The mode of chaperoning of dithiothreitol-denatured alpha-lactalbumin by alpha-crystallin.

Molecular chaperones prevent the aggregation of partially folded or misfolded forms of protein. alpha-crystallin performs such a function in the ocular lens. To gain insight into the mechanism of the anti-aggregation activity of alpha-crystallin, we performed dynamic light scattering (DLS) measurements investigating its interaction with partially denatured alpha-lactalbumin over a 24 hr period. Analyses were conducted as a function of the concentration of alpha-lactalbumin as well as the bovine alpha-crystallin/alpha-lactalbumin ratio. Additional studies of the systems were performed by HPLC and SDS gel electrophoresis. The particle distribution patterns derived from the DLS data indicated that the chaperoned complex (lactalbumin plus crystallin) is a loose fluffy globular entity. After the complex becomes saturated with lactalbumin, it appears to release the partially denatured lactalbumin which may aggregate into high molecular weight moieties. These eventually may precipitate out of solution. On longer standing, 24hr and over, the chaperoned complex as well as the lactalbumin aggregates become more compact. The chaperoned complex (alpha-crystallin plus alpha-lactalbumin) is in dynamic equilibrium both with the monomeric and the aggregated alpha-lactalbumin population.     

On the equilibrium between monomeric alpha-lactalbumin and the chaperoning complex of alpha-crystallin

In chaperoning dithiothreitol-denatured alpha-lactabumin, alpha-crystallin forms a chaperoning complex. In order to study the kinetics of such chaperoning it needs to be established whether the formation of the chaperoning complex is a reversible or irreversible process. The chaperoning reaction was studied by dynamic light scattering as a function of concentration and weight ratio of alpha-lactalbumin/alpha-crystallin. HPLC and subsequent SDS-PAGE gel electrophoresis experiments established that the chaperoning complex formed contains both alpha-crystallin and alpha-lactalbumin. Upon rechromatographing the chaperoning complex, the presence of monomeric alpha-lactalbumin has been demonstrated in addition to the chaperoning complex itself. This and equilibrium dialysis experiments demonstrated conclusively the existence of an equilibrium between monomeric partially denatured alpha-lactalbumin and the chaperoning complex made of alpha-lactalbumin and alpha-crystallin.     

The selective inhibition of serpin aggregation by the molecular chaperone, alpha-crystallin, indicates a nucleation-dependent specificity

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that prevent the misfolding and aggregation of proteins. However, specific details about their substrate specificity and mechanism of chaperone action are lacking. alpha1-Antichymotrypsin (ACT) and alpha1-antitrypsin (alpha1-AT) are two closely related members of the serpin superfamily that aggregate through nucleation-dependent and nucleation-independent pathways, respectively. The sHsp alpha-crystallin was unable to prevent the nucleation-independent aggregation of alpha1-AT, whereas alpha-crystallin inhibited ACT aggregation in a dose-dependent manner. This selective inhibition of ACT aggregation coincided with the formation of a stable high molecular weight alpha-crystallin-ACT complex with a stoichiometry of 1 on a molar subunit basis. The kinetics of this interaction occur at the same rate as the loss of ACT monomer, suggesting that the monomeric species is bound by the chaperone. 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (Bis-ANS) binding and far-UV circular dichroism data suggest that alpha-crystallin interacts specifically with a non-native conformation of ACT. The finding that alpha-crystallin does not interact with alpha1-AT under these conditions suggests that alpha-crystallin displays a specificity for proteins that aggregate through a nucleation-dependent pathway, implying that the dynamic nature of both the chaperone and its substrate protein is a crucial factor in the chaperone action of alpha-crystallin and other sHsps.     

