distorted chromophore to the protein, producing a more relaxed pB' chromophore structure. The C=O stretching mode downshifts from 1660 to 1651 cm(-1) in the pB'-to-pB transition, indicating the reformation of a hydrogen bond to the carbonyl oxygen. Based on reported x-ray data, this suggests that the chromophore ring flips during the transition from pB' to pB. These results confirm the existence and importance of the pB' intermediate in photoactive yellow protein receptor activation.

Picosecond dynamics of G-protein coupled receptor activation in rhodopsin from time-resolved UV resonance Raman spectroscopy.

The protein response to retinal chromophore isomerization in the visual pigment rhodopsin is studied using picosecond time-resolved UV resonance Raman spectroscopy. High signal-to-noise Raman spectra are obtained using a 1 kHz Ti:Sapphire laser apparatus that provides <3 ps visible (466 nm) pump and UV (233 nm) probe pulses. When there is no time delay between the pump and probe events, tryptophan modes W18, W16, and W3 exhibit decreased Raman scattering intensity. At longer pump-probe time delays of +5 and +20 ps, both tryptophan (W18, W16, W3, and W1) and tyrosine (Y1 + 2xY16a, Y7a, Y8a) peak intensities drop by up to 3%. These intensity changes are attributed to decreased hydrophobicity in the microenvironment near at least one tryptophan and one tyrosine residue that likely arise from weakened interaction with the beta-ionone ring of the chromophore following cis-to-trans isomerization. Examination of the crystal structure suggests that W265 and Y268 are responsible for these signals. These UV Raman spectral changes are nearly identical to those observed for the rhodopsin-to-Meta I transition, implying that impulsively driven protein motion by the isomerizing chromophore during the 200 fs primary transition drives key structural changes that lead to protein activation.

Time-resolved resonance Raman analysis of chromophore structural changes in the formation and decay of rhodopsin's BSI intermediate.

Time-resolved resonance Raman microchip flow experiments are performed to obtain the vibrational spectrum of the chromophore in rhodopsin's BSI intermediate and to probe structural changes in the bathorhodopsin-to-BSI and BSI-to-lumirhodopsin transitions. Kinetic Raman spectra from 250 ns to 3 micros identify the key vibrational features of BSI. BSI exhibits relatively intense HOOP modes at 886 and 945 cm(-1) that are assigned to C(14)H and C(11)H=C(12)H A(u) wags, respectively. This result suggests that in the bathorhodopsin-to-BSI transition the highly strained all-trans chromophore has relaxed in the C(10)-C(11)=C(12)-C(13) region, but is still distorted near C(14). The low frequency of the 11,12 A(u) HOOP mode in BSI compared with that of lumirhodopsin and metarhodopsin I indicates weaker coupling between the 11H and 12H wags due to residual distortion of the BSI chromophore near C(11)=C(12). The C=NH(+) stretching mode in BSI at 1653 cm(-1) exhibits a normal deuteriation induced downshift of 23 cm(-1), implying that there is no significant structural rearrangement of the Schiff base counterion region in the transition of bathorhodopsin to BSI. However, a dramatic Schiff base environment change occurs in the BSI-to-lumirhodopsin transition, because the 1638 cm(-1) C=NH(+) stretching mode in lumirhodopsin is unusually low and shifts only 7 cm(-1) in D(2)O, suggesting that it has essentially no H-bonding acceptor. With these data we can for the first time compare and discuss the room temperature resonance Raman vibrational structure of all the key intermediates in visual excitation.