A peptide sequence—YSGVCHTDLHAWHGDWPLPVK [40–60]—in yeast alcohol dehydrogenase prevents the aggregation of denatured substrate proteins (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2002
a-Crystallin, the major eye lens protein and a member of the small heat-shock protein family, has been shown to protect the aggregation of several proteins and enzymes under denaturing conditions. The region(s) in the denaturing proteins that interact with a-crystallin during chaperone action has not been identified. Determination of these sites would explain the wide chaperoning action (promiscuity) of acrystallin. In the present study, using two different methods, we have identified a sequence in yeast alcohol dehydrogenase (ADH) that binds to a-crystallin during chaperone-like action. The first method involved the incubation of a-crystallin with ADH peptides at 48 jC for 1 h followed by separation and analysis of bound peptides. In the second method, a-crystallin was first derivatized with a photoactive trifunctional cross-linker, sulfosuccinimidyl-2[6-(biotinamido)-2-( p-azidobenzamido)-hexanoamido]ethyl-1,3di-thiopropionate (sulfo-SBED), and then complexed with ADH at 48 jC for 1 h in the dark. The complex was photolyzed and digested with protease, and the biotinylated peptide fragments were isolated using an avidin column and then analyzed. The amino acid sequencing and mass spectral analysis revealed the sequence YSGVCHTDLHAWHGDWPLPVK (yeast ADH 40 -60 ) as the a-crystallin binding site in ADH. The interaction was further confirmed by demonstrating complex formation between a-crystallin and a synthetic peptide representing the binding site of ADH. D
Mechanism of thermal aggregation of yeast alcohol dehydrogenase I
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2008
Kinetics of thermal aggregation of yeast alcohol dehydrogenase I (yADH) have been studied using dynamic light scattering at a fixed temperature (56°C) and under the conditions where the temperature was elevated at a constant rate (1 K/min). The initial parts of the dependences of the hydrodynamic radius on time (or temperature) follow the exponential law. At rather high values of time splitting of the population of aggregates into two components occurs. It is assumed that such peculiarities of the kinetics of thermal aggregation of yADH are due to the presence of a sequence -YSGVCHTDLHAWHGDWPLPVKin the polypeptide chain possessing chaperone-like activity. Thermodynamic parameters for thermal denaturation of yADH have been calculated from the differential scanning calorimetry data.
Enzyme and Microbial Technology, 2007
The present communication reports on changes in the secondary and tertiary structures of native and apo-yeast alcohol dehydrogenase upon heating at 50 • C, as evident from circular dichroism (CD) studies. The presence of sugars provided significant protection with trehalose being the most effective. Exposure of hydrophobic clusters in the protein molecule upon heat denaturation was confirmed by fluorescence studies using 1-anilinonaphthalene-8-sulfonate (ANS) as a hydrophobic reporter probe. All sugars, and especially trehalose, reduced the affinity of both forms of the enzyme for this probe. The effectiveness of sugars in diminishing ANS fluorescence enhancement is in accordance with their ability to protect aggregation of the proteins, reported earlier [Miroliaei M, Nemat-Gorgani M. Sugars protect native and apo yeast alcohol dehydrogenase against irreversible thermoinactivation. Enzyme Microb Technol 2001;29:554-9]. It is concluded that prevention of the mechanisms of irreversible thermoinactivation may occur with retention of the secondary and tertiary structural properties of the proteins.
European Journal of Biochemistry, 1995
The lack of crystal structure for tetrameric yeast alcohol dehydrogenases (ADHs) has precluded, until now, the identification of the residues involved in subunit contacts. In order to address this question, we have characterized the thermal stability and dissociation propensity of native ADH I and ADH I1 isozymes as well as of several chimeric (ADH I-ADH 11) enzymes. Three groups of substitutions affecting the thermostability have been identified among the 24 substitutions observed between isozymes I and 11. The first group contains a Cys277+Ser substitution, located at the interface between subunits in a threedimensional model of ADH I, based on the crystallographic structure of the dimeric horse liver ADH. In the second group, the Asp236-Asn substitution is located in the same interaction zone on the model. The stabilizing effect of this substitution can result from the removal of a charge repulsion between subunits. It is shown that the effect of these two groups of substitutions correlates with changes in dissociation propensities. The third group contains the Metl68+Arg substitution that increases the thermal stability, probably by the formation of an additional salt bridge between subunits through the putative interface. These data suggest that at least part of the subunit contacts observed in horse liver ADH are located at homologous positions in yeast ADHs.