Lactose synthase: effect of alpha-lactalbumin on substrate activity of N-acylglucosamines

N-Acetyl-, N-propionyl-, N-butyryl- and N-valerylglucosamines were synthesized as topographical probes to localize further the interaction site of alpha-lactalbumin on galactosyltransferase. All these compounds were found to be substrates for galactosyltransferase with Km values in the millimolar range. In the presence of alpha-lactalbumin, the Michaelis-Menten constants were diminished. However, the effect on the initial rates of these reactions varied. Thus, at low N-acylglucosamine concentrations, alpha-lactalbumin activated the enzyme activity, but at high concentrations, alpha-lactalbumin became inhibitory. This mixed-type inhibition kinetics indicated that a quaternary complex between galactosyltransferase, alpha-lactalbumin, Mn2+-UDPgalactose and N-acylglucosamine existed during the catalytic process. The ability of these N-acylglucosamine substrates to bind to lactose synthase complex was further substantiated by the physical association of galactosyltransferase onto the solid-bound alpha-lactalbumin in the presence of any one of these compounds. The data revealed that the presence of the N-acyl group up to five carbons in length did not interfere with the interaction between alpha-lactalbumin and galactosyltransferase, suggesting that alpha-lactalbumin was not bound in the vicinity of the C-2 region of the monosaccharide site. The inhibitory effect of alpha-lactalbumin on N-acyllactosamine formation is probably a consequence of conformational changes of galactosyltransferase.     

The molecular chaperone, alpha-crystallin, inhibits amyloid formation by apolipoprotein C-II.

Under lipid-free conditions, human apolipoprotein C-II (apoC-II) exists in an unfolded conformation that over several days forms amyloid ribbons. We examined the influence of the molecular chaperone, alpha-crystallin, on amyloid formation by apoC-II. Time-dependent changes in apoC-II turbidity (at 0.3 mg/ml) were suppressed potently by substoichiometric subunit concentrations of alpha-crystallin (1-10 microg/ml). alpha-Crystallin also inhibits time-dependent changes in the CD spectra, thioflavin T binding, and sedimentation coefficient of apoC-II. This contrasts with stoichiometric concentrations of alpha-crystallin required to suppress the amorphous aggregation of stressed proteins such as reduced alpha-lactalbumin. Two pieces of evidence suggest that alpha-crystallin directly interacts with amyloidogenic intermediates. First, sedimentation equilibrium and velocity experiments exclude high affinity interactions between alpha-crystallin and unstructured monomeric apoC-II. Second, the addition of alpha-crystallin does not lead to the accumulation of intermediate sized apoC-II species between monomer and large aggregates as indicated by gel filtration and sedimentation velocity experiments, suggesting that alpha-crystallin does not inhibit the relatively rapid fibril elongation upon nucleation. We propose that alpha-crystallin interacts stoichiometrically with partly structured amyloidogenic precursors, inhibiting amyloid formation at nucleation rather than the elongation phase. In doing so, alpha-crystallin forms transient complexes with apoC-II, in contrast to its chaperone behavior with stressed proteins.     

Kinetic and structural characterization of adsorption-induced unfolding of bovine alpha -lactalbumin.

Conformational changes of bovine alpha-lactalbumin induced by adsorption on a hydrophobic interface are studied by fluorescence and circular dichroism spectroscopy. Adsorption of bovine alpha-lactalbumin on hydrophobic polystyrene nanospheres induces a non-native state of the protein, which is characterized by preserved secondary structure, lost tertiary structure, and release of calcium. This partially denatured state therefore resembles a molten globule state, which is an intermediate in the folding of bovine alpha-lactalbumin. Stopped-flow fluorescence spectroscopy reveals two kinetic phases during adsorption with rate constants k(1) approximately 50 s(-1) and k(2) approximately 8 s(-1). The rate of partial unfolding is remarkably fast and even faster than unfolding induced by the addition of 5.4 m guanidinium hydrochloride to native alpha-lactalbumin. The large unfolding rates exclude the possibility that unfolding of bovine alpha-lactalbumin to the intermediate state occurs before adsorption takes place. Stopped-flow fluorescence anisotropy experiments show that adsorption of bovine alpha-lactalbumin on polystyrene nanospheres occurs within the dead time (15 ms) of the experiment. This shows that the kinetic processes as determined by stopped-flow fluorescence spectroscopy are not affected by diffusion or association processes but are solely caused by unfolding of bovine alpha-lactalbumin induced by adsorption on the polystyrene surface. A scheme is presented that incorporates the results obtained and describes the adsorption of bovine alpha-lactalbumin.     

