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Theory and Simulations of Polyelectrolytes International Summer School June 23-25 2016, Lomonosov Moscow State University Natural polyelectrolytes Alexey Shaytan, PHD [email protected] 23 June 2016 https://goo.gl/cKnnbN Outline • Introduction: main class of natural polyelectrolytes • Key differences between natural and synthetic polymers • Main concepts and advances in molecular biology • Detailed discussion of main classes of natural polyelectrolytes and their complexes with examples: –Nucleic acids (DNA, RNA) –Proteins –Polysaccharides –Lipids What are natural polyelectrolytes? Natural polyelectrolytes are biomacromolecules which are at the same time polyelectrolytes IUPAC definitions* Polyelectrolyte molecule: A macromolecule in which a substantial portion of the constitutional units have ionizable or ionic groups, or both. Macromolecule = Polymer molecule: A molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass. Biomacromolecule: Macromolecule (including proteins, nucleic acids, and polysaccharides) formed by living organisms. Biopolymer: Substance composed of one type of biomacromolecules. Polymer: A substance composed of macromolecules. Common definitions A macromolecule is a very large molecule. *Terminology for biorelated polymers and applications (IUPAC Recommendations 2012) Classes of natural polyelectrolytes? Are the same as four classes of biopolymers Biopolymer classes • • • • Nucleic acids (DNA, RNA) Proteins Polysaccharides Lipids* DNA and RNA – are always polyelectrolytes. In other classes – depends on composition. Proteins on average have 20% of charged groups (in vertebrates). Phospholipids (membrane lipids) – 1-2 charges per lipid molecule. Polysaccharides may be uncharged or have highest density of all known biopolymers (heparin). *Not always considered as macromolecules or polymers. Natural polyelectrolytes: main locations Animal cell Bacterial cell RNA DNA Polysaccharides • • • Lipids Extra cellular matrix Cartilage, skin, etc. Bacterial cell envelopes • Membranes Virus Proteins • everywhere Diagram is not comprehensive and shows only example locations of biomacromolecules From monomers to polymers Class Monomers Monomer types # Topology nucleotide DNA/RNA 4 A,T/U,G,C Linear 20 Linear Many, +many modifications Linear or branched Amino acid Protein Monosaccharide Polysaccharide Lipid Lipid Many, Depends on organism Aggregates of individual molecules, e.g. Lipid bilayers Central dogma of molecular biology DNA replication In living organisms * Translation: Ribosomes make protein from mRNA Transcription: RNA polymerase copies DNA to mRNA *Image from: https://commons.wikimedia.org/wiki/File:Peptide_syn.png Central dogma of molecular biology https://en.wikipedia.org/wiki/Protein_production#/media/File:Genetic_code.svg Practical synthesis of biopolymers Obtaining and generating DNA DNA in vitro transcription • Solid-phase DNA synthesis RNA • Polymerase chain reaction (PCR) Expression system Protein purification Protein • Molecular cloning: use bacteria to copy DNA Folding and self-assembly Proteins, RNA, and DNA may self-assemble into 3D structures with extremely high precision Small soluble proteins spontaneously fold into unique 3D structure Ribosome 30S subunit formed by RNA and proteins DNA origami Box 1: protein folding Levinthal's paradox: very large number of degrees of freedom in an unfolded polypeptide chain, the molecule has an astronomical number of possible conformations. An estimate 10^143 was made in one of his papers. Sequences are optimized for folding. Folding funnel Folding pathways with intermediates billions of years Evolution, selection, mutations Mutations in DNA/RNA during copying Over billions of years nature performed vast sampling of sequence space for DNA, RNA and proteins Big numbers and sequence space • Atoms in visible universe ~10^81 • Number of theoretical proteins 100 amino acids in length ~10^130 • Cells in human body ~4*10^13 • Bacteria on Earth ~5*10^30 Box 2: Antibodies and immune system Antibodies may bind to other molecules with high affinity Generated by immune system via rapid sampling of protein sequence space Box 3: Aptamers, in vitro evolution Aptamer - oligonucleotide or peptide that binds to a specific target molecule. RNA