Publications by authors named "Alexey K Surin"

22 Publications

  • Page 1 of 1

Manual mass spectrometry de novo sequencing of the anionic host defense peptides of the Cuban Treefrog Osteopilus septentrionalis.

Rapid Commun Mass Spectrom 2021 Apr;35(7):e9061

Chemistry Department, Lomonosov Moscow State University, Leninskie Gory 1/3, Moscow, 119991, Russia.

Rationale: Host defense peptides accumulated in the skin glands of the animals constitute the basis of the adaptive and immune system of amphibians. The peptidome of the Cuban frog Osteopilus septentrionalis was established using tandem mass spectrometry as the best analytical tool to elucidate the sequence of these peptides.

Methods: Manual interpretation of complementary collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), and electron transfer dissociation (ETD) tandem mass spectra recorded with an Orbitrap Elite mass spectrometer in liquid chromatography/mass spectrometry (LC/MS) mode was used to sequence the peptide components of the frog skin secretion, obtained by mild electrostimulation.

Results: Although the vast majority of amphibian peptides discovered so far are cationic, surprisingly only anionic peptides were identified in the skin secretion of the Cuban frog Osteopilus septentrionalis. Mass spectrometry allowed the sequences to be established of 16 representatives of new peptide families: septenins 1 and septenins 2. The highest sequence coverage when dealing with these anionic peptides was obtained with CID normalized collision energy 35 and HCD normalized collision energy 28.

Conclusions: Mirror-symmetrical peptides are sequenced using N-terminal acetylation. Acetylated Ser is reliably distinguished from isomeric Glu by the loss of ketene from b-ions containing the corresponding residue. Calculations of the physicochemical and structural properties of the discovered anionic septenins 1 and 2 allowed the mechanism of their interaction with microbe cells to be postulated.
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http://dx.doi.org/10.1002/rcm.9061DOI Listing
April 2021

Myosin Binding Protein-C Forms Amyloid-Like Aggregates In Vitro.

Int J Mol Sci 2021 Jan 13;22(2). Epub 2021 Jan 13.

Laboratory of the Structure and Functions of Muscle Proteins, Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, 142290 Pushchino, Russia.

This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5-10 min) formation of large (>2 μm) aggregates. sMyBP-C oligomers formed both at the initial 5-10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7-10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-β quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (<26%), alternating ordered/disordered regions in the protein molecule, and S-S bonds providing for general stability.
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http://dx.doi.org/10.3390/ijms22020731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828380PMC
January 2021

Antimicrobial and Amyloidogenic Activity of Peptides. Can Antimicrobial Peptides Be Used against SARS-CoV-2?

Int J Mol Sci 2020 Dec 15;21(24). Epub 2020 Dec 15.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Russia.

At present, much attention is paid to the use of antimicrobial peptides (AMPs) of natural and artificial origin to combat pathogens. AMPs have several points that determine their biological activity. We analyzed the structural properties of AMPs, as well as described their mechanism of action and impact on pathogenic bacteria and viruses. Recently published data on the development of new AMP drugs based on a combination of molecular design and genetic engineering approaches are presented. In this article, we have focused on information on the amyloidogenic properties of AMP. This review examines AMP development strategies from the perspective of the current high prevalence of antibiotic-resistant bacteria, and the potential prospects and challenges of using AMPs against infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
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http://dx.doi.org/10.3390/ijms21249552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765370PMC
December 2020

New Model for Stacking Monomers in Filamentous Actin from Skeletal Muscles of .