Identification of a region in alcohol dehydrogenase that binds to alpha-crystallin during chaperone action

alpha-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 alpha-crystallin during chaperone action has not been identified. Determination of these sites would explain the wide chaperoning action (promiscuity) of alpha-crystallin. In the present study, using two different methods, we have identified a sequence in yeast alcohol dehydrogenase (ADH) that binds to alpha-crystallin during chaperone-like action. The first method involved the incubation of alpha-crystallin with ADH peptides at 48 degrees C for 1 h followed by separation and analysis of bound peptides. In the second method, alpha-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 degrees C 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 alpha-crystallin binding site in ADH. The interaction was further confirmed by demonstrating complex formation between alpha-crystallin and a synthetic peptide representing the binding site of ADH.     

Effects of a helix substitution on the folding mechanism of bovine alpha-lactalbumin

The structure, stability, and unfolding-refolding kinetics of a chimeric protein, in which the amino acid sequence of the flexible loop region (residues 105-110) comes from equine lysozyme and the remainder of the sequence comes from bovine alpha-lactalbumin were studied by circular dichroism spectroscopy and stopped-flow measurements, and the results were compared with those of bovine alpha-lactalbumin. The substitution of the flexible loop in bovine alpha-lactalbumin with the helix D of equine lysozyme destabilizes the molten globule state, although the native state is significantly stabilized by substitution of the flexible loop region. The kinetic refolding and unfolding experiments showed that the chimeric protein refolds significantly faster and unfolds substantially slower than bovine alpha-lactalbumin. To characterize the transition state between the molten globule and the native states, we investigated the guanidine hydrochloride concentration dependence of the rate constants of refolding and unfolding. Despite the significant differences in the stabilities of both the molten globule and native states between the chimeric protein and bovine alpha-lactalbumin, the free energy level of the transition state is not affected by the amino acid substitution in the flexible loop region. Our results suggest that the destabilization in the molten globule state of the chimeric protein is caused by the disruption of the non-native interaction in the flexible loop region and that the disruption of the non-native interaction reduces the free energy barrier of refolding. We conclude that the non-native interaction in the molten globule state may act as a kinetic trap for the folding of alpha-lactalbumin.     

Fitting yeast and mammalian prion aggregation kinetic data with the Finke-Watzky two-step model of nucleation and autocatalytic growth

Recently, we reported 14 amyloid protein aggregation kinetic data sets that were fit using the "Ockham's razor"/minimalistic Finke-Watzky (F-W) two-step model of slow nucleation (A --> B, rate constant k 1) and fast autocatalytic growth (A + B --> 2B, rate constant k 2), yielding quantitative (average) rate constants for nucleation ( k 1) and growth ( k 2), where A is the monomeric protein and B is the polymeric protein [Morris, A. M., et al. (2008) Biochemistry 47, 2413-2427]. Herein, we apply the F-W model to 27 representative prion aggregation kinetic data sets obtained from the literature. Each prion data set was successfully fit with the F-W model, including three different yeast prion proteins (Sup35p, Ure2p, and Rnq1p) as well as mouse and human prions. These fits yield the first quantitative rate constants for the steps of nucleation and growth in prion aggregation. Examination of a Sup35p system shows that the same rate constants are obtained for nucleation and for growth within experimental error, regardless of which of six physical methods was used, a unique set of important control experiments in the protein aggregation literature. Also provided herein are analyses of several factors influencing the aggregation of prions such as glutamine/asparagine rich regions and the number of oligopeptide repeats in the prion domain. Where possible, verification or refutation of previous correlations to glutamine/asparagine regions, or the number of repeat sequences, in literature aggregation kinetics is given in light of the quantitative rate constants obtained herein for nucleation and growth during prion aggregation. The F-W model is then contrasted to four literature mechanisms that address the molecular picture of prion transmission and propagation. Key limitations of the F-W model are listed to prevent overinterpretation of the data being analyzed, limitations that derive ultimately from the model's simplicity. Finally, possible avenues of future research are suggested.     

