Deoxycholate induced tetramer of αA-crystallin and sites of phosphorylation: Fluorescence correlation spectroscopy and femtosecond solvation dynamics
16 Apr 2012-Journal of Chemical Physics (American Institute of Physics)-Vol. 136, Iss: 15, pp 155101-155101
TL;DR: The steady state emission maximum and average solvation time of acrylodan labeled at cysteine 131 position of αA-crystallin is markedly affected on addition of NaDC, while the tryptophan (trp-9) becomes more exposed, which suggests that NaDC binds near the cys-131 and makes the terminal region ofαA- Crystallin exposed.
Abstract: Structure and dynamics of acrylodan labeled αA-crystallin tetramer formed in the presence of a bile salt (sodium deoxycholate, NaDC) has been studied using fluorescence correlation spectroscopy (FCS) and femtosecond up-conversion techniques. Using FCS it is shown that, the diffusion constant (Dt) of the αA-crystallin oligomer (mass ∼800 kDa) increases from ∼35 μm2 s−1 to ∼68 μm2 s−1. This corresponds to a decrease in hydrodynamic radius (rh) from ∼6.9 nm to ∼3.3 nm. This corresponds to about 10-fold decrease in molecular mass to ∼80 kDa and suggests formation of a tetramer (since mass of αA-crystallin monomer is ∼20 kDa). The steady state emission maximum and average solvation time (〈τs〉) of acrylodan labeled at cysteine 131 position of αA-crystallin is markedly affected on addition of NaDC, while the tryptophan (trp-9) becomes more exposed. This suggests that NaDC binds near the cys-131 and makes the terminal region of αA-crystallin exposed. This may explain the enhanced auto-phosphorylation activity of αA-crystallin near the terminus of the 173 amino acid protein (e.g., at the threonine 13, serine 45, or serine 169 and 172) and suggests that phosphorylation at ser-122 (close to cys-131) is relatively less important.
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TL;DR: The observation indicates that after intercalation inside the hydrophobic pocket, neutral ellipticine molecules experience similar confinement in all the bile salts.
Abstract: The entrapment of neutral and cationic species of an anticancer drug, namely ellipticine and their dynamic features in different bile salt aggregates have been investigated for the first time using steady state and time-resolved fluorescence spectroscopy. Because ellipticine exists in various prototropic forms under physiological conditions, we performed comparative photophysical and dynamical studies on these prototropic species in different bile salts varying in their head groups and hydrophobic skeletons. We found that the initial interaction between ellipticine and bile salts is governed by the electrostatic forces where cationic ellipticine is anchored to the head groups of bile salts. Bile salts having conjugated head groups are better candidates to bind with the cationic species than those having the non-conjugated ones. The fact implies that binding of cationic species to different bile salts depends on the pKa of the corresponding bile acids. The hydrophobic interaction dominates at higher concentrations of bile salts due to formation of aggregates and results in entrapment of neutral ellipticine molecules according to their hydrophobicity indices. Thus bile salts act as multisite drug carriers. The rotational relaxation parameters of cationic ellipticine were found to be dependent on head groups and the number of hydroxyl groups on the hydrophilic surface of bile salts. Cationic ellipticine exhibits a faster rotational relaxation in the tri-hydroxy bile salt aggregates than in di-hydroxy bile salts. We interpreted this observation from the fact that tri-hydroxy bile salts hold a higher number of water molecules in their hydrophilic surface offering a less viscous environment for ellipticine compared to di-hydroxy bile salts. Surprisingly, the neutral ellipticine molecules display almost the same rotational relaxation in all the bile salts. The observation indicates that after intercalation inside the hydrophobic pocket, neutral ellipticine molecules experience similar confinement in all the bile salts.
11 citations
TL;DR: This article presents a review of work done with respect to the interaction of ATP, peptide generated from lens crystallin and other proteins and some bivalent metal ions with α-crystallin and discusses the role of these interactions on its structure and function and cataract formation.
9 citations
TL;DR: The data clearly reveals that structural breakdown of α‐crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation.
Abstract: α-Crystallin is a multimeric eye lens protein having molecular chaperone-like function which is crucial for lens transparency. The stability and unfolding-refolding properties of α-crystallin plays important roles for its function. We undertook a multi probe based fluorescence spectroscopic approach to explore the changes in the various levels of organization of this protein at different urea concentration. Steady state fluorescence studies reveal that at 0.6M urea a compact structural intermediate is formed which has a native-like secondary structure with enhanced surface exposure of hydrophobic groups. At 2.8M urea the tertiary interactions are largely collapsed with partial collapse of secondary and quaternary structure. The surface solvation probed by picosecond time resolved fluorescence of acrylodan labeled α-crystallin revealed dry native-like core of α-crystallin at 0.6M urea compared to enhanced water penetration at 2.8M urea and extensive solvation at 6M urea. Activation energy for the subunit exchange decreased by 22 kJ mol−1 on changing urea concentration from 0 to 0.6M compared with over 75 kJ mol−1 on changing urea concentration from 0 to 2.8M. Light scattering and analytical ultracentrifugation techniques were used to determine size and oligomerization of the unfolding intermediates. The data indicated swelling but no oligomer breakdown at 0.6M urea. At 2.8M urea the oligomeric size is considerably reduced and a monomer is produced at 6M urea. The data clearly reveals that structural breakdown of α-crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 549–560, 2014.
