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Fundamentals of protein and nucleic acid structure Lecture 2 Structural Bioinformatics Dr. Avraham Samson 81-871 Tree of Life All known life forms use the same building blocks suggesting there was a common ancestor The tree of life is not that simple! Gene transfer across kingdoms continually occurs (cyanobacteria became chlorophylls, proteobacteria mitochondria, viruses) Inside living cells 29 atoms of life Most common elements: H, O, C, N, P, S (97% of organism weight) Most common ions: Ca2+, K+, Na+, Mg2+, Cl- Forces affecting structure: • • • • • H-bonding Van der Waals Electrostatics Hydrophobicity Disulfide Bridges d 2.6 Å < d < 3.1Å 150° < θ < 180° Forces affecting structure: • • • • • H-bonding Van der Waals Electrostatics Hydrophobicity Disulfide Bridges Repulsion דחייה Attraction משיכה d 3 Å < d < 4Å Forces affecting structure: • • • • • H-bonding Van der Waals Electrostatics Hydrophobicity Disulfide Bridges Coulomb’s law d d = 2.8 Å “IONIC BOND” קשר יוני “SALT BRIDGE” גשר מלח E = Energy k = constant D = Dielectric constant (vacuum = 1; H2O = 80) q1 & q2 = electronic charges (Coulombs) r = distance (Å) Forces affecting structure: • • • • • H-bonding Van der Waals Electrostatics Hydrophobicity Disulfide Bridges Forces affecting structure: • • • • • H-bonding Van der Waals Electrostatics Hydrophobicity Disulfide Bridges Other names: cystine bridge disulfide bridge Hair contains lots of disulfide bonds which are broken and reformed by heat10 Levels of protein structure Primary: Amino acid sequence Secondary: Local fold pattern of small subsequence Tertiary: Fold of entire protein chain Quaternary: Complex of multiple chains Primary (1o) structure The amino acid sequence is the primary structure Primary (1o) structure 20 amino acids 13 Primary (1o) structure 14 Primary (1o) structure 15 pKa = pH of 50% dissociation 16 Primary (1o) structure Amino acid nomenclature 17 Primary (1o) structure • Amino acids polymerize through peptide bonds to form polypeptides 18 Primary (1o) structure • N-terminal is the start of a polypeptide chain • Amino acids are also called residues 19 Primary (1o) structure side chains שייר צדדי backbone שלד 20 Primary (1o) structure • Post translational modification are important because they can change the function of proteins (i.e. phosphorylation, acetylation, hydroxylation, carbohydrate and lipid modifications) N-terminal acetylation O-phosphotyrosine hydroxyproline γ-carboxyglutamate 21 Amino acids chirality Enantiomers –mirror images - תמונת ראי- אננטיומרים chiral center C מרכז כיראלי Dextro-Laevus in Latin 22 Most proteins: only L amino acids C chiral center is L configuration איך לקבוע קונפיגורציה LוD- •סדר את האטומים לפי עדיפות על פי הכללים הבאים כלל .1אטום עם מספר אטומי יותר גבוה בעל סדר עדיפות יותר גבוה. )(I > Cl > O > N> C > H כלל .2אם האטומים זהים ,העדיפות על פי האטומים המתמירים )(C(CH3)3 > CH(CH3)2 > CH2CH3 > CH3 •שים את האטום עם סדר העדיפות הכי נמוך מאחור. • קבע את כיוון החץ ,מסדר עדיפות הכי גבוה לככוון הכי נמוך .אם החץ: 23רמז יותר קל בשביל לזכור ) CORN :תירס( עם כיוון השעון קונפיגורציה D נגד כיוון השעון קונפיגורציה L Bond angles Torsion angles w, φ, and ψ • Unlike w, the two backbone dihedral angles φ and ψ are free to rotate • This rotation freedom allows protein folding w Dihedral angles: -180o < φ < +180o -180o < ψ < +180o w is 0o or 180o 25 Peptide bond is planar Cα, C, O, N, H, Cα all lie in the same plane 26 Torsion angle (w) is usually trans Steric hindrance Question: What other residues can be cis? 27 Except for X-Pro bond in which cis is preferred Steric hindrance Steric hindrance Steric hindrance allows both cis and trans (4:1 ratio) 28 Ramachandran Diagrams Preferred regions • Steric hindrance dictates torsion angle preference • Ramachandran plot show preferred regions of φ and ψ dihedral angles 30 Secondary (2o) structure Helices • -helix • 310-helix • p-helix b Sheets • Antiparallel • Parallel Turns • b,g,,and p Unstructured α-helix is the most common 3.6 residues per turn (number of residues in one full rotation of 360°) 5.4 Å pitch (translation along axis for one full rotation of 360°) Hydrogen bond: i→i+4 Secondary (2o) structure Helices • -helix • 310-helix • p-helix b Sheets • Antiparallel • Parallel Turns • b,g,,and p Unstructured 310-helices are rare in proteins: 3.1 residues per turn (number of residues in one full rotation of 360°) 6 Å pitch (translation along axis for one full rotation of 360°) Hydrogen bond: i→i+3 Secondary (2o) structure Helices • -helix • 310-helix • p-helix b Sheets • Antiparallel • Parallel Turns • b,g,,and p Unstructured π -helices are rare in proteins 4.3 residues per turn (number of residues in one full rotation of 360°) 6 Å pitch (translation along axis for one full rotation of 360°) Hydrogen bond: i→i+5 Hydrogen bonding in helices • CO of residue (n) forms an h-bond with NH of residue: – (n+4) → α-helix – (n+3) → 310-helix – (n+5) → π-helix 34 Ribbon diagram of α-helix 35 Ramachandran of α-helix 36 Secondary (2o) structure Helices • -helix • 310-helix • p-helix b Sheets • Antiparallel • Parallel Turns • b,g,,and p Unstructured In antiparallel b-sheets •Adjacent β-strands run in opposite directions •Hydrogen bonds (dashed lines) between NH and CO stabilize the structure •The side chains (in green) are above and below the sheet Secondary (2o) structure Helices • -helix • 310-helix • p-helix b Sheets • Antiparallel • Parallel Turns • b,g,,and p Unstructured In parallel b-sheets •Adjacent β-strands run in same direction •Hydrogen bonds (dashed lines) between NH and CO stabilize the structure •The side chains (in green) are above and below the sheet Ribbon diagram of β sheet • In addition to being purely parallel or antiparallel, β sheets can be mixed, with strands running in both parallel and antiparallel directions • Arrow pointing to C-terminal end 39 Ramachandran of β-sheet 40 Secondary (2o) structure Helices • -helix • 310-helix • p-helix b Sheets • Antiparallel • Parallel Turns • b,g,,and p Unstructured CO of residue i forms h-bonds with