Alpha-crystallin binds to the aggregation-prone molten-globule state of alkaline protease: implications for preventing irreversible thermal denaturation

Alpha-crystallin, the major eye-lens protein with sequence homology with heat-shock proteins (HSPs), acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain more insight into its chaperoning ability, we used a protease as the model system that is known to require a propeptide (intramolecular chaperone) for its proper folding. The protease ("N" state) from Conidiobolus macrosporus (NCIM 1298) unfolds at pH 2.0 ("U" state) through a partially unfolded "I" state at pH 3.5 that undergoes transition to a molten globule-(MG) like "I(A)" state in the presence of 0.5 M sodium sulfate. The thermally-stressed I(A) state showed complete loss of structure and was prone to aggregation. Alpha-crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The alpha-crystallin-bound I(A) state exhibited native-like secondary and tertiary structure showing the interaction of alpha-crystallin with the MG state of the protease. 8-Anilinonaphthalene sulphonate (ANS) binding studies revealed the involvement of hydrophobic interactions in the formation of the complex of alpha-crystallin and protease. Refolding of acid-denatured protease by dilution to pH 7.5 resulted in aggregation of the protein. Unfolding of the protease in the presence of alpha-crystallin and its subsequent refolding resulted in the generation of a near-native intermediate with partial secondary and tertiary structure. Our studies represent the first report of involvement of a molecular chaperone-like alpha-crystallin in the unfolding and refolding of a protease. Alpha-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of the alkaline protease and keeps it in a near-native, folding-competent intermediate state.     

Conversion of human alpha-lactalbumin to an apo-like state in the complexes with basic poly-amino acids: toward understanding of the molecular mechanism of antitumor action of HAMLET

It was recently shown that alpha-lactalbumin associated with oleic acid (HAMLET) interacts with core histones thereby triggering apoptosis of tumor cells (J. Biol. Chem. 2003, 278, 42131). In previous work, we revealed that monomeric alpha-lactalbumin in the absence of fatty acids can also interact with histones and, moreover, with basic poly-amino acids (poly-Lys and poly-Arg) that represent simple models of histone proteins (Biochemistry 2004, 43, 5575). Association of alpha-lactalbumin with histone or poly-Lys(Arg) essentially changes its properties. In the present work, the character of the changes in structural properties and conformational stability of alpha-lactalbumin in the complex with poly-Lys(Arg) has been studied in detail by steady-state fluorescence, circular dichroism, and differential scanning calorimetry. Complex formation strongly depends on ionic strength, confirming its electrostatic nature. Experiments with the poly-amino acids of various molecular masses demonstrated a direct proportionality between the number of alpha-lactalbumin molecules bound per poly-Lys(Arg) and the surface area of the poly-amino acid random coil. The binding of the poly-amino acids to Ca2+-saturated human alpha-lactalbumin decreases its thermal stability down to the level of its free apo-form and decreases Ca2+-affinity by 4 orders of magnitude. The conformational state of alpha-lactalbumin in a complex with poly-Lys(Arg), named alpha-LActalbumin Modified by Poly-Amino acid (LAMPA), differs from all other alpha-lactalbumin states characterized to date, representing an apo-like (molten globule-like) state with substantially decreased affinity for calcium ion. The requirement for efficient conversion of alpha-lactalbumin to the LAMPA state is a poly-Lys(Arg) chain consisting of several tens of amino acid residues.     