Structure and function of yeast alcohol dehydrogenase
Journal of the Serbian Chemical Society, 2000
1. Introduction 2. Isoenzymes of YADH 3. Substrate specificity 4. Kinetic mechanism 5. Primary structure 6. The active site 7. Mutations in the yeast enzyme 8. Chemical mechanism 9. Binding of coenzymes 10. Hydride transfer This article has been corrected. Link to the correction 10.2298/JSC0008609E
Binding of coenzymes to yeast alcohol dehydrogenase
Journal of the Serbian Chemical Society, 2010
In this work, the binding of coenzymes to yeast alcohol dehydrogenase (EC 1.1.1.1) were investigated. The main criterions were the change in the standard free energies for individual reaction steps, the internal equilibrium constants and the overall changes in the reaction free energies. The calculations were performed for the wild type enzyme at pH 6-9 and for 15 different mutant type enzymes, with single or double point mutations, at pH 7.3. The abundance of theoretical and experimental data enabled the binding of coenzymes to enzyme to be assessed in depth.
Role of partial protein unfolding in alcohol-induced protein aggregation
Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of disease-related as well as nondisease-related proteins. Here we probed the biophysical mechanisms underlying alcohol-induced protein aggregation, in particular the role of partial protein unfolding in aggregation. We have studied aggregation mechanisms due to benzyl alcohol which is used in numerous biochemical and biotechnological applications. We chose cytochrome c as a model protein, for the reason that various optical and structural probes are available to monitor its global and partial unfolding reactions. Benzyl alcohol induced the aggregation of cytochrome c in isothermal conditions and decreased the temperature at which the protein aggregates. However, benzyl alcohol did not perturb the overall native conformation of cytochrome c. Instead, it caused partial unfolding of a local protein region around the methionine residue at position 80. Site-specific optical probes, two-dimensional NMR titrations, and hydrogen exchange all support this conclusion. The protein aggregation temperature varied linearly with the melting temperature of the Met80 region. Stabilizing the Met80 region by heme iron reduction drastically decreased protein aggregation, which confirmed that the local unfolding of this region causes protein aggregation. These results indicate that a possible mechanism by which alcohols induce protein aggregation is through partial rather than complete unfolding of native proteins.
International Journal of Biological Macromolecules, 2008
-Casein (-CN) showing properties of intrinsically unstructured proteins (IUP) displays many similarities with molecular chaperones and shows anti-aggregation activity in vitro. Chaperone activities of bovine and camel -CN were studied using alcohol dehydrogenase (ADH) as a substrate. To obtain an adequate relevant information about the chaperone capacities of studied caseins, three different physical parameters including chaperone constant (k c , M −1 ), thermal aggregation constant (k T , • C −1 ) and aggregation rate constant (k t , min −1 ) were measured. Bovine -CN displays greater chaperone activity than camel -CN. Fluorescence studies of 8-anilino-1-naphthalenesulfonic acid (ANS) binding demonstrated that bovine -CN is doted with larger effective hydrophobic surfaces at all studied temperatures than camel -CN. Greater relative hydrophobicity of bovine -CN than camel -CN may be a factor responsible for stronger interactions of bovine -CN with the aggregation-prone pre denatured molecular species of the substrate ADH, which resulted in greater chaperone activity of bovine -CN. (T. Haertlé). oligomers, refolding, targeting, and the degradation of defective proteins or their undesired forms . It has been proposed that molecular chaperones form several structurally unrelated protein families . Although no consensus sequence has been identified among different families of chaperones, some of their common features are good separation of hydrophilic (increasing solubility) and hydrophobic (binding hydrophobic substrates) domains (i.e. amphiphilicity), presence in aqueous media in form of large oligomers and sometimes in forms of micellelike-associated structures. High frequency of prolyl residues and absence of cystyl residues in their primary structures bestow more open and flexible structures to chaperones . For example ␣-crystallin, a small heat-shock protein of the eye lens is 0141-8130/$ -see front matter
Archives of biochemistry and biophysics, 2016
Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 Å where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 Å to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxyl...