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Struktura polimerów i biopolimerów (3) Andrzej Koliński Pracownia Teorii Biopolimerów Wydział Chemii, Uniwersytet Warszawski [email protected] http://www.biocomp.chem.uw.edu.pl Łańcuch idealny (swobodnie związany): <R2> ~ n <S2>= <R2> /6 oraz Łańcuch rzeczywisty (atermiczny): <R2> ~ n6/5 Współczynnik ekspansji: α2 = <R2>/<R2o> α5−α3 = const. (1−Q/T) n1/2 (Teoria Flory,ego) Skrajne wartości T R 2 1/ 2 = aN R 3/ 5 T = Q: R 2 1/ 2 = aN1 / 2 2 1/ 2 aN 1/ 3 Polymer solutions and melts Polymer solutions and melts polymer melts many-chain problem 1 23 tagged chain m “tagged chain“ in a “matrix“ rm 0 n rn N-1 N Polymer entanglements De Gennes „reptation” theory De Gennes „reptation” theory • Originally proposed for polymer diffusion in crosslinked gels • Then extend on polymer solutions • Explains some experimental facts (3.0 and -2.0 exponents for the viscosity and diffusion chain length dependences) • Wrong exponent for viscosity (3.0 versus experimental 3.43.6) • Does not differentiate between the critical points for viscosity and diffusion • Completely wrong for cyclic polymers The problem can be resolved by properly designed computer simulations SIMULATIONS OF MULTIPLE CHAIN SYSTEMS – FAILURE OF THE REPTATION MODEL - Coarse-grained polymer model - Many chains in a periodic box - Monte Carlo dynamics - The snapshots of single chains shown for clarity - Lateral mode of motion dominates - The measured dynamic properties agree with experiment SIMULATIONS OF MULTIPLE CHAIN SYSTEMS – FAILURE OF THE REPTATION MODEL - Coarse-grained polymer model - Many chains in a periodic box - Monte Carlo dynamics - The snapshots of single chains shown for clarity - Lateral mode of motion dominates - The measured dynamic properties agree with experiment A. Kolinski, J. Skolnick and R. Yaris, "Monte Carlo Studies on the Long Time Dynamic Properties of Dense Cubic Lattice Multichain Systems. I. The Homopolymeric Melt", J. Chem. Phys. 86:71647174 (1987). A. Kolinski, J. Skolnick and R. Yaris, "Monte Carlo Studies on the Long Time Dynamic Properties of Dense Cubic Lattice Multichain Systems. II. Probe Polymer in a Matrix of Different Degrees of Polymerization", J. Chem. Phys. 86:7174-7180 (1987). Polymer motion in concentrated solutions and melts 1. Rouse-like motion of segments 2. Some segments move slower, due to the dragging of other chains Stress Polymers: Deformation I II III IV Strain I. Chain unfolding, unwinding, unwrapping or uncoupling (low energy) II. Chain sliding (low energy) III. Bond stretching, side group ordering (high energy) IV. Bond breaking (high energy) Stress Polymers: Deformation Ceramics Metals Polymers Strain •Lower elastic modulus, yield and ultimate properties •Greater post-yield deformability •Greater failure strain (Strain-Stress > odkształcenie-naprężenie) Polymers: Viscoelasticity • Dependency of stress-strain behavior on time and loading rate • Due to mobility of chains with each other • Crosslinking may affect viscoelastic response Stress increasing loading rate Strain Polymers: Thermal Properties decreasing temperature or increasing crystallinity log(Modulus) Stress • In the liquid/melt state enough thermal energy for random motion (Brownian motion) of chains • Motions decrease as the melt is cooled • Motion ceases at “glass transition temperature” • Polymer hard and glassy below Tg, rubbery above Tg Tg Tm semicrystalline crosslinked linear amorphous Strain Temperature Viscoelastic Deformation Glassy Materials and Tg Rigid Brittle Solid below Tg Viscous Deformable liquid above Tg F A d v dx η is the viscosity of the fluid Q o exp R T Viscoelastic Modulus versus Temperature Fracture of Polymers •Thermoset and Thermoplastic materials below Tg behave as brittle solids and fail by cracking. The cracks are sharp. •Crystalline Thermoplastic Resins above Tg yield and undergo ductile failure. •Noncrystalline Thermoplastic resins above Tg undergo Crazing and Cracking. • Crazing - Orientation of polymer chains across the opening of a “cracklike” feature. The work of crazing is 1000 times larger than the surface work to create a crack. •Cracking then occurs down the middle of the craze. Craze Formation Crack Propagation Through Crazed Area Cracking Example in Polymers Polymers and Biopolymers Molecular Biology and Structure of Biopolymers nature of the genetic code maintenance of genes through DNA replication transcription of information from DNA to