Int J Mol Sci 2020 Nov 6;21(21). Epub 2020 Nov 6.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

To date, some scientific evidence (limited proteolysis, mass spectrometry analysis, electron microscopy (EM)) has accumulated, which indicates that the generally accepted model of double-stranded of filamentous actin (F-actin) organization in eukaryotic cells is not the only one. This entails an ambiguous understanding of many of the key cellular processes in which F-actin is involved. For a detailed understanding of the mechanism of F-actin assembly and actin interaction with its partners, it is necessary to take into account the polymorphism of the structural organization of F-actin at the molecular level. Using electron microscopy, limited proteolysis, mass spectrometry, X-ray diffraction, and structural modeling we demonstrated that F-actin presented in the EM images has no double-stranded organization, the regions of protease resistance are accessible for action of proteases in F-actin models. Based on all data, a new spatial model of filamentous actin is proposed, and the F-actin polymorphism is discussed.
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http://dx.doi.org/10.3390/ijms21218319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664232PMC
November 2020

Desmin Interacts Directly with Mitochondria.

Int J Mol Sci 2020 Oct 30;21(21). Epub 2020 Oct 30.

Institute of Protein Research of Russian Academy of Sciences, Vavilova st., 34, 119334 Moscow, Russia.

Desmin intermediate filaments (IFs) play an important role in maintaining the structural and functional integrity of muscle cells. They connect contractile myofibrils to plasma membrane, nuclei, and mitochondria. Disturbance of their network due to desmin mutations or deficiency leads to an infringement of myofibril organization and to a deterioration of mitochondrial distribution, morphology, and functions. The nature of the interaction of desmin IFs with mitochondria is not clear. To elucidate the possibility that desmin can directly bind to mitochondria, we have undertaken the study of their interaction in vitro. Using desmin mutant Des(Y122L) that forms unit-length filaments (ULFs) but is incapable of forming long filaments and, therefore, could be effectively separated from mitochondria by centrifugation through sucrose gradient, we probed the interaction of recombinant human desmin with mitochondria isolated from rat liver. Our data show that desmin can directly bind to mitochondria, and this binding depends on its N-terminal domain. We have found that mitochondrial cysteine protease can disrupt this interaction by cleavage of desmin at its N-terminus.
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http://dx.doi.org/10.3390/ijms21218122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663591PMC
October 2020

Antimicrobial and Amyloidogenic Activity of Peptides Synthesized on the Basis of the Ribosomal S1 Protein from Thermus Thermophilus.

Int J Mol Sci 2020 09 2;21(17). Epub 2020 Sep 2.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

Controlling the aggregation of vital bacterial proteins could be one of the new research directions and form the basis for the search and development of antibacterial drugs with targeted action. Such approach may be considered as an alternative one to antibiotics. Amyloidogenic regions can, like antibacterial peptides, interact with the "parent" protein, for example, ribosomal S1 protein (specific only for bacteria), and interfere with its functioning. The aim of the work was to search for peptides based on the ribosomal S1 protein from , exhibiting both aggregation and antibacterial properties. The biological system of the response of Gram-negative bacteria to the action of peptides was characterized. Among the seven studied peptides, designed based on the S1 protein sequence, the R23I (modified by the addition of HIV transcription factor fragment for bacterial cell penetration), R23T (modified), and V10I (unmodified) peptides have biological activity that inhibits the growth of cells, that is, they have antimicrobial activity. But, only the R23I peptide had the most pronounced activity comparable with the commercial antibiotics. We have compared the proteome of peptide-treated and intact cells. These important data indicate a decrease in the level of energy metabolism and anabolic processes, including the processes of biosynthesis of proteins and nucleic acids. Under the action of 20 and 50 μg/mL R23I, a decrease in the number of proteins in cells was observed and S1 ribosomal protein was absent. The obtained results are important for understanding the mechanism of amyloidogenic peptides with antimicrobial activity and can be used to develop new and improved analogues.
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http://dx.doi.org/10.3390/ijms21176382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504387PMC
September 2020

Amyloidogenic Propensities of Ribosomal S1 Proteins: Bioinformatics Screening and Experimental Checking.

Int J Mol Sci 2020 Jul 22;21(15). Epub 2020 Jul 22.