aptamer binding vitamin B molecule SELEX method to generate RNA/DNA aptamers Macular degeneration Macugen – aptamer binds VEGF protein for treatment of AMD Key features of biopolymers What can we learn from nature? • Self-assembly • Precise non-covalent interactions between macromolecules • Program 3D structure via sequence • Library generation and artificial selection • Conversion of various polymers (DNA<->RNA->protein) • Use living systems as vehicles to produce polymers Why study biopolymers? We expect technological revolutions to happen in biotech soon. • Next-generation sequencing • Gene editing • Optogenetics Why study biopolymers? Synthetic biology – rational engineering of new organisms. • New layer of abstraction • Standardization of biological parts Electrical engineering Transistor Synthetic biology Gene Logic element Regulatory elements Integrated circuit Gene circuit Chassis Model organism Registry of standard biological parts pats.igem.org Part II – detailed discussion Nucleic acids Nucleic acids X=OH for RNA X=H for DNA X nucleoside nucleotide DNA bases: A-T, G-C RNA bases: A,U,G,C Uracil replaces Thymine in RNA Nucleic acids DNA forms A-DNA B-DNA RNA secondary structure Z-DNA RNA has A-from in double helix RNA hairpin Nucleic acids: RNA vs DNA Sugar puckering Only conformation adopted by RNA RNA has less conformational entropy, lower penalty for adopting various 3D structures A or B conformation depends on sugar puckering, which is affected by presence of 2’ OH group in RNA Chromatin Complex organism Bacteria Eukaryotic cell Cell nucleus 6 µm vs • Chromatin = DNA + proteins + RNA • Compacts DNA ~1000 000 times • • • • Nucleus - control center of the cell Turns genes on and off Responds to stimuli Has epigenetic memory Human DNA length: 2 meters Total body DNA length: 80 billion km 2 nm + + 10 nm 2x Tetramer = Core Histones Nucleosome structure 147 bp 2x 30 nm 300 nm Nucleosome core particle (NCP) Histone Tails 700 nm Linker DNA + Linker Histone Nucleosome = Nucleosome Chromatosome 1400 nm Felsenfeld and Groudine. Nature, 2003 3D-print your own nucleosome: Nucleosome LEGO project github.com/molsim/nuclLEGO 26 Nucleosome structure AK Shaytan, GA Armeev, A Goncearenco, VB Zhurkin, D Landsman, AR Panchenko, JMB, 2016 Part II – detailed discussion Proteins Proteins Amino acid Peptide bond Proteins: structure It is believed that hydrophobic collapse is the main driving force for protein folding Proteins: hydrophobic/hydrophilic balance For globular soluble proteins charged residues are exposed on the surface - charged Among totally non exposed residues charged residues are at ~6% Charged amino acid frequency in vertebrates (ASP,GLU,LYS,ARG) ~23% + charged hydrophobic Shaytan AK, Shaitan KV, Khokhlov AR. Biomacromolecules 2009, 10,1224-1237 Example: self-assembling fibrils EF-C peptide Amino-acid sequence: Gln-Cys-Lys-Ile-Lys-Gln-Ile-Ile-Asn-Met-Trp-Gln Nanofibrils (d=4 nm, l=100-400nm) + Viral vector + Cell = Up to 100 fold viral transduction enhancement Yolamanova M, Meier C, Shaytan AK, et al., Nature Nanotechnol. 2013, 8(2):130-6. Protein design Molecular mechanics force fields Problems: • Total free energy of folding is a sum of many opposing components (hydrophobic, electrostatic, polar, entropic) • Native proteins are marginally stable 5-15 kcal/mol between folded and unfolded state • Conformational space is huge! (Leventhal’s paradox) Protein design De novo protein design is possible due to advances in computational biology Part II – detailed discussion Polysaccharides Polysaccharides Polymers of monosaccharides (simple sugars) General formula for simple sugar Cyclic isomers of glucose Stereo isomers of glucose Amylose a polymer o glucose Amylopectin – branched form Polyelectrolytes among polysaccharides Glycosaminoglycans – essential components of extracellular matrix contributes to the tensile strength of cartilage, tendons, ligaments Heparin has the highest negative charge density of any known biological molecule. Agarose Part II – detailed discussion Lipids Lipids Hydrophobic or amphiphilic small molecules Lipid membranes Lipid membranes 5nm Atomistic models M. Bozdaganyan, maser thesis, 2010 Ion channels in membranes M.A. Kasimova, master thesis, 2011 10 ns M. Jensen, PNAS, 2010 Thank you for attention! Questions?