The effect of pH on the kinetics of iron release from diferric ovotransferrin induced by pyrophosphate

Pyrophosphate-induced iron release from diferric ovotransferrin follows biphasic kinetics in the pH range from 6.6 to 8.6 except at pH 8.0 where the kinetics become monophasic. The rates of formation of the four molecular species, Fe2OT, FeOTN, FeOTC, and ApoOT, were studied by urea gel electrophoresis and the four microscopic rate constants were calculated at various pH values. Below pH 8.0, these intrinsic rate constants for iron release from Fe2OT follow the order k2N greater than k1N greater than k2C greater than k1C. Each constant diminishes almost proportionally with an increase in pH with the faster rate constants being affected more by the fall in hydrogen ions than the slower ones. Around pH 8.0 the four rates are approximately equal, resulting in monophasic kinetics. However, the rate constants from the C-site become faster than that from the N-site at pH above 8.2. At low pH, there is a marked preference for iron to be released from the N-site rather than from the C-site and such preference becomes less distinct as pH increases. A rather weak positive cooperativity between the two sites is demonstrated in pH between 6.8 and 7.8. The ligand responsible for the transition from biphasic to monophasic kinetics at pH 8.0 is not known. It is possible that there are different anions such as [CO3(2-)] and [HCO3-] at the two iron-binding sites, which might explain the preferential rates of iron release from these sites during protonation.     

Kinetics of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle: mechanism of protective action of alpha-crystallin

The kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle have been studied by dynamic light scattering (0.08M Hepes, pH 6.8, containing 0.1M NaCl; 48 degrees C). The hydrodynamic radius of the start aggregates determined from the initial linear parts of the dependences of the hydrodynamic radius (R(h)) on time was found to be 16.7 +/- 1.0 nm. At rather high values of time, the R(h) value for the protein aggregates becomes proportional to t(1/1.8) = t(0.56) suggesting that the aggregation process proceeds in the regime of diffusion-limited cluster-cluster aggregation. In the presence of alpha-crystallin, a protein possessing the chaperone-like activity, the process of protein aggregation switches to the regime of reaction-limited cluster-cluster aggregation as indicated by the exponential dependence of the R(h) value on time. It was shown that the addition of alpha-crystallin raises the rate of thermal inactivation of Phb. These data in combination with the results of the study of interaction of Phb with alpha-crystallin by analytical ultracentrifugation suggest that alpha-crystallin interacts with the intermediates of unfolding of the Phb molecule.     

Analysis of the RNA chaperoning activity of the hepatitis C virus core protein on the conserved 3'X region of the viral genome

The core protein of hepatitis c virus (HCV) is a structural protein with potent RNA chaperoning activities mediated by its hydrophilic N-terminal domain D1, which is thought to play a key role in HCV replication. To further characterize the core chaperoning properties, we studied the interactions between core D1 and the conserved HCV 3'X genomic region required for genome replication. To this end, we monitored the real-time annealing kinetics of native and mutated fluorescently labelled 16-nt palindromic sequence (DLS) and 27-nt Stem Loop II (SL2) from X with their respective complementary sequences. Core D1 and peptides consisting of the core basic domains were found to promote both annealing reactions and partly switch the loop-loop interaction pathway, which predominates in the absence of peptide, towards a pathway involving the stem termini. The chaperone properties of the core D1 peptides were found to be mediated through interaction of their basic clusters with the oligonucleotide phosphate groups, in line with the absence of high affinity site for core on HCV genomic RNA. The core ability to facilitate the interconversion between different RNA structures may explain how this protein regulates RNA structural transitions during HCV replication.     

Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction

The MutT pyrophosphohydrolase, in the presence of Mg2+, catalyzes the hydrolysis of nucleoside triphosphates by nucleophilic substitution at Pbeta, to yield the nucleotide and PP(i). The best substrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of its Km being 540-fold lower than that of dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PP(i) dissociates first from the enzyme-product complex (k3), followed by NMP (k4), leaving a product-binding form of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step (k5) [Saraswat, V., et al. (2002) Biochemistry 41, 15566-15577]. Single- and multiple-turnover kinetic studies of the hydrolysis of dGTP and 8-oxo-dGTP and global fitting of the data to this mechanism have yielded all of the nine rate constants. Consistent with an "iso" mechanism, single-turnover studies with dGTP and 8-oxo-dGTP hydrolysis showed slow apparent second-order rate constants for substrate binding similar to their kcat/Km values, but well below the diffusion limit (approximately 10(9) M(-1) s(-1)): k(on)app = 7.2 x 10(4) M(-1) s(-1) for dGTP and k(on)app = 2.8 x 10(7) M(-1) s(-1) for 8-oxo-dGTP. These low k(on)app values are fitted by assuming a slow iso step (k5 = 12.1 s(-1)) followed by fast rate constants for substrate binding: k1 = 1.9 x 10(6) M(-1) s(-1) for dGTP and k1 = 0.75 x 10(9) M(-1) s(-1) for 8-oxo-dGTP (the latter near the diffusion limit). With dGTP as the substrate, replacing Mg2+ with Mn2+ does not change k1, consistent with the formation of a second-sphere MutT-M2+-(H2O)-dGTP complex, but slows the iso step (k5) 5.8-fold, and its reverse (k(-5)) 25-fold, suggesting that the iso step involves a change in metal coordination, likely the dissociation of Glu-53 from the enzyme-bound metal so that it can function as the general base. Multiple-turnover studies with dGTP and 8-oxo-dGTP show bursts of product formation, indicating partially rate-limiting steps following the chemical step (k2). With dGTP, the slow steps are the chemical step (k2 = 10.7 s(-1)) and the iso step (k5 = 12.1 s(-1)). With 8-oxo-dGTP, the slow steps are the release of the 8-oxo-dGMP product (k4 = 3.9 s(-1)) and the iso step (k5 = 12.1 s(-1)), while the chemical step is fast (k2 = 32.3 s(-1)). The transient kinetic studies are generally consistent with the steady state kcat and Km values. Comparison of rate constants and free energy diagrams indicate that 8-oxo-dGTP, at low concentrations, is a better substrate than dGTP because it binds to MutT 395-fold faster, dissociates 46-fold slower, and has a 3.0-fold faster chemical step. The true dissociation constants (KD) of the substrates from the E-form of MutT, which can now be obtained from k(-1)/k1, are 3.5 nM for 8-oxo-dGTP and 62 microM for dGTP, indicating that 8-oxo-dGTP binds 1.8 x 10(4)-fold tighter than dGTP, corresponding to a 5.8 kcal/mol lower free energy of binding.     

Analysis of rate constants governing the exchange of guanine nucleotides bound to EF-Tu catalysed by EF-Ts.

The kinetics of the heterologous exchange of GDP bound to EF-Tu by free GTP catalysed by EF-Ts have been analysed with a view to correlating results obtainable with different computational procedures. The affinity of EF-Ts for EF-Tu.GTP was found to be somewhat less than previously proposed by Romero et al. (Biochemistry 260, 6167:1985) though still greater than for EF-Tu.GDP. There is a close interrelationship between the constants for the binding of GTP to EF-Tu.EF-Ts and of EF-Ts to EF-Tu.GTP. The declining fractional rate of exchange observed by Romero et al. during displacement of GDP by GTP appears to be dependent on the ratio of the rate constants (k-1 + k-2)k4/k1k-2 as defined in the text, not on that of K4/K1 as they proposed.     

Control of replication of plasmid R1: formation of an initial transient complex is rate-limiting for antisense RNA--target RNA pairing.