Institute of Protein Research, Russian Academy of Sciences, Pushchino 142290, Moscow Region, Russia.

Structural S1 domains belong to the superfamily of oligosaccharide/oligonucleotide-binding fold domains, which are highly conserved from prokaryotes to higher eukaryotes and able to function in RNA binding. An important feature of this family is the presence of several copies of the structural domain, the number of which is determined in a strictly limited range from one to six. Despite the strong tendency for the aggregation of several amyloidogenic regions in the family of the ribosomal S1 proteins, their fibril formation process is still poorly understood. Here, we combined computational and experimental approaches for studying some features of the amyloidogenic regions in this protein family. The FoldAmyloid, Waltz, PASTA 2.0 and Aggrescan programs were used to assess the amyloidogenic propensities in the ribosomal S1 proteins and to identify such regions in various structural domains. The thioflavin T fluorescence assay and electron microscopy were used to check the chosen amyloidogenic peptides' ability to form fibrils. The bioinformatics tools were used to study the amyloidogenic propensities in 1331 ribosomal S1 proteins. We found that amyloidogenicity decreases with increasing sizes of proteins. Inside one domain, the amyloidogenicity is higher in the terminal parts. We selected and synthesized 11 amyloidogenic peptides from the and ribosomal S1 proteins and checked their ability to form amyloids using the thioflavin T fluorescence assay and electron microscopy. All 11 amyloidogenic peptides form amyloid-like fibrils. The described specific amyloidogenic regions are actually responsible for the fibrillogenesis process and may be potential targets for modulating the amyloid properties of bacterial ribosomal S1 proteins.
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http://dx.doi.org/10.3390/ijms21155199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432502PMC
July 2020

Determination of amyloid core regions of insulin analogues fibrils.

Prion 2020 01;14(1):149-162

Institute of Protein Research, Russian Academy of Sciences , Pushchino, Russian Federation.

A rapid-acting insulin lispro and long-acting insulin glargine are commonly used for the treatment of diabetes. Clinical cases have described the formation of injectable amyloidosis with these insulin analogues, but their amyloid core regions of fibrils were unknown. To reveal these regions, we have analysed the hydrolyzates of insulin fibrils and its analogues using high-performance liquid chromatography and mass spectrometry methods and found that insulin and its analogues have almost identical amyloid core regions that intersect with the predicted amyloidogenic regions. The obtained results can be used to create new insulin analogues with a low ability to form fibrils.

Abbreviations: a.a., amino acid residues; HPLC-MS, high-performance liquid chromatography/mass spectrometry; m/z, mass-to-charge ratio; TEM, transmission electron microscopy.
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http://dx.doi.org/10.1080/19336896.2020.1776062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518739PMC
January 2020

Interaction of two strongly divergent archaellins stabilizes the structure of the Halorubrum archaellum.

Microbiologyopen 2020 07 21;9(7):e1047. Epub 2020 Apr 21.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.

Halophilic archaea from the genus Halorubrum possess two extraordinarily diverged archaellin genes, flaB1 and flaB2. To clarify roles for each archaellin, we compared two natural Halorubrum lacusprofundi strains: One of them contains both archaellin genes, and the other has the flaB2 gene only. Both strains synthesize functional archaella; however, the strain, where both archaellins are present, is more motile. In addition, we expressed these archaellins in a Haloferax volcanii strain from which the endogenous archaellin genes were deleted. Three Hfx. volcanii strains expressing Hrr. lacusprofundi archaellins produced functional filaments consisting of only one (FlaB1 or FlaB2) or both (FlaB1/FlaB2) archaellins. All three strains were motile, although there were profound differences in the efficiency of motility. Both native and recombinant FlaB1/FlaB2 filaments have greater thermal stability and resistance to low salinity stress than single-component filaments. Functional supercoiled Hrr. lacusprofundi archaella can be composed of either single archaellin: FlaB2 or FlaB1; however, the two divergent archaellin subunits provide additional stabilization to the archaellum structure and thus adaptation to a wider range of external conditions. Comparative genomic analysis suggests that the described combination of divergent archaellins is not restricted to Hrr. lacusprofundi, but is occurring also in organisms from other haloarchaeal genera.
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http://dx.doi.org/10.1002/mbo3.1047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349177PMC
July 2020

Should the Treatment of Amyloidosis Be Personified? Molecular Mechanism of Amyloid Formation by Aβ Peptide and Its Fragments.

