Corroborative studies on chain packing characteristics of biological medium-chain-length poly-3-hydroxyalkanoates with different monomeric composition.

Medium-chain-length poly-3-hydroxyalkanoates (mcl-PHAs) with varied monomeric compositions were biosynthesized by producer bacteria fed with different fatty acids as carbon source. Octanoic-, lauric-, stearic-, and oleic acids were used to produce four types of mcl-PHAs viz. PHA-OC, PHA-LA, PHA-ST, and PHA-OL, respectively. The mcl-PHAs as film-casted preparations exhibit distinct traits e.g., PHA-OC and PHA-ST films are less flexible than PHA-LA while PHA-OL is a sticky, glue-like material; PHA-ST is opaque whereas PHA-OC, PHA-LA, and PHA-OL displayed transparent layers. The observation is attributed to polymer chain packing and side chain crystallization. A structure-property investigation of these biopolymers was carried out employing different spectroscopic and microscopic analyses in addition to thermal analyses. Comparative analyses of the results were applied in the interpretation and discussion of structure-property relationship.

Measuring the Ultrafast Correlation Dynamics of a Multilevel System Using the Center Line Slope Analysis in Two-Dimensional Electronic Spectroscopy.

In a two-dimensional (2D) optical spectrum of a multilevel system, there are diagonal peaks and off-diagonal cross-peaks that correlate the different levels. The time-dependent properties of these diagonal peaks and cross-peaks contain much information about the dynamics of the multilevel system. The time-dependent diagonal peakshape that depends on the spectral diffusion dynamics of the associated transition and characterized by the frequency-fluctuation correlation function (FFCF) is well studied. However, the time-dependent peakshape of a cross-peak that provides the correlation dynamics between different transitions is much less studied or understood. We derived the third-order nonlinear response functions that describe the cross-peaks in a 2D electronic spectrum of a multilevel system that arise from processes sharing a common ground state and/or from internal conversion and population transfer. We can use the center line slope (CLS) analysis to characterize the cross-peaks in conjunction with the diagonal peaks. This allows us to recover the frequency-fluctuation cross-correlation functions (FXCFs) between two transitions. The FXCF and its subsidiary quantities such as the initial correlation and the initial covariance between different transitions are important for studying the correlation effects between states in complex systems, such as energy-transfer processes. Furthermore, knowledge of how various molecular processes over different timescales affect simultaneously different transitions can also be obtained from the measured FXCF. We validated and tested our derived equations and analysis process by studying, as an example, the 2D electronic spectra of metal-free phthalocyanine in solution. We measured and analyzed the diagonal peaks of the Qx and Qy transitions and the cross-peaks between these two transitions of this multilevel electronic system and obtained the associated FFCFs and FXCFs. In this model system, we measured negative components of FXCF over the tens of picosecond timescale. This suggests that in phthalocyanine, the Qx and Qy transitions coupling with the solvent molecule motion are anticorrelated to each other.

Revealing the excitation energy transfer network of Light-Harvesting Complex II by a phenomenological analysis of two-dimensional electronic spectra at 77 K.

Energy equilibration in light-harvesting antenna systems normally occurs before energy is transferred to a reaction center. The equilibration mechanism is a characteristic of the excitation energy transfer (EET) network of the antenna. Characterizing this network is crucial in understanding the first step of photosynthesis. We present our phenomenology-based analysis procedure and results in obtaining the excitonic energy levels, spectral linewidths, and transfer-rate matrix of Light-Harvesting Complex II directly from its 2D electronic spectra recorded at 77 K with waiting times between 100 fs to 100 ps. Due to the restriction of the models and complexity of the system, a unique EET network cannot be constructed. Nevertheless, a recurring pattern of energy transfer with very similar overall time scales between spectral components (excitons) is consistently obtained. The models identify a "bottleneck" state in the 664-668 nm region although with a relatively shorter lifetime (∼4-6 ps) of this state compared to previous studies. The model also determines three terminal exciton states at 675, 677-678, and 680-681 nm that are weakly coupled to each other. The excitation energy equilibration between the three termini is found to be independent of the initial excitation conditions, which is a crucial design for the light-harvesting complexes to ensure the energy flow under different light conditions and avoid excitation trapping. We proposed two EET schemes with tentative pigment assignments based on the interpretation of the modeling results together with previous structure-based calculations and spectroscopic observables.

Measuring Ultrafast Spectral Diffusion and Correlation Dynamics by Two-Dimensional Electronic Spectroscopy.

The frequency fluctuation correlation function (FFCF) measures the spectral diffusion of a state's transition while the frequency fluctuation cross-correlation function (FXCF) measures the correlation dynamics between the transitions of two separate states. These quantities contain a wealth of information on how the chromophores or excitonic states interact and couple with its environment and with each other. We summarize the experimental implementations and theoretical considerations of using two-dimensional electronic spectroscopy to characterize FFCFs and FXCFs. Applications can be found in systems such as the chlorophyll pigment molecules in light-harvesting complexes and CdSe nanomaterials.

Temperature Dependence of the Energy Transfer in LHCII Studied by Two-Dimensional Electronic Spectroscopy.

We measured two-dimensional electronic spectra of light-harvesting complex II (LHCII) at various temperatures (77, 110, 150, 230, and 295 K) under conditions free from singlet-singlet annihilation. We elucidated the temperature-dependent excitation energy transfer dynamics in the Chl a manifold of LHCII. Global analysis revealed that the dynamics can be summarized in distinct time scales from 200 fs up to 15 ps. While the fastest dynamics with a decay time of ∼0.2-0.3 ps are relatively temperature-independent, the lifetimes and relative contributions of slower components showed considerable temperature dependence. The slowest time scale of equilibration with the lowest-energy Chl a increased from ∼5 ps at 295 K to ∼15 ps at 77 K. The final excited state is independent of initial excitation at 230 K and above, whereas static energy disorder is apparent at lower temperatures. A clear temperature dependence of uphill energy transfer processes was also discerned, which is consistent with the detailed-balance condition.