The replication frequency of plasmid R1 is determined by the availability of the initiator protein RepA. Synthesis of RepA is negatively controlled by an antisense RNA, CopA, which forms a duplex with the upstream region of the RepA mRNA, CopT. We have previously shown that the in vitro formation of the CopA-CopT duplex follows second-order kinetics and occurs in at least two steps. The first step is the formation of a transient (kissing) complex, which is subsequently converted to a persistent duplex. Here, we investigate the details of the reaction scheme and determine the rate constants of the pathway from the free RNAs to the complete duplex. Using a shortened CopA RNA (CopI) we have been able to determine the association and dissociation rate constants (k1,k-1) for the kissing complex (which are inferred to be the same for CopI-T and CopA-T), and measured the hybridization rate constant k2 (for CopA-T k2 is at least 1000-fold greater than for CopI-T). The analysis of CopA derivatives of mutant and wild-type origin shows that the rate of formation of the kissing complex is rate-limiting for the overall pairing reaction between CopA and CopT, both in vitro and in vivo. The biological implications of the kinetically irreversible RNA-RNA binding reaction scheme are discussed.     

Oligomerization-dependent changes in the thermodynamic properties of the TPR-MET receptor tyrosine kinase

Although oligomerization of receptor tyrosine kinases (RTKs) is necessary for receptor activation and signaling, a quantitative understanding of how oligomerization mediates these critical processes does not exist. We present a comparative thermodynamic analysis of functionally active dimeric and functionally inactive monomeric soluble analogues of the c-MET RTK, which clearly reveal that oligomerization regulates the binding affinity and binding kinetics of the kinase toward ATP and tyrosine-containing peptide substrates. Thermodynamic binding data for oligomeric c-MET were obtained from the dimeric TPR-MET oncoprotein, a functionally active fusion derivative of the c-MET RTK. This naturally occurring oncoprotein contains the cytoplasmic domain of c-MET fused to a coiled coil dimerization domain from the nuclear pore complex. Comparative data were obtained from a soluble monomeric kinase compromising the c-MET cytoplasmic domain (cytoMET). Significantly, under equilibrium binding conditions, the oligomeric phosphorylated kinase showed a significantly lower dissociation constant (K(d,dimer) = 11 microM) for a tyrosine-containing peptide derived from the C-terminal tail of the c-MET RTK when compared to the phosphorylated monomeric kinase cytoMET (K(d,monomer) = 140 microM). Surprisingly, equilibrium dissociation constants measured for the kinase and ATP were independent of the oligomerization state of the kinase (approximately 10 microM). Stopped-flow analysis of peptide substrate binding showed that the association rate constants (k(2)) differed 2-fold and dissociation rate constants (k(-2)) differed 10-fold when phosphorylated TPR-MET was compared to phosphorylated cytoMET. ATP binding abrogated the differences in k(2) rates observed between the two oligomeric states of the c-MET cytoplasmic domain. These results clearly imply that oligomerization induces important thermodynamic and conformational changes in the substrate binding regions of the c-MET protein and provide quantitative mechanistic insights into the necessary role of oligomerization in RTK activation.     

Eye lens alphaA- and alphaB-crystallin: complex stability versus chaperone-like activity.

The major lens protein alpha-crystallin is composed of two related types of subunits, alphaA- and alphaB-crystallin, of which the former is essentially lens-restricted, while the latter also occurs in various other tissues. With regard to their respective chaperone capacities, it has been reported that homomultimeric alphaA-crystallin complexes perform better in preventing thermal aggregation of proteins, while alphaB-crystallin complexes protect more efficiently against reduction-induced aggregation of proteins. Here, we demonstrate that this seeming discrepancy is solved when the reduction assay is performed at increasing temperatures: above 50 degrees C alphaA- performs better than alphaB-crystallin also in this assay. This inversion in protective capacity might relate to the greater resistance of alphaA-crystallin to heat denaturation. Infrared spectroscopy, however, revealed that this is not due to a higher thermostability of alphaA-crystallin's secondary structure. Also the accessible hydrophobic surfaces do not account for the chaperoning differences of alphaA- and alphaB-crystallin, since regardless of the experimental temperature alphaB-crystallin displays a higher hydrophobicity. It is argued that the greater complex stability of alphaA-crystallin, as evident upon urea denaturation, and the higher chaperone capacity of alphaB-crystallin at physiological temperatures reflect the evolutionary compromise to obtain an optimal functioning of heteromeric alpha-crystallin as a lens protein.