Effects of Distal Pocket Mutations on the Geminate Recombination of NO with Leghemoglobin on the Picosecond Time Scale

The picosecond NO geminate rebinding kinetics of wild-type leghemoglobin, a monomeric plant hemoglobin with structural similarity to myoglobin, and six mutant proteins at the distal histidine (H61G, H61A, H61V, H61L, H61R, H61F) are investigated. All of the mutant proteins yield rebinding kinetics that are initially more rapid than that of the wild-type protein. At long times, the rebinding of H61F becomes slower than that of wild-type leghemoglobin. The H61V, H61L, and H61G mutant proteins give extraordinarily rapid and complete geminate rebinding. On a 40 ps time scale, distal effects are overwhelmingly evident for all of the mutants considered. That binding is both rapid and, in several cases, essentially single-exponential is suggestive of the nature of the barrier induced by the distal modification:  it must be such that the ligand is prohibited from reorienting with respect to, and diffusing sufficiently far from, the heme iron so that a distribution of return paths is not offered to it. Over the past 20 years, the relative importance attributed to the proximal and the distal sides in modulating geminate ligand binding has varied considerably. Our results with leghemoglobin are discussed in terms of the relative contributions of proximal and distal effects to geminate rebinding kinetics

A Comparative Femtosecond Coherence Study of the Unligated Monomeric Hemeproteins Myoglobin and Leghemoglobin

Impulsive optical excitation has been performed on wild type, unligated leghemoglobin for the first time to compare the induced vibrational coherence with that observed in myoglobin. Both proteins were excited at the Soret maxima and probed at red and blue edges of the Soret band. The resulting kinetic traces were modulated by low-frequency vibrations. Leghemoglobin shows a decrease in vibrational amplitude compared with myoglobin. The possible cause for the amplitude differences is discussed in terms of contributions from both ground- and excited-state vibrational coherences and ground-state heterogeneity

Fluorescence of Dietary Porphyrins as a Basis for Real-Time Detection of Fecal Contamination on Meat

Digestion of green plants in the gastrointestinal (GI) tract produces degradation products from chlorophyll that cause ingesta and feces to be highly fluorescent. This property was exploited for development and construction of instruments to noninvasively detect minute quantities of feces on meat samples in real time. The presence of feces on meat products is a primary source of foodborne pathogens, such as Escherichia coli O157:H7 and Salmonella. This new technology provides a rapid and accurate alternative to the practice of visual inspection and augments more time-consuming biological testing methods. This innovation can assist meat processors and government inspectors in their efforts to provide safe and wholesome food to consumers

Interaction of Glutathione S-Transferase with Hypericin: A Photophysical Study

The photophysics of hypericin have been studied in its complex with two different isoforms, A1-1 and P1-1, of the protein glutathione S-transferase (GST). One molecule of hypericin binds to each of the two GST subunits. Comparisons are made with our previous results for the hypericin/human serum albumin complex (Photochem. Photobiol. 1999, 69, 633−645). Hypericin binds with high affinity to the GSTs:  0.65 μM for the A1-1 isoform and 0.51 μM for the P1-1 isoform (Biochemistry 2004, 43, 12761−12769). The photophysics and activity of hypericin are strongly modulated by the binding protein. Intramolecular hydrogen-atom transfer is suppressed in both cases. Most importantly, while there is significant singlet oxygen generation from hypericin bound to GST A1-1, binding to GST P1-1 suppresses singlet oxygen generation to almost negligible levels. The data are rationalized in terms of a simple model in which the hypericin photophysics depends entirely upon the decay of the triplet state by two competing processes, quenching by oxygen to yield singlet oxygen and ionization, the latter of these two are proposed to be modulated by A1-1 and P1-1

Dynamic Solvation in Room-Temperature Ionic Liquids

The dynamic solvation of the fluorescent probe, coumarin 153, is measured in five room-temperature ionic liquids using different experimental techniques and methods of data analysis. With time-resolved stimulated-emission and time-correlated single-photon counting techniques, it is found that the solvation is comprised of an initial rapid component of ∼55 ps. In all the solvents, half or more of the solvation is completed within 100 ps. The remainder of the solvation occurs on a much longer time scale. The emission spectra of coumarin 153 are nearly superimposable at all temperatures in a given solvent unless they are obtained using the supercooled liquid, suggesting that the solvents have an essentially glassy nature. The physical origin of the two components is discussed in terms of the polarizability of the organic cation for the faster one and the relative diffusional motion of the cations and the anions for the slower one. A comparison of the solvation response functions obtained from single-wavelength and from spectral-reconstruction measurements is provided. Preliminary fluorescence-upconversion measurements are presented against which the appropriateness of the single-wavelength method for constructing solvation correlation functions and the use of stimulated-emission measurements is considered. These measurements are consistent with the trends mentioned above, but a comparison indicates that the presence of one or more excited states distorts the stimulated-emission kinetics such that they do not perfectly reproduce the spontaneous emission data. Fluorescence-upconversion results indicate an initial solvation component on the order of ∼7 ps

Fluorescence Correlation Spectroscopic Study of Serpin Depolymerization by Computationally Designed Peptides