J Alzheimers Dis Rep 2018 Oct 24;2(1):181-199. Epub 2018 Oct 24.

Institute of Protein Research, Russian Academy of Science, Pushchino, Moscow Region, Russia.

Aβ and Aβ peptides are believed to be associated with Alzheimer's disease. Aggregates (plaques) of Aβ fibrils are found in the brains of humans affected with this disease. The mechanism of formation of Aβ fibrils has not been studied completely, which hinders the development of a correct strategy for therapeutic prevention of this neurodegenerative disorder. It has been found that the most toxic samples upon generation of fibrils are different oligomeric formations. Based on different research methods used for studying amyloidogenesis of Aβ and Aβ peptides and its amyloidogenic fragments, we have proposed a new mechanism of formation of amyloid fibrils. In accord with this mechanism, the main building unit for fibril generation is a ring-like oligomer. Association of ring-like oligomers results in the formation of fibrils of different morphologies. Our model implies that to prevent development of Alzheimer's disease a therapeutic intervention is required at the earliest stages of amyloidogenesis-at the stage of formation of ring-like oligomers. Therefore, the possibility of a personified approach for prevention not only of Alzheimer's disease development but also of other neurodegenerative diseases associated with the formation of fibrils is argued.
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http://dx.doi.org/10.3233/ADR-180063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218156PMC
October 2018

Salt-dependent regulation of archaellins in Haloarcula marismortui.

Microbiologyopen 2019 05 1;8(5):e00718. Epub 2018 Oct 1.

Molecular Biology of Archaea, Faculty of Biology, University of Freiburg, Freiburg, Germany.

Microorganisms require a motility structure to move towards optimal growth conditions. The motility structure from archaea, the archaellum, is fundamentally different from its bacterial counterpart, the flagellum, and is assembled in a similar fashion as type IV pili. The archaellum filament consists of thousands of copies of N-terminally processed archaellin proteins. Several archaea, such as the euryarchaeon Haloarcula marismortui, encode multiple archaellins. Two archaellins of H. marismortui display differential stability under various ionic strengths. This suggests that these proteins behave as ecoparalogs and perform the same function under different environmental conditions. Here, we explored this intriguing system to study the differential regulation of these ecoparalogous archaellins by monitoring their transcription, translation, and assembly into filaments. The salt concentration of the growth medium induced differential expression of the two archaellins. In addition, this analysis indicated that archaellation in H. marismortui is majorly regulated on the level of secretion, by a still unknown mechanism. These findings indicate that in archaea, multiple encoded archaellins are not completely redundant, but in fact can display subtle functional differences, which enable cells to cope with varying environmental conditions.
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http://dx.doi.org/10.1002/mbo3.718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6528647PMC
May 2019

To Be Fibrils or To Be Nanofilms? Oligomers Are Building Blocks for Fibril and Nanofilm Formation of Fragments of Aβ Peptide.

Langmuir 2018 02 30;34(6):2332-2343. Epub 2018 Jan 30.

Institute of Protein Research, Russian Academy of Sciences , Pushchino 142290, Russia.