Evidence of Increased Hydrophobicity and Dynamics inside the Tail Region of Glycolipid Self-Assemblies Using 2-n-Alkyl-Pyrene Derivatives to Probe Different Locations.

New designer biofluorophores are being increasingly used in the investigation of complex cellular processes. In this study, we utilized new derivatives of pyrene (Py), i.e., 2-n-alkyl-pyrenes (Py-C4 and Py-C8), in order to probe different regions inside the hydrophobic tail of n-dodecyl ß-d-maltoside (ßMal-C12) in two different phases (cubic ↔ lamellar). Although the sensitivity to the local environment is reduced compared to that of Py, attaching C4 and C8 at the 2-position of Py can provide a possible means to probe the local hydrophobicity in different parts of the tail region. The absence of excimer fluorescence and the ratio of the vibronic fluorescence peak intensities (I1/I3) in a lipid environment indicate the existence of Py as monomers in the hydrophobic region, similar to hydrophobic solvation, yet close to the headgroup region. When Py is replaced by Py-C4 and Py-C8, there is a small increase in hydrophobicity (reduction in I1/I3) as the Py moiety is pulled deeper inside the tail region of both cubic and lamellar phases. The larger space of the tail region in the lamellar phase is reflected as more local hydrophobicity measured by the probes which can penetrate deep inside, whereas the curved structure of the cubic phase limits the available space for the probes. Three fluorescence lifetime components were measured in lipid, indicating the heterogeneous nature of the hydrophobic region. In the lamellar phase, a large reduction in the average lifetime value, led by the long decay component, was measured for Py-C4 (reduction by 25%) and Py-C8 (45%) compared to that of the parent Py. This observation suggests the presence of a mechanism of interaction more collisional than static between the Py moiety and the tail region of the bilayer unit due to the ample space provided by the lamellar phase as the probe is buried deeper inside the hydrophobic region. A much smaller effect was observed in the cubic phase and was correlated with the tight environment around the probes, which stems from the increased curvature of the cubic phase. The current results provide a deeper understanding of the hydrophobic region during phase transition of lipid self-assembly which is important for better control during the process of membrane-protein crystallization.

Measuring the Ultrafast Correlation Dynamics between the Q x and Q y Bands in Chlorophyll Molecules.

We use two-dimensional electronic spectroscopy to measure the ultrafast correlation dynamics between the Q x and Q y transitions in chlorophyll molecules. We derive a variation to the center line slope method to quantify the frequency fluctuation cross-correlation function, C xy( Tw). Compared with the frequency fluctuation correlation function of the Q y transition, we observe that there is only a minimal correlation between the Q x and Q y transition, even at the ultrashort timescale of ∼100 fs, which then decays to zero in a time scale of ∼2 ps.

Vancomycin Determination by Disrupting Electron-Transfer in a Fluorescence Turn-On Squaraine-Anthraquinone Triad.

A highly sensitive and selective probe for Vancomycin (Van) in aqueous and serum samples is developed in this study. The probe is based on a triad consisting of a near-infrared squaraine dye (Seta-640) conjugated to two anthraquinone molecules via Lys-d-Ala-d-Ala peptides. In the absence of Van, the close proximity and efficient electron-transfer from the excited Seta-640 dye to anthraquinone result in significant fluorescence quenching of the dye ("off"-state). When Van is added, the antibiotic molecules bind with high affinity to the -d-Ala-d-Ala ligands in a 2:1 stoichiometry (Van:triad), resulting in fluorescence recovery that is as high as 30 times ("on"-state). Even though bound Van enhances the fluorescence by reducing the rate of (intrinsic) polarity-induced nonradiative decay process, this effect plays only a minor role. Instead, the main reason behind the observed fluorescence recovery after drug binding is the effective inhibition of electron-transfer; plausibly arising from a steric-induced lengthening of the spatial separation between electron donor and acceptor. The probe has detection limits of 7.0 and 96.9 nM in buffer and human serum, respectively, operates in the clinically relevant range, is insensitive to Van crystalline degradation product (CDP-1), and is easy to operate by using a commonly available fluorescence spectrometer.

Detecting local heterogeneity and ionization ability in the head group region of different lipidic phases using modified fluorescent probes.

Local heterogeneity in lipid self-assembly is important for executing the cellular membrane functions. In this work, we chemically modified 2-(2'-hydroxyphenyl)benzoxazole (HBO) and attached a C8 alkyl chain in two different locations to probe the microscopic environment of four lipidic phases of dodecyl ß-maltoside. The fluorescence change in HBO and the new probes (HBO-1 and HBO-2) shows that in all phases (micellar, hexagonal, cubic and lamellar) three HBO tautomeric species (solvated syn-enol, anionic, and closed syn-keto) are stable. The formation of multi tautomers reflects the heterogeneity of the lipidic phases. The results indicate that HBO and HBO-1 reside in a similar location within the head group region, whereas HBO-2 is slightly pushed away from the sugar-dominated area. The stability of the solvated syn-enol tautomer is due to the formation of a hydrogen bond between the OH group of the HBO moiety and an adjacent oxygen atom of a sugar unit. The detected HBO anions was proposed to be a consequence of this solvation effect where a hydrogen ion abstraction by the sugar units is enhanced. Our results point to a degree of local heterogeneity and ionization ability in the head group region as a consequence of the sugar amphoterism.