mRNA translation of mRNA into protein. DNA mRNA protein J. Skolnick, J. Fetrow and A. Kolinski, "Structural genomics and its importance for gene function analysis", Nature Biotechnology, 18:283-287 (2000) Problem „drugiej części” kodu genetycznego Sequences and structures KNOWN STRUCTURES ~ 100,000 FR NEW FOLDS ? COMPARATIVE MODELING KNOWN SEQUENCES ~ 50,000,000 Ścieżka zwijania CI2 S. Kmiecik & A. Kolinski. „Characterization of Protein Folding Pathways by Reducedspace Modeling.” Proceedings of the National Academy of Sciences of the USA, 104(30):12330-5, 2007 Literatura J. Setubal, J. Meidanis: „Introduction to computational molecular biology” P. C. Turner, et al.: „Instatnt notes in molecular biology” C. Branden, J. Tooze: „Introduction to protein structure” Scheme of a cell: an introduction Nucleic acids (DNA and RNA) • Form the genetic material of all living organisms. • Found mainly in the nucleus of a cell (hence “nucleic”) • Contain phosphoric acid as a component (hence “acid”) • They are made up of nucleotides. Nucleotides • A nucleotide has 3 components – Sugar (ribose in RNA, deoxyribose in DNA) – Phosphoric acid – Nitrogen base • • • • Adenine (A) Guanine (G) Cytosine (C) Thymine (T) or Uracil (U) Purines & Pyrimidines Nucleic acids are polymers of nucleotides. Each nucleotide includes a base that is either a purine (adenine or guanine), or a pyrimidine (cytosine, uracil, or thymine). Nucleoside bases found in RNA: O NH2 N N N N H adenine (A) N HN H2N N guanine (G) O NH2 N H NH N N H cytosine (C) O N H uracil (U) O Some nucleic acids contain modified bases. Examples: Nucleoside bases found in RNA: O NH2 N N N N H N HN H2N adenine (A) O NH2 N H N NH N N H guanine (G) N H O cytosine (C) O uracil (U) Examples of modified bases found in tRNA: O NH2 CH3 NH2 + H3C N +N N N H N HN H2N N N H O + CH3 N N H HN O NH N H O 1-methyladenine (m1A) 7-methylguanine (m7G) 3-methylcytosine (m3C) pseudouracil () In a nucleotide, e.g., adenosine monophosphate (AMP), the base is bonded to a ribose sugar, which has a phosphate in ester linkage to the 5' hydroxyl. NH2 NH2 N N N adenine N N N H 2 O 3P HO 5' CH2 ribose adenine H O 3' OH H OH 2' adenosine N N O CH2 H 1' H N N N N 4' NH2 H O H H OH H OH adenosine monophosphate (AMP) Nucleic acids have a backbone of alternating Pi & ribose moieties. Phosphodiester linkages form as the 5' phosphate of one nucleotide forms an ester link with the 3' OH of the adjacent nucleotide. A short stretch of RNA is shown. NH2 adenine N N 5' end O O P NH2 N N cytosine 5' O CH2 4' O H O H 1' H ribose O O O 5' CH2 O O H H H O P O ribose H OH 3' O nucleic acid N H OH 2' 3' P N O O (etc) 3' end H cytosine (C) N N O O H guanine (G) N N N G H N N H C NH H H G C base pair in tRNA Hydrogen bonds link 2 complementary nucleotide bases on separate nucleic acid strands, or on complementary portions of the same strand. Conventional base pairs: A & U (or T); C & G. In the diagram at left, H-bonds are in red. Bond lengths are inexact. The image at right is based on X-ray crystallography of tRNAGln. H atoms are not shown. Nucleotides Nitrogenous Base Phosphate Group Sugar Nitrogenous Base Phosphate Group Sugar DNA RNA A G A A=T G=C G C C G G A A C TU C T U G G Watson and Crick DNA Proteins • Composed of a chain of amino acids. 20 possible groups R | H2N--C--COOH | H Fig. 6.1 Fig. 6.2. Acidic and basic amino acids. Fig. 6.2. Neutral, non-polar (hydrophobic) amino acids. Fig. 6.2. Neutral, polar (hydrophilic) amino acids. Proteins R | H2N--C--COOH | H R | H2N--C--COOH | H Amino acids are joined to form unbranched polypeptides by a peptide bond. Covalent bond between the carboxyl group of one amino acid and amino group of the next amino acid. Fig. 6.3 N-terminus C-terminus 5’ (DNA) 3’ (DNA) Dipeptide This is a peptide bond R O R | II | H2N--C--C--NH--C--COOH | | H H Peptide bond Protein structure • Linear sequence of amino acids folds to form a complex 3-D structure. • The structure of a protein is intimately connected to its function. Proteins show four hierarchical levels of structural organization: 1. Primary structure = amino acid sequence 2. 3. Secondary structure = folding and twisting of a single polypeptide chain. Result of weak H-bonds and electrostatic interactions e.g., -helix (coiled) and -pleated sheet (zig-zag). Tertiary structure = three dimensional shape (or conformation) of a polypeptide chain. 