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A Lomakin

Publications and source records attributed to A Lomakin.

At least 19 recordsLinked to original sources
PubMed2001

Enhanced crystallization of the Cys18 to Ser mutant of bovine gammaB crystallin.

The cysteine residues of the gamma crystallins, a family of ocular lens proteins, are involved in the aggregation and phase separation of these proteins. Both these phenomena are implicated in cataract formation. We have used bovine gammaB crystallin as a model system to study the role of the individual cysteine residues in the aggregation and phase separation of the gamma crystallins. Here, we compare the thermodynamic and kinetic behavior of the recombinant wild-type protein (WT) and the Cys18 to Ser (C18S) mutant. We find that the solubilities of the two proteins are similar. The kinetics of crystallization, however, are different. The WT crystallizes slowly enough for the metastable liquid-liquid coexistence to be easily observed. C18S, on the other hand, crystallizes rapidly; the metastable coexisting liquid phases of the pure mutant do not form. Nevertheless, the coexistence curve of C18S can be determined provided that crystallization is kinetically suppressed. In this way we found that the coexistence curve coincides with that of the WT. Despite the difference in the kinetics of crystallization, the two proteins were found to have the same crystal forms and almost identical X-ray structures. Our results demonstrate that even conservative point mutations can bring about dramatic changes in the kinetics of crystallization. The implications of our findings for cataract formation and protein crystallization are discussed.

Amino Acid Substitution↗
PubMed2001

Amyloid beta-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins.

Assembly of the amyloid beta-protein (Abeta) into neurotoxic oligomers and fibrils is a seminal event in Alzheimer's disease. Understanding the earliest phases of Abeta assembly, including prenucleation and nucleation, is essential for the development of rational therapeutic strategies. We have applied a powerful new method, photoinduced cross-linking of unmodified proteins (PICUP), to the study of Abeta oligomerization. Significant advantages of this method include an extremely short reaction time, enabling the identification and quantification of short lived metastable assemblies, and the fact that no pre facto structural modification of the native peptide is required. Using PICUP, the distribution of Abeta oligomers existing prior to assembly was defined. A rapid equilibrium was observed involving monomer, dimer, trimer, and tetramer. A similar distribution was seen in studies of an unrelated amyloidogenic peptide, whereas nonamyloidogenic peptides yielded distributions indicative of a lack of monomer preassociation. These results suggest that simple nucleation-dependent polymerization models are insufficient to describe the dynamic equilibria associated with prenucleation phases of Abeta assembly.

Amyloid beta-Peptides↗
PubMed2001

Crystal cataracts: human genetic cataract caused by protein crystallization.

Several human genetic cataracts have been linked recently to point mutations in the gammaD crystallin gene. Here we provide a molecular basis for lens opacity in two genetic cataracts and suggest that the opacity occurs because of the spontaneous crystallization of the mutant proteins. Such crystallization of endogenous proteins leading to pathology is an unusual event. Measurements of the solubility curves of crystals of the Arg-58 to His and Arg-36 to Ser mutants of gammaD crystallin show that the mutations dramatically lower the solubility of the protein. Furthermore, the crystal nucleation rate of the mutants is enhanced considerably relative to that of the wild-type protein. It should be noted that, although there is a marked difference in phase behavior, there is no significant difference in protein conformation among the three proteins.

Cataract↗
PubMed2000

Molecular basis of a progressive juvenile-onset hereditary cataract.