7 citations
TL;DR: The most interesting observation of the present work is that the viscosities of different RTIL-cosolvent mixtures can efficiently control the cis-trans isomerization kinetics of the anionic fluorophore merocyanine 540 and the translational diffusion of a hydrophobic probe.
Abstract: Literature reports provide ample evidence of the dynamical studies of various fluorophores in different room-temperature ionic liquid (RTIL)–cosolvent mixtures. However, most of the experimental and simulation studies reveal that ∼50% of the spectral relaxation dynamics is fast and cannot be resolved using traditional time correlated single photon counting (TCSPC) measurements. Our group has also investigated the dynamics of a solvatochromic probe coumarin 153 (C153) in a RTIL–cosolvent mixture using a TCSPC setup (S. Sarkar, R. Pramanik, C. Ghatak, P. Setua and N. Sarkar, J. Phys. Chem. B, 2010, 114, 2779–2789). Consequently, a major portion of the solvation dynamics remained undetected and moreover we could not monitor the dynamics beyond 0.4 mole fraction of the cosolvents. Thus in this study, we have rekindled our interest to sufficiently capture the rotational anisotropy and solvation dynamics of C153 beyond 0.4 mole fraction of the cosolvents in the RTIL–cosolvent mixture employing femtosecond fluorescence upconversion measurements. Additionally, we have utilized another RTIL with a higher alkyl chain length and viscosity to obtain a comprehensive and quantitative picture of the role of viscosity on the dynamics of the probe molecule. The most interesting observation of the present work is that the viscosities of different RTIL–cosolvent mixtures can efficiently control the cis–trans isomerization kinetics of the anionic fluorophore merocyanine 540 (MC 540) and the translational diffusion of a hydrophobic probe. The optimization of geometrical structures of [EmimOs]– and [EmimOs]–cosolvent mixtures followed by frequency analyses in both gas and solution phases have been carried out using quantum chemical calculations with the aid of density functional theory (DFT) methods. The computation based on the bond distances, electron densities and non-covalent interactions (NCI) has also been used to investigate the existence of the hydrogen-bond (H-bond). Again to comprehend van der Waals interactions and the conventional hydrogen-bond, the evolution of NCI plots are simulated. Therefore, the detailed experimental and theoretical studies presented in this manuscript lead to the inference that addition of the conventional solvents finely tunes the physicochemical properties of RTILs and broadens their scope of applications in the fields of chemistry and biology.
4 citations
24 Sep 2016
References
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Book•
01 Jan 1983
TL;DR: This book describes the fundamental aspects of fluorescence, the biochemical applications of this methodology, and the instrumentation used in fluorescence spectroscopy.
Abstract: Fluorescence methods are being used increasingly in biochemical, medical, and chemical research. This is because of the inherent sensitivity of this technique. and the favorable time scale of the phenomenon of fluorescence. 8 Fluorescence emission occurs about 10- sec (10 nsec) after light absorp tion. During this period of time a wide range of molecular processes can occur, and these can effect the spectral characteristics of the fluorescent compound. This combination of sensitivity and a favorable time scale allows fluorescence methods to be generally useful for studies of proteins and membranes and their interactions with other macromolecules. This book describes the fundamental aspects of fluorescence. and the biochemical applications of this methodology. Each chapter starts with the -theoreticalbasis of each phenomenon of fluorescence, followed by examples which illustrate the use of the phenomenon in the study of biochemical problems. The book contains numerous figures. It is felt that such graphical presentations contribute to pleasurable reading and increased understand ing. Separate chapters are devoted to fluorescence polarization, lifetimes, quenching, energy transfer, solvent effects, and excited state reactions. To enhance the usefulness of this work as a textbook, problems are included which illustrate the concepts described in each chapter. Furthermore, a separate chapter is devoted to the instrumentation used in fluorescence spectroscopy. This chapter will be especially valuable for those perform ing or contemplating fluorescence measurements. Such measurements are easily compromised by failure to consider a number of simple principles."
28,073 citations
TL;DR: The iterative threading assembly refinement (I-TASSER) server is an integrated platform for automated protein structure and function prediction based on the sequence- to-structure-to-function paradigm.