4. Determined by the genetic code of the mRNA. Function of R groups contained in the polypeptide. Quaternary structure = association between polypeptides in multi-subunit proteins (e.g., hemoglobin). Occurs only with two or more polypeptides. Fig. 6.4 DNA in action • Questions about DNA as the carrier of genetic information: – How is the information stored in DNA? – How is the stored information used ? The need for an intermediary • Fact 1 : Ribosomes are the sites of protein synthesis. • Fact 2 : Ribosomes are found in the cytoplasm. 2 types of cells: Prokaryotes v.s.Eukaryotes The need for an intermediary The need for an intermediary • Fact 1 : Ribosomes are the sites of protein synthesis. • Fact 2 : Ribosomes are found in the cytoplasm. • Question : How does information ‘flow’ from DNA to protein? The Intermediary • Ribonucleic acid (RNA) is the “messenger”. • The “messenger RNA” (mRNA) can be synthesized on a DNA template. • Information is copied (transcribed) from one strand of DNA to mRNA. (TRANSCRIPTION) Next question… • How do I interpret the information carried by mRNA? • Think of the sequence as a sequence of “triplets”. • Think of AUGCCGGGAGUAUAG as AUGCCG-GGA-GUA-UAG. • Each triplet (codon) maps to an amino acid. The Genetic Code • f : codon amino acid • 1968 Nobel Prize in medicine – Nirenberg and Khorana • Important – The genetic code is universal! • It is also redundant / degenerate. How was the genetic code deciphered? 1. Cell-free , protein synthesizing machinery isolated from E. coli. (ribosomes, tRNAs, protein factors, radio-labeled amino acids). Synthetic mRNA containing only one type of base: UUU = Phe, CCC = Pro, AAA = Lys, GGG = ? 2. Synthetic copolymers (CCC, CCA, CAC, ACC, CAA, ACA, AAC, AAA): Pro, Lys (already defined) + Asp, Glu, His, & Thr Proportion (%AC) varied to determine exactly which codon specified which amino acid. 3. Synthetic polynucleotides of known composition: UCU CUC UCU CUC Ser Leu Ser Leu 1968: Robert Holley (Cornell), H. G. Khorana (Wisconsin-Madison), and Marshal Nirenberg (NIH). The Genetic Code Fig. 6.8 Translation • The sequence of codons is translated to a sequence of amino acids. • Transfer RNA (tRNA) – a different type of RNA – matches amino acids to codons in mRNA. – Freely float in the cytoplasm. – Every amino acid has its own type of tRNA that binds to it alone. • Anti-codon – codon binding crucial. • Show animation tRNA tRNA tRNA tRNA 3d structure The gene and the genome • A sequence of nucleotides on the DNA that encodes a polypeptide is called a gene. • Genome = Set of all genes in the organism + junk stuff (the entire DNA content). More complexity • The RNA message is sometimes “edited”. • Exons are nucleotide segments whose codons will be expressed. • Introns are intervening segments (genetic gibberish) that are snipped out. • Exons are spliced together to form mRNA. Splicing frgjjthissentencehjfmkcontainsjunkelm thissentencecontainsjunk Central Dogma of Molecular Biology DNA RNA Protein Phenotype • • Transcription : DNA RNA • Translation : RNA Protein Central dogma ZOOM IN tRNA transcription DNA rRNA snRNA translation mRNA POLYPEPTIDE Information flow Transcription – key steps DNA • Initiation • Elongation • Termination Transcription – key steps DNA • Initiation • Elongation • Termination Transcription – key steps DNA • Initiation • Elongation • Termination DNA + RNA Promoters • Promoters are sequences in the DNA just upstream of transcripts that define the sites of initiation. Promoter 5’ 3’ • The role of the promoter is to attract RNA polymerase to the correct start site so transcription can be initiated. Step 2-Initiation-requirements: 1. 2. 3. 4. 5. 6. mRNA Ribosome Initiator tRNA (fMet tRNA in prokaryotes) 3 Initiation factors (IF1, IF2, IF3) Mg2+ GTP (guanosine triphosphate) Fig. 6.17 Fig. 6.18 Genes can be switched on and off • In an adult multicellular organism, there is a wide variety of cell types seen in the adult. eg, muscle, nerve and blood cells. • The different cell types contain the same DNA though. • This differentiation arises because different cell types express different genes. Regulation of genes • What turns genes on and off? • When is a gene turned on or off? • Where (in which cells) is a gene turned on? • How many copies of the gene product are produced? Regulatory sequences • These are binding sites for proteins, often short stretches of DNA (~25 nucleotides). • Inexactly repeating patterns (“motifs”). • Motifs stand out as highly conserved regions in a multiple sequence alignment. Regulatory sequences