In a recent paper, patients with a progressive juvenile-onset hereditary cataract have been reported to have a point mutation in the human gammaD crystallin gene (Stephan, D. A., Gillanders, E., Vanderveen, D., Freas-Lutz, D., Wistow, G., Baxevanis, A. D., Robbins, C. M., VanAuken, A., Quesenberry, M. I., Bailey-Wilson, J., et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1008-1012). This mutation results in the substitution of Arg-14 in the native protein by a Cys residue. It is not understood how this mutation leads to cataract. We have expressed recombinant wild-type human gammaD crystallin (HGD) and its Arg-14 to Cys mutant (R14C) in Escherichia coli and show that R14C forms disulfide-linked oligomers, which markedly raise the phase separation temperature of the protein solution. Eventually, R14C precipitates. In contrast, HGD slowly forms only disulfide-linked dimers and no oligomers. These data strongly suggest that the observed cataract is triggered by the thiol-mediated aggregation of R14C. The aggregation profiles of HGD and R14C are consistent with our homology modeling studies that reveal that R14C contains two exposed cysteine residues, whereas HGD has only one. Our CD, fluorescence, and differential scanning calorimetric studies show that HGD and R14C have nearly identical secondary and tertiary structures and stabilities. Thus, contrary to current views, unfolding or destabilization of the protein is not necessary for cataractogenesis.

Adolescent↗
PubMed2000

Determinants of the peptide-induced conformational change in the human class II major histocompatibility complex protein HLA-DR1.

The human class II major histocompatibility complex protein HLA-DR1 has been shown previously to undergo a distinct conformational change from an open to a compact form upon binding peptide. To investigate the role of peptide in triggering the conformational change, the minimal requirements for inducing the compact conformation were determined. Peptides as short as two and four residues, which occupy only a small fraction of the peptide-binding cleft, were able to induce the conformational change. A mutant HLA-DR1 protein with a substitution in the beta subunit designed to fill the P1 pocket from within the protein (Gly(86) to Tyr) adopted to a large extent the compact, peptide-bound conformation. Interactions important in stabilizing the compact conformation are shown to be distinct from those responsible for high affinity binding or for stabilization of the complex against thermal denaturation. The results suggest that occupancy of the P1 pocket is responsible for partial conversion to the compact form but that both side chain and main chain interactions contribute to the full conformational change. The implications of the conformational change to intracellular antigen loading and presentation are discussed.

Amino Acid Sequence↗
PubMed1999

Amyloid beta-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates.

Alzheimer's disease is characterized by extensive cerebral amyloid deposition. Amyloid deposits associated with damaged neuropil and blood vessels contain abundant fibrils formed by the amyloid beta-protein (Abeta). Fibrils, both in vitro and in vivo, are neurotoxic. For this reason, substantial effort has been expended to develop therapeutic approaches to control Abeta production and amyloidogenesis. Achievement of the latter goal is facilitated by a rigorous mechanistic understanding of the fibrillogenesis process. Recently, we discovered a novel intermediate in the pathway of Abeta fibril formation, the amyloid protofibril (Walsh, D. M., Lomakin, A., Benedek, G. B., Condron, M. M., and Teplow, D. B. (1997) J. Biol. Chem. 272, 22364-22372). We report here results of studies of the assembly, structure, and biological activity of these polymers. We find that protofibrils: 1) are in equilibrium with low molecular weight Abeta (monomeric or dimeric); 2) have a secondary structure characteristic of amyloid fibrils; 3) appear as beaded chains in rotary shadowed preparations examined electron microscopically; 4) give rise to mature amyloid-like fibrils; and 5) affect the normal metabolism of cultured neurons. The implications of these results for the development of therapies for Alzheimer's disease and for our understanding of fibril assembly are discussed.

Alzheimer Disease↗
PubMed1999

Aeolotopic interactions of globular proteins.

Protein crystallization, aggregation, liquid-liquid phase separation, and self-assembly are important in protein structure determination in the industrial processing of proteins and in the inhibition of protein condensation diseases. To fully describe such phase transformations in globular protein solutions, it is necessary to account for the strong spatial variation of the interactions on the protein surface. One difficulty is that each globular protein has its own unique surface, which is crucial for its biological function. However, the similarities amongst the macroscopic properties of different protein solutions suggest that there may exist a generic model that is capable of describing the nonuniform interactions between globular proteins. In this paper we present such a model, which includes the short-range interactions that vary from place to place on the surface of the protein. We show that this aeolotopic model [from the Greek aiolos ("variable") and topos ("place")] describes the phase diagram of globular proteins and provides insight into protein aggregation and crystallization.

Computer Simulation↗
PubMed1999

Self-assembly of helical ribbons.

The self-assembly of helical ribbons is examined in a variety of multicomponent enantiomerically pure systems that contain a bile salt or a nonionic detergent, a phosphatidylcholine or a fatty acid, and a steroid analog of cholesterol. In almost all systems, two different pitch types of helical ribbons are observed: high pitch, with a pitch angle of 54 +/- 2 degrees, and low pitch, with a pitch angle of 11 +/- 2 degrees. Although the majority of these helices are right-handed, a small proportion of left-handed helices is observed. Additionally, a third type of helical ribbon, with a pitch angle in the range 30-47 degrees, is occasionally found. These experimental findings suggest that the helical ribbons are crystalline rather than liquid crystal in nature and also suggest that molecular chirality may not be the determining factor in helix formation. The large yields of helices produced will permit a systematic investigation of their individual kinetic evolution and their elastic moduli.

Bile Acids and Salts↗
PubMed1999

A conformational change in the human major histocompatibility complex protein HLA-DR1 induced by peptide binding.

To investigate a conformational change accompanying peptide binding to class II MHC proteins, we probed the structure of a soluble version of the human class II MHC protein HLA-DR1 in empty and peptide-loaded forms. Peptide binding induced a large decrease in the effective radius of the protein as determined by gel filtration, dynamic light scattering, and analytical ultracentrifugation. It caused a substantial increase in the cooperativity of thermal denaturation and induced alterations in MHC polypeptide backbone structure as determined by circular dichroism. These changes suggest a condensation of the protein around the bound peptide. An antibody specific for beta58-69 preferentially bound the empty protein, indicating that the peptide-induced conformational change involves the beta-subunit helical region. The conformational change may have important implications for the mechanisms of intracellular antigen presentation pathways.

Amino Acid Sequence↗
PubMed1999

Monitoring protein assembly using quasielastic light scattering spectroscopy.

This article discussed the principles and practice of QLS with respect to protein assembly reactions. Particles undergoing Brownian motion in solution produce fluctuations in scattered light intensity. We have described how the temporal correlation function of these fluctuations can be measured and how mathematical analysis of the correlation function provides information about the distribution of diffusion coefficients of the particles. We have explained that deconvolution of the correlation function is an "ill-posed" problem and therefore that careful attention must be paid to the assumptions incorporated into data analysis procedures. We have shown how the Stokes-Einstein relationship can be used to convert distributions of diffusion coefficients into distributions of particle size. In the case of fibrillar polymers, this process allows direct determination of fibril length, enabling nucleation and elongation rates to be calculated. Finally, we have used examples from studies of A beta fibrillogenesis to illustrate the power these quantitative capabilities provide for understanding the molecular mechanisms of the fibrillogenesis reaction and for guiding the development of fibrillogenesis inhibitors.

Amino Acid Sequence↗
PubMed1998

Oligomerization and phase separation in globular protein solutions.

We have chemically crosslinked a globular protein, gamma IIIb-crystallin, to produce a system of well-defined oligomers: monomers, dimers, trimers and a mixture of higher n-mers. Gel electrophoresis, size exclusion chromatography, quasielastic light scattering spectroscopy, and electrospray ionization mass spectrometry were used to characterize the oligomers formed. The liquid-liquid phase separation boundaries of the various oligomers were measured. We find that at a given concentration the phase separation temperature strongly increases with the molecular weight of the oligomers. This phase behavior is very similar to previous findings for gamma II-crystallin, for which oxidation-induced oligomerization is accompanied by an increase in the phase separation temperature. These findings imply that for phase separation, the detailed changes of the surface properties of the proteins are less important than the purely steric effects of oligomerization.

Animals↗
PubMed1998

Temperature dependence of amyloid beta-protein fibrillization.

