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Mikhail A Karymov

Publications and source records attributed to Mikhail A Karymov.

7 recordsLinked to original sources
PubMed2005

Protein interactions and misfolding analyzed by AFM force spectroscopy.

Protein misfolding is conformational transition dramatically facilitating the assembly of protein molecules into aggregates of various morphologies. Spontaneous formation of specific aggregates, mostly amyloid fibrils, was initially believed to be limited to proteins involved in the development of amyloidoses. However, recent studies show that, depending on conditions, the majority of proteins undergo structural transitions leading to the appearance of amyloidogenic intermediates followed by aggregate formation. Various techniques have been used to characterize the protein misfolding facilitating the aggregation process, but no direct evidence as to how such a conformational transition increases the intermolecular interactions has been obtained as of yet. We have applied atomic force microscopy (AFM) to follow the interaction between protein molecules as a function of pH. These studies were performed for three unrelated and structurally distinctive proteins, alpha-synuclein, amyloid beta-peptide (Abeta) and lysozyme. It was shown that the attractive force between homologous protein molecules is minimal at physiological pH and increases dramatically at acidic pH. Moreover, the dependence of the pulling forces is sharp, suggesting a pH-dependent conformational transition within the protein. Parallel circular dichroism (CD) measurements performed for alpha-synuclein and Abeta revealed that the decrease in pH is accompanied by a sharp conformational transition from a random coil at neutral pH to the more ordered, predominantly beta-sheet, structure at low pH. Importantly, the pH ranges for these conformational transitions coincide with those of pulling forces changes detected by AFM. In addition, protein self-assembly into filamentous aggregates studied by AFM imaging was shown to be facilitated at pH values corresponding to the maximum of pulling forces. Overall, these results indicate that proteins at acidic pH undergo structural transition into conformations responsible for the dramatic increase in interprotein interaction and promoting the formation of protein aggregates.

Amino Acid Sequence↗
PubMed2005

Forcing nonamyloidogenic beta-synuclein to fibrillate.

The fibrillation and aggregation of alpha-synuclein is a key process in the formation of intracellular inclusions, Lewy bodies, in substantia nigral neurons and, potentially, in the pathology of Parkinson's disease and several other neurodegenerative disorders. Alpha-synuclein and its homologue beta-synuclein are both natively unfolded proteins that colocalize in presynaptic terminals of neurons in many regions of the brain, including those of dopamine-producing cells of the substantia nigra. Unlike its homologue, beta-synuclein does not form fibrils and has been shown to inhibit the fibrillation of alpha-synuclein. In this study, we demonstrate that fast and efficient aggregation and fibrillation of beta-synuclein can be induced in the presence of a variety of factors. Certain metals (Zn(2+), Pb(2+), and Cu(2+)) induce a partially folded conformation of beta-synuclein that triggers rapid fibrillation. In the presence of these metals, mixtures of alpha- and beta-synucleins exhibited rapid fibrillation. The metal-induced fibrillation of beta-synuclein was further accelerated by the addition of glycosaminoglycans or high concentrations of macromolecular crowding agents. Beta-synuclein also rapidly formed soluble oligomers and fibrils in the presence of pesticides, whereas the addition of low concentrations of organic solvents induced formation of amorphous aggregates. These new findings demonstrate the potential effect of environmental pollutants in generating an amyloidogenic, and potentially neurotoxic, conformation, in an otherwise benign protein.

Amyloid↗
PubMed2005

Effects of nitration on the structure and aggregation of alpha-synuclein.

Substantial evidence suggests that the aggregation of the presynaptic protein alpha-synuclein is a key step in the etiology of Parkinson's disease (PD). Although the molecular mechanisms underlying alpha-synuclein aggregation remain unknown, oxidative stress has been implicated in the pathogenesis of PD. Here, we report the effects of tyrosine nitration on the propensity of human recombinant alpha-synuclein to fibrillate in vitro. The properties of nitrated alpha-synuclein were investigated using a variety of biophysical and biochemical techniques, which revealed that nitration led to formation of a partially folded conformation with increased secondary structure relative to the intrinsically disordered structure of the monomer, and to oligomerization at neutral pH. The degree of self-association was concentration-dependent, but at 1 mg/mL, nitrated alpha-synuclein was predominantly an octamer. At low pH, small-angle X-ray scattering data indicated that the nitrated protein was monomeric. alpha-Synuclein fibrillation at neutral pH was completely inhibited by nitrotyrosination and is attributed to the formation of stable soluble oligomers. The presence of heparin or metals did not overcome the inhibition; however, the inhibitory effect was eliminated at low pH. The addition of nitrated alpha-synuclein inhibited fibrillation of non-modified alpha-synuclein at neutral pH. Potential implications of these findings to the etiology of Parkinson's disease are discussed.

Animals↗
PubMed2003

"Antiparallel" DNA loop in gal repressosome visualized by atomic force microscopy.

DNA looping is often involved in positive and negative regulation of gene transcription in both prokaryotes and eukaryotes. The transcription of the gal operon of Escherichia coli from two overlapping promoters P1 and P2 is negatively regulated via Gal repressosome assembly. It involves binding of two dimeric Gal repressor proteins (GalR) to two operators, O(E) and O(I), flanking the two promoters, and formation of 113 bp DNA loop due to tetramerization of the two bound GalR dimers. The process requires negatively supercoiled DNA and the presence of the histone-like protein HU. Previous modeling of the repressosome based on evaluation of DNA elastic energy suggested a mutual antiparallel, rather than parallel, orientation of the two gal operators in an under-twisted DNA loop. To visualize the Gal loop by atomic force microscopy (AFM), plasmid DNA molecules were constructed with increased distance between the two operators. The AFM results demonstrated the formation of an antiparallel DNA loop in the Gal repressosome consistent with our earlier hypothesis. Importantly, the overall shape of the GalR mediated loop proved to be indistinguishable from that in the chimerical loop of the same size containing two lac operators (instead of two gal operators) and formed by LacI. In addition, a possibility of the gal operon repression mediated by GalR in the absence of HU was shown in the new DNA constructs. Implications of these findings for the DNA structural organization in bacterial nucleoid are discussed.

Bacterial Proteins↗
PubMed2003

Assembly of single chromatin fibers depends on the tension in the DNA molecule: magnetic tweezers study.

We have used magnetic tweezers to study in real time chaperone-mediated chromatin assembly/disassembly at the level of single chromatin fibers. We find a strong dependence of the rate of assembly on the exerted force, with strong inhibition of assembly at forces exceeding 10 pN. During assembly, and especially at higher forces, occasional abrupt increases in the length of the fiber were observed, giving a clear indication of reversibility of the assembly process. This result is a clear demonstration of the dynamic equilibrium between nucleosome assembly and disassembly at the single chromatin fiber level.

Chromatin↗
PubMed2002

Selective requirements for histone H3 and H4 N termini in p300-dependent transcriptional activation from chromatin.

The N-terminal tails of the core histones play important roles in transcriptional regulation, but their mechanism(s) of action are poorly understood. Here, pure chromatin templates assembled with varied combinations of recombinant wild-type and mutant core histones have been employed to ascertain the role of individual histone tails, both in overall acetylation patterns and in transcription. In vitro assays show an indispensable role for H3 and H4 tails, especially major lysine substrates, in p300-dependent transcriptional activation, as well as activator-targeted acetylation of promoter-proximal histone tails by p300. These results indicate, first, that constraints to transcription are imposed by nucleosomal histone components other than histone N-terminal tails and, second, that the histone N-terminal tails have selective roles, which can be modulated by targeted acetylation, in transcriptional activation by p300.

Acetylation↗