Abstract: The iterative threading assembly refinement (I-TASSER) server is an integrated platform for automated protein structure and function prediction based on the sequence-to-structure-to-function paradigm. Starting from an amino acid sequence, I-TASSER first generates three-dimensional (3D) atomic models from multiple threading alignments and iterative structural assembly simulations. The function of the protein is then inferred by structurally matching the 3D models with other known proteins. The output from a typical server run contains full-length secondary and tertiary structure predictions, and functional annotations on ligand-binding sites, Enzyme Commission numbers and Gene Ontology terms. An estimate of accuracy of the predictions is provided based on the confidence score of the modeling. This protocol provides new insights and guidelines for designing of online server systems for the state-of-the-art protein structure and function predictions. The server is available at http://zhanglab.ccmb.med.umich.edu/I-TASSER.
5,792 citations
TL;DR: The I-TASSER server has been developed to generate automated full-length 3D protein structural predictions where the benchmarked scoring system helps users to obtain quantitative assessments of the I- TASSER models.
Abstract: Prediction of 3-dimensional protein structures from amino acid sequences represents one of the most important problems in computational structural biology. The community-wide Critical Assessment of Structure Prediction (CASP) experiments have been designed to obtain an objective assessment of the state-of-the-art of the field, where I-TASSER was ranked as the best method in the server section of the recent 7th CASP experiment. Our laboratory has since then received numerous requests about the public availability of the I-TASSER algorithm and the usage of the I-TASSER predictions. An on-line version of I-TASSER is developed at the KU Center for Bioinformatics which has generated protein structure predictions for thousands of modeling requests from more than 35 countries. A scoring function (C-score) based on the relative clustering structural density and the consensus significance score of multiple threading templates is introduced to estimate the accuracy of the I-TASSER predictions. A large-scale benchmark test demonstrates a strong correlation between the C-score and the TM-score (a structural similarity measurement with values in [0, 1]) of the first models with a correlation coefficient of 0.91. Using a C-score cutoff > -1.5 for the models of correct topology, both false positive and false negative rates are below 0.1. Combining C-score and protein length, the accuracy of the I-TASSER models can be predicted with an average error of 0.08 for TM-score and 2 A for RMSD. The I-TASSER server has been developed to generate automated full-length 3D protein structural predictions where the benchmarked scoring system helps users to obtain quantitative assessments of the I-TASSER models. The output of the I-TASSER server for each query includes up to five full-length models, the confidence score, the estimated TM-score and RMSD, and the standard deviation of the estimations. The I-TASSER server is freely available to the academic community at http://zhang.bioinformatics.ku.edu/I-TASSER
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4,754 citations
TL;DR: It is shown that alpha-crystallin refracts light and protects proteins from aggregation in the transparent eye lens and that in nonlens cells alpha-Crystallin may have other functions in addition to its capacity to suppress aggregation of proteins.
Abstract: The alpha-crystallins (alpha A and alpha B) are major lens structural proteins of the vertebrate eye that are related to the small heat shock protein family. In addition, crystallins (especially alpha B) are found in many cells and organs outside the lens, and alpha B is overexpressed in several neurological disorders and in cell lines under stress conditions. Here I show that alpha-crystallin can function as a molecular chaperone. Stoichiometric amounts of alpha A and alpha B suppress thermally induced aggregation of various enzymes. In particular, alpha-crystallin is very efficient in suppressing the thermally induced aggregation of beta- and gamma-crystallins, the two other major mammalian structural lens proteins. alpha-Crystallin was also effective in preventing aggregation and in refolding guanidine hydrochloride-denatured gamma-crystallin, as judged by circular dichroism spectroscopy. My results thus indicate that alpha-crystallin refracts light and protects proteins from aggregation in the transparent eye lens and that in nonlens cells alpha-crystallin may have other functions in addition to its capacity to suppress aggregation of proteins.
1,822 citations
TL;DR: In this paper, a probe molecule coumarin 153 (Cu153) and picosecond spectroscopic techniques were used to examine the solvation dynamics in polar liquids and showed that the frequency of the electronic spectrum of this probe provides a convenient measure of solvation energetics.
Abstract: Solvation dynamics in polar liquids have been examined using the probe molecule coumarin 153 (Cu153) and picosecond spectroscopic techniques. Steady‐state absorption and fluorescence spectra of Cu153 as a function of solvent show that the frequency of the electronic spectrum of this probe provides a convenient measure of solvation energetics. Both nonspecific dipolar and to a smaller degree H‐bonding solute–solvent interactions are involved. Time‐correlated single photon counting was used to observe time‐dependent shifts of the fluorescence spectrum of Cu153 in a variety of alcohols, propylene carbonate, and N‐methylpropionamide solvents as a function of temperature. These time‐dependent spectral shifts provide a direct measure of the time dependence of the solvation process. Theoretical models that treat the solvent as a dielectric continuum do not adequately account for the observed solvation dynamics. In the solvents studied, such theories predict a single exponential shift of the fluorescence spectrum...
1,127 citations