Fibrillogenesis of the amyloid beta-protein (Abeta) is believed to play a central role in the pathogenesis of Alzheimer's disease. Previous studies of the kinetics of Abeta fibrillogenesis showed that the rate of fibril elongation is proportional to the concentration of monomers. We report here the study of the temperature dependence of the Abeta fibril elongation rate constant, ke, in 0.1 M HCl. The rate of fibril elongation was measured at Abeta monomer concentrations ranging from 50 to 400 microM and at temperatures from 4 degreesC to 40 degreesC. Over this temperature range, ke increases by two orders of magnitude. The temperature dependence of ke follows the Arrhenius law, ke = A exp (-EA/kT). The preexponential factor A and the activation energy EA are approximately 6 x 10(18) liter/(mol.sec) and 23 kcal/mol, respectively. Such a high value of EA suggests that significant conformational changes are associated with the binding of Abeta monomers to fibril ends.

Amyloid beta-Peptides↗
PubMed1998

A theoretical analysis of specificity of nucleic acid interactions with oligonucleotides and peptide nucleic acids (PNAs).

We treat theoretically the problem of the specificity of interaction between nucleic acid and an oligonucleotide, its analog or its mimic (such as peptide nucleic acid, or PNA). We consider simplest models with only essential details using numerical solutions of kinetic equations and the kinetic Monte Carlo method. In our first model, describing the formation of complementary duplex, we demonstrate anti-correlation between specificity and affinity for nucleic acid/oligonucleotide interaction. We analyze in detail one notable exception. Homopyrimidine PNAs exhibit very high affinity to DNA forming extraordinarily stable DNA/(PNA)2 triplexes with the complementary DNA strand. At the same time, such PNAs show remarkable sequence specificity of binding to duplex DNA. We formulate a theoretical model for the two-step process of PNA interaction with DNA. The calculations demonstrate that two-stage binding may secure both high affinity and very high specificity of PNA interaction with DNA. Our computer simulations define the range of parameter values in which high specificity is achieved. These findings are of great importance for numerous applications of PNA and for design of future drugs which specifically interact with DNA.

Base Composition↗
PubMed1998

Aggregation in aqueous solutions of bovine lens gamma-crystallins: special role of gamma(s).

PURPOSE: To study the aggregation of bovine gamma-crystallins in aqueous solutions and the effect of gamma(s)-crystallin on the aggregation of other gamma-crystallins. METHODS: Aggregation in aqueous solutions of gamma(s)-, gammaII-, and gammaIVa-crystallin was monitored by quasielastic light scattering. Aggregation in mixtures of gamma(s)- and gammaII-crystallin, and gamma(s)- and gammaIVa-crystallin, was also monitored by light scattering. RESULTS: All of the gamma-crystallins studied formed large aggregates (or "megamers") in aqueous solutions. However, each protein differed in the relative rates of formation of megamers. Gamma(s)-crystallin formed megamers much more slowly than gammaII- and gammaIVa-crystallin. In solutions containing mixtures of gammaII and gamma(s), and gammaIVa and gamma(s), gamma(s)-crystallin significantly suppressed the aggregation of gammaII- and gammaIVa-crystallin. Megamerization seemed to be associated with thiol oxidation in these proteins. CONCLUSIONS: Gamma-crystallins undergo aggregation in which a small fraction of the proteins form a few large aggregates, whereas the larger proportion of the proteins remain monomeric. This suggests that the megamerization is preceded by an initial modification of the protein. The modification is associated with the thiol groups, and only such modified protein species participate in megamerization. The presence of gamma(s) significantly slows the megamerization of gammaII- and gammaIVa-crystallin. This fact, together with the previous finding that gamma(s) strongly reduces the phase separation temperatures of the gamma-crystallins, suggests that gamma(s)-crystallin plays an important role in suppressing the formation of light-scattering elements and helps maintain lens transparency.

Animals↗
PubMed1997

Amyloid beta-protein fibrillogenesis. Detection of a protofibrillar intermediate.