Members of the serine proteinase inhibitor (serpin) family play important roles in the inflammatory and coagulation cascades. Interaction of a serpin with its target proteinase induces a large conformational change, resulting in insertion of its reactive center loop (RCL) into the main body of the protein as a new strand within beta-sheet A. Intermolecular insertion of the RCL of one serpin molecule into the beta-sheet A of another leads to polymerization, a widespread phenomenon associated with a general class of diseases known as serpinopathies. Small peptides are known to modulate the polymerization process by binding within beta-sheet A. Here, we use fluorescence correlation spectroscopy (FCS) to probe the mechanism of peptide modulation of alpha(1)-antitrypsin (alpha(1)-AT) polymerization and depolymerization, and employ a statistical computationally-assisted design strategy (SCADS) to identify new tetrapeptides that modulate polymerization. Our results demonstrate that peptide-induced depolymerization takes place via a heterogeneous, multi-step process that begins with internal fragmentation of the polymer chain. One of the designed tetrapeptides is the most potent antitrypsin depolymerizer yet found

Picosecond time resolved spectroscopy used as a tool to probe excited state photophysics of biologically and environmentally relevant systems

This dissertation focuses on the applicability of a variety of time-resolved spectroscopic techniques to probe the excited state photophysics of a range of biologically and environmentally relevant systems.;Hypericin, a biologically active perylene quinone and an efficient photosensitizing agent has a wide spectrum of light induced biological activities. However, it is highly insoluble in water. Insertion of hypericin in the well-characterized AOT reverse micelles gives us the opportunity to study its excited-state photophysics as a function of varying pH. Hypomycin B, another biologically active perylene quinone is unique in the sense that it has only one hydroxyl group peri to a carbonyl group as opposed to four in hypericin. Transient absorption data failed to reveal any stimulated emission for hypomycin B. This result has been discussed in the context of the number of H-atoms that may be transferred in the excited state for hypericin and whether the transfer is stepwise or concerted.;Geminate recombination of ligands in heme proteins is mainly affected either by the proximal pocket effects or by mutations on the distal side of the heme pocket. Our transient absorption kinetics of different proximal mutants of both sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) reveal very little influence of the proximal pocket on the geminate recombination of NO. However, the first ever study of distal pocket mutations in Lba, show profound effects of the distal amino acid(s) on NO recombination on the geminate timescale.;Solvation dynamics of five different room temperature ionic liquids using coumarin 153 as the fluorescent probe reveal an initial phase of rapid solvation due to the polarizability of the cationic part of the ionic liquids. The amplitude of the rapidly decaying components correlates well with the viscosity of the ionic liquids. Finally, the complex of coumarin 153 with apomyoglobin has been exhaustively characterized. This is the first step towards analyzing the contribution of the individual amino acid residues in the heme pocket towards the total protein dielectric response.</p

Heterogeneous And Anomalous Diffusion Inside Lipid Tubules

Self-assembled lipid tubules with crystalline bilayer walls are promising candidates for controlled drug delivery vehicles on the basis of their ability to release preloaded biological molecules in a sustained manner. While a previous study has shown that the release rate of protein molecules from lipid tubules depends on the associated molecular mass, suggesting that the pertinent diffusion follows the well-known Stokes - Einstein relationship, only a few attempts have been made toward investigating the details of molecular diffusion in the tubule interior. Herein, we have characterized the diffusion rates of several molecules encapsulated in lipid tubules formed by 1,2-bis(10,12- tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) using the techniques of fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). Our results show that the mobility of these molecules depends not only on their positions in the DC8,9PC tubules but also on their respective concentrations. While the former indicates that the interior of the DC8,9PC tubules is heterogeneous in terms of diffusion, the latter further highlights the possibility of engineering specific conditions for achieving sustained release of a drug molecule over a targeted period of time. In addition, our FCS results indicate that the molecular diffusions inside the crystalline bilayer walls of the DC 8,9PC tubules strongly deviate from the normal, stochastic processes, with features characterizing not only anomalous subdiffusions but also motions that are superdiffusive in nature. © 2007 American Chemical Society

Crowding-Induced Quenching of Intrinsic Tryptophans of Serum Albumins: A Residue-Level Investigation of Different Conformations

Macromolecular crowding has been known to influence the global conformational landscape of proteins in various ways. However, only a few studies have focused their attention on the local perturbations that can occur in the presence of crowding agents. Here we have used the sensitivity of the tryptophan (Trp) fluorescence of two homologous serum albumins (BSA and HSA) to its (Trp) surroundings to monitor the local changes in the immediate proximity of the intrinsic fluorophore. Using the tool of fluorescence quenching we have shown that the commonly used synthetic macromolecular crowders (e.g., Dextran 6, Dextran 40, Ficoll 70, and PEG 8000) can bring about dramatic conformational modulations in the two proteins. Moreover, the nature of perturbation was observed to be largely dependent on the specific crowding agent used, with Dextran 6 showing the maximum effect while PEG 8000 showed the least. Additionally the extent of local structure modulation was found to be the largest either in the native state of the proteins or under near-native conditions, signifying the important role that the surrounding amino acids play in determining the fluorescence of the Trp residues. Also, surprisingly, although BSA and HSA show ∼76% sequence homology and have almost identical structural disposition in the native state, their individual responses to the crowder-induced perturbation were found to be quite different