To identify the key stages in the amyloid fibril formation we studied the aggregation of amyloidogenic fragments of Aβ peptide, Aβ(16-25), Aβ(31-40), and Aβ(33-42), using the methods of electron microscopy, X-ray analysis, mass spectrometry, and structural modeling. We have found that fragments Aβ(31-40) and Aβ(33-42) form amyloid fibrils in the shape of bundles and ribbons, while fragment Aβ(16-25) forms only nanofilms. We are the first who performed 2D reconstruction of amyloid fibrils by the Markham rotation technique on electron micrographs of negatively stained fragments of Aβ peptide. Combined analysis of the data allows us to speculate that both the fibrils and the films are formed via association of ring-shaped oligomers with the external diameter of about 6 to 7 nm, the internal diameter of 2 to 3 nm, and the height of ∼3 nm. We conclude that such oligomers are the main building blocks in fibrils of any morphology. The interaction of ring oligomers with each other in different ways makes it possible to explain their polymorphism. The new mechanism of polymerization of amyloidogenic proteins and peptides, described here, could stimulate new approaches in the development of future therapeutics for the treatment of amyloid-related diseases.
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http://dx.doi.org/10.1021/acs.langmuir.7b03393DOI Listing
February 2018

The Mechanism Underlying Amyloid Polymorphism is Opened for Alzheimer's Disease Amyloid-β Peptide.

J Alzheimers Dis 2016 09;54(2):821-30

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.

It has been demonstrated using Aβ40 and Aβ42 recombinant and synthetic peptides that their fibrils are formed of complete oligomer ring structures. Such ring structures have a diameter of about 8-9 nm, an oligomer height of about 2- 4 nm, and an internal diameter of the ring of about 3-4 nm. Oligomers associate in a fibril in such a way that they interact with each other, overlapping slightly. There are differences in the packing of oligomers in fibrils of recombinant and synthetic Aβ peptides. The principal difference is in the degree of orderliness of ring-like oligomers that leads to generation of morphologically different fibrils. Most ordered association of ring-like structured oligomers is observed for a recombinant Aβ40 peptide. Less ordered fibrils are observed with the synthetic Aβ42 peptide. Fragments of fibrils the most protected from the action of proteases have been determined by tandem mass spectrometry. It was shown that unlike Aβ40, fibrils of Aβ42 are more protected, showing less ordered organization compared to that of Aβ40 fibrils. Thus, the mass spectrometry data agree with the electron microscopy data and structural models presented here.
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http://dx.doi.org/10.3233/JAD-160405DOI Listing
September 2016

Structural model of amyloid fibrils for amyloidogenic peptide from Bgl2p-glucantransferase of S. cerevisiae cell wall and its modifying analog. New morphology of amyloid fibrils.

Biochim Biophys Acta 2016 11 6;1864(11):1489-99. Epub 2016 Aug 6.

Institute of Protein Research, Russian Academy of Science, 142290 Pushchino, Moscow Region, Russia. Electronic address:

We performed a comparative study of the process of amyloid formation by short homologous peptides with a substitution of aspartate for glutamate in position 2 - VDSWNVLVAG (AspNB) and VESWNVLVAG (GluNB) - with unblocked termini. Peptide AspNB (residues 166-175) corresponded to the predicted amyloidogenic region of the protein glucantransferase Bgl2 from the Saccharomyces cerevisiae cell wall. The process of amyloid formation was monitored by fluorescence spectroscopy (FS), electron microscopy (EM), tandem mass spectrometry (TMS), and X-ray diffraction (XD) methods. The experimental study at pH3.0 revealed formation of amyloid fibrils with similar morphology for both peptides. Moreover, we found that the morphology of fibrils made of untreated ammonia peptide is not mentioned in the literature. This morphology resembles snakes lying side by side in the form of a wave without intertwining. Irrespective of the way of the peptide preparation, the rate of fibril formation is higher for AspNB than for GluNB. However, preliminary treatment with ammonia highly affected fibril morphology especially for AspNB. Such treatment allowed us to obtain a lag period during the process of amyloid formation. It showed that the process was nucleation-dependent. With or without treatment, amyloid fibrils consisted of ring-like oligomers with the diameter of about 6nm packed either directly ring-to-ring or ring-on-ring with a slight shift. We also proposed the molecular structure of amyloid fibrils for two studied peptides.
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http://dx.doi.org/10.1016/j.bbapap.2016.08.002DOI Listing
November 2016

X-ray diffraction and electron microscopy data for amyloid formation of Aβ40 and Aβ42.

Data Brief 2016 Sep 20;8:108-13. Epub 2016 May 20.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

The data presented in this article are related to the research article entitled "One of the possible mechanisms of amyloid fibrils formation based on the sizes of primary and secondary folding nuclei of Aβ40 and Aβ42" (Dovidchenko et al., 2016) [1]. Aβ peptide is one of the most intensively studied amyloidogenic peptides. Despite the huge number of articles devoted to studying different fragments of Aβ peptide there are only several papers with correct kinetics data, also there are a few papers with X-ray data, especially for Aβ42. Our data present X-ray diffraction patterns both for Aβ40 and Aβ42 as well for Tris-HCl and wax. Moreover, our data provide kinetics of amyloid formation by recombinant Аβ40 and synthetic Аβ42 peptides by using electron microscopy.
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http://dx.doi.org/10.1016/j.dib.2016.05.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889875PMC
September 2016

Insulin and Lispro Insulin: What is Common and Different in their Behavior?

Curr Protein Pept Sci 2017 ;18(1):57-64

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

There are different insulin analogues with various pharmacokinetic characteristics, such as, rapid-acting, long-acting, or intermediate-acting analogues. Since insulin tends to form amyloid aggregates, it is of particular interest to measure characteristic times of formation of amyloid aggregates and compare those to action times for insulin and its analogues. For the study we have chosen one of the insulin analogues - insulin Lispro, which is a fast acting insulin analog. It is usually thought of amyloid aggregation as a nucleation-dependent process. We have estimated the size of the primary nucleus to be one monomer and the size of the secondary nucleus to be around zero in both insulin and Lispro insulin aggregation processes. The main structural element of insulin and Lispro insulin amyloid fibrils is a rounded ring oligomer of about 6-7 nm in diameter, about 2-3 nm in height and about 2 nm in diameter of the hole. Fibrils of several μm in length are produced due to interaction of such oligomers. The packing of ring oligomers in fibrils differs because of the difference in their orderliness. Though the initial stages of fibril formation (monomer, oligomer) are similar, the further process depends on the unique sequence of each peptide. Namely the sequence affects the final morphology of mature amyloids. These observations allow us to conclude that formation of fibrils by short peptides occurs via and by means of oligomer ring structures. Such an important issue as the nature of polymorphism of insulin amyloid fibrils has been settled by us. The role of early oligomeric aggregates in such processes as nucleation and aggregation of amyloid fibrils has been examined.
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http://dx.doi.org/10.2174/1389203717666160526122421DOI Listing
February 2017

One of the possible mechanisms of amyloid fibrils formation based on the sizes of primary and secondary folding nuclei of Aβ40 and Aβ42.

J Struct Biol 2016 06 22;194(3):404-14. Epub 2016 Mar 22.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia. Electronic address:

In the presented paper, theoretical as well as electron microscopy and X-ray diffraction experimental approaches were employed for studding the process of Aβ amyloid formation. Using quantitative estimates of a number of monomers which form the nuclei of amyloid fibrils the sizes of folding nuclei of amyloid fibrils for Aβ40 and 42 have been determined for the first time. We have shown that the size of the primary nucleus of Aβ42 peptide fibrils corresponds to 3 monomers, the size of the secondary nucleus for this peptide is 2 monomers. Applying the same analysis to Aβ40 we conclude that the size of the primary nucleus is 2 monomers, and the size of the secondary nucleus is one monomer. Summation of our theoretical and experimental results has allowed us to propose a new model of the structural organization of amyloid fibrils. Our model suggests that the generation of fibrils takes place along the following simplified pathway: a monomer→a ring oligomer→a mature fibril consisting of ring oligomers. These data shed more light upon our understanding of what sizes of the oligomers could represent main targets for future therapies (tetramers for Aβ42 and trimers for Aβ40), and aid in the development of inhibitors of Aβ40 and 42 oligomer formation.
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http://dx.doi.org/10.1016/j.jsb.2016.03.020DOI Listing
June 2016

Studies of Polymorphism of Amyloid-β42 Peptide from Different Suppliers.

J Alzheimers Dis 2015 ;47(3):583-93

Institute of Protein Research, Russian Academy of Science, Pushchino, Moscow Region, Russia.

The aim of this study was to investigate the process of amyloidogenesis of amyloid-β (Aβ)42 peptide, by means of fluorescence spectroscopy, electron microscopy, X-ray diffraction, and mass spectrometry. It has been repeatedly reported in the literature that the process of fibril formation by Aβ42 peptide depends considerably not only upon the specific conditions (ionic conditions, pH, temperature, mixing, etc.), as well as the manufacturing route (synthetic or recombinant), but also on the methods of synthesis and purification. We have, for the first time, systematically analyzed samples of Aβ42 peptide supplied by five different companies (Anaspec, Invitrogen, Enzo, Sigma-Aldrich, and SynthAssist) and obtained evidence of significant variability, including lot to lot variations. All studied samples formed amyloid-like fibrils at pH3-6, and the fibrils contained cross-β structures. Samples from Anaspec, Invitrogen, and Enzo formed one particular type of amyloid-like fibrils, while the samples from Sigma-Aldrich and SynthAssist formed another distinct type of fibrils. The observed polymorphism emphasizes the capacity of the Aβ42 peptide to act as a prion agent with varying structural characteristics. The presented data have allowed us to propose a possible mechanism of formation of amyloid-like fibrils.
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http://dx.doi.org/10.3233/JAD-150147DOI Listing
July 2016

Ion coalescence in Fourier transform mass spectrometry: should we worry about this in shotgun proteomics?

Eur J Mass Spectrom (Chichester) 2015 ;21(3):459-70

V.L. Talrose Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, 38 Leninsky Pr., Bld. 2, Moscow 119334, Russia. Moscow Institute of Physics and Technology (State University), 9 Institutskii Per., Dolgoprudny 141700, Moscow region, Russia.

Coupling of motion of the ion clouds with close m/z values is a well-established phenomenon for ion- trapping mass analyzers. In Fourier transform ion cyclotron resonance mass spectrometry it is known as ion coalescence. Recently, ion coalescence was demonstrated and semiquantitatively characterized for the Orbitrap mass analyzer as well. When it occurs, the coalescence negatively affects the basic characteristics of a mass analyzer. Specifically, the dynamic range available for the high resolving power mass measurements reduces. In shotgun proteomics, another potentially adverse effect of ion coalescence is interference of the isotopic envelopes for the coeluting precursor ions of close m/z values, subjected to isolation before fragmentation. In this work we characterize coalescence events for synthetic peptide mixtures with fully and partially overlapping (13)C-isotope envelopes including pairs of peptides with glutamine deamidation. Furthermore, we demonstrate that fragmentation of the otherwise coalesced peptide ion clouds may remove the locking between them owing to the total charge redistribution between more ion species in the mass spectrum. Finally, we estimated the possible scale of the coalescence phenomenon for shotgun proteomics by considering the fraction of coeluted peptide pairs with the close masses using literature data for the yeast proteome. It was found that up to one tenth of all peptide identifications with the relative mass differences of 20 ppm or less in the corresponding pairs may potentially experience the coalescence of the (13)C-isotopic envelopes. However, sample complexity in a real proteomics experiment and precursor ion signal splitting between many channels in tandem mass spectrometry drastically increase the threshold for coalescence, thus leading to practically coalescence-free proteomics based on Fourier transform mass spectrometry.
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http://dx.doi.org/10.1255/ejms.1356DOI Listing
October 2015

How to determine the size of folding nuclei of protofibrils from the concentration dependence of the rate and lag-time of aggregation. II. Experimental application for insulin and LysPro insulin: aggregation morphology, kinetics, and sizes of nuclei.

J Phys Chem B 2014 Feb 24;118(5):1198-206. Epub 2014 Jan 24.

Institute of Protein Research , Russian Academy of Sciences, 4 Institutskaya str., Pushchino, Moscow Region, 142290, Russia.

Insulin is a commonly used protein for studies of amyloidogenesis. There are a few insulin analogues with different pharmacokinetic characteristics, in particular the onset and duration of action. One of them is LysPro insulin. The behavior of LysPro insulin in the process of amyloid formation has not been studied in detail yet. To quantitatively investigate the differences between insulin and LysPro insulin in the aggregation reaction, we used thioflavin T fluorescence assay, electron microscopy, X-ray diffraction methods, and theoretical modeling. Kinetic experimental data for both insulin samples demonstrated the increase of the lag-time for LysPro insulin at low concentrations of monomers, particularly at 2 and 4 mg/mL, which corresponds to the pharmaceutical concentration. However, the morphology of insulin and LysPro insulin fibrils and their X-ray diffraction patterns is identical. Mature fibrils reach 10-12 μm in length and about 3-4 nm in diameter. The obtained analytical solution allow us to determine the sizes of the primary and secondary nuclei from the experimentally obtained concentration dependences of the time of growth and the ratio of the lag-time duration to the time of growth of amyloid protofibrils. In the case of insulin and LysPro insulin, we have exponential growth of amyloid protofibrils following the "bifurcation + lateral growth" scenario. In accord with the developed theory and the experimental data, we obtained that the size of the primary nucleus is equal to one monomer and the size of the secondary nucleus is zero in both insulin and LysPro insulin.
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http://dx.doi.org/10.1021/jp4083568DOI Listing
February 2014

A novel web server predicts amino acid residue protection against hydrogen-deuterium exchange.

Bioinformatics 2013 Jun 24;29(11):1375-81. Epub 2013 Apr 24.

Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.

Motivation: To clarify the relationship between structural elements and polypeptide chain mobility, a set of statistical analyses of structures is necessary. Because at present proteins with determined spatial structures are much less numerous than those with amino acid sequence known, it is important to be able to predict the extent of proton protection from hydrogen-deuterium (HD) exchange basing solely on the protein primary structure.

Results: Here we present a novel web server aimed to predict the degree of amino acid residue protection against HD exchange solely from the primary structure of the protein chain under study. On the basis of the amino acid sequence, the presented server offers the following three possibilities (predictors) for user's choice. First, prediction of the number of contacts occurring in this protein, which is shown to be helpful in estimating the number of protons protected against HD exchange (sensitivity 0.71). Second, probability of H-bonding in this protein, which is useful for finding the number of unprotected protons (specificity 0.71). The last is the use of an artificial predictor. Also, we report on mass spectrometry analysis of HD exchange that has been first applied to free amino acids. Its results showed a good agreement with theoretical data (number of protons) for 10 globular proteins (correlation coefficient 0.73). We pioneered in compiling two datasets of experimental HD exchange data for 35 proteins.

Availability: The H-Protection server is available for users at http://bioinfo.protres.ru/ogp/

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btt168DOI Listing
June 2013

Backbone carbonyl group basicities are related to gas-phase fragmentation of peptides and protein folding.

Angew Chem Int Ed Engl 2007 ;46(9):1481-4

Laboratory for Biological and Medical Mass Spectrometry, BMC, Uppsala University, Box 583, 75123 Uppsala, Sweden.

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http://dx.doi.org/10.1002/anie.200603881DOI Listing
May 2007