Fibrillogenesis of the amyloid beta-protein (Abeta) is a seminal pathogenetic event in Alzheimer's disease. Inhibiting fibrillogenesis is thus one approach toward disease therapy. Rational design of fibrillogenesis inhibitors requires elucidation of the stages and kinetics of Abeta fibrillogenesis. We report results of studies designed to examine the initial stages of Abeta oligomerization. Size exclusion chromatography, quasielastic light scattering spectroscopy, and electron microscopy were used to characterize fibrillogenesis intermediates. After dissolution in 0.1 M Tris-HCl, pH 7.4, and removal of pre-existent seeds, Abeta chromatographed almost exclusively as a single peak. The molecules composing the peak had average hydrodynamic radii of 1.8 +/- 0.2 nm, consistent with the predicted size of dimeric Abeta. Over time, an additional peak, with a molecular weight >100,000, appeared. This peak contained predominantly curved fibrils, 6-8 nm in diameter and <200 nm in length, which we have termed "protofibrils." The kinetics of protofibril formation and disappearance are consistent with protofibrils being intermediates in the evolution of amyloid fibers. Protofibrils appeared during the polymerization of Abeta-(1-40), Abeta-(1-42), and Abeta-(1-40)-Gln22, peptides associated with both sporadic and inherited forms of Alzheimer's disease, suggesting that protofibril formation may be a general phenomenon in Abeta fibrillogenesis. If so, protofibrils could be attractive targets for fibrillogenesis inhibitors.

Alzheimer Disease↗
PubMed1997

Kinetic theory of fibrillogenesis of amyloid beta-protein.

Prior quasielastic light scattering (QLS) studies of fibrillogenesis of synthetic amyloid beta-protein (Abeta)-(1-40) at low pH have suggested a kinetic model in which: (i) fibrillogenesis requires a nucleation step; (ii) nuclei are produced by Abeta micelles in addition to seeds initially present; and (iii) fibril elongation occurs by irreversible binding of Abeta monomers to the fibril ends. Here we present the full mathematical formulation of this model. We describe the temporal evolution of the concentrations of Abeta monomers and micelles as well as the concentration and size distribution of fibrils. This formulation enables deduction of the fundamental parameters of the model-e.g., the nucleation and elongation rate constants kn and ke-from the time dependency of the apparent diffusion coefficient measured by QLS. The theory accurately represents the experimental observations for Abeta concentrations both below and above c*, the critical concentration for Abeta micelle formation. We suggest that the method of QLS in combination with this theory can serve as a powerful tool for understanding the molecular factors that control Abeta plaque formation.

Amyloid beta-Peptides↗
PubMed1996

On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants.

We have studied the fibrillogenesis of synthetic amyloid beta-protein-(1-40) fragment (A beta) in 0.1 M HCl. At low pH, A beta formed fibrils at a rate amenable to detailed monitoring by quasi-elastic light-scattering spectroscopy. Examination of the fibrils with circular dichroism spectroscopy and electron microscopy showed them to be highly similar to those found in amyloid plaques. We determined the hydrodynamic radii of A beta aggregates during the entire process of fibril nucleation and growth. Above an A beta concentration of approximately 0.1 mM, the initial rate of elongation and the final size of fibrils were independent of A beta concentration. Below an A beta concentration of 0.1 mM, the initial elongation rate was proportional to the peptide concentration, and the resulting fibrils were significantly longer than those formed at higher concentration. We also found that the surfactant n-dodecylhexaoxyethylene glycol monoether (C12E6) slowed nucleation and elongation of fibrils in a concentration-dependent manner. Our observations are consistent with a model of A beta fibrillogenesis that includes the following key steps: (i) peptide micelles form above a certain critical A beta concentration, (ii) fibrils nucleate within these micelles or on heterogeneous nuclei (seeds), and (iii) fibrils grow by irreversible binding of monomers to fibril ends. Interpretation of our data enabled us to determine the sizes of fibril nuclei and A beta micelles and the rates of fibril nucleation (from micelles) and fibril elongation. Our approach provides a powerful means for the quantitative assay of A beta fibrillogenesis.

Amyloid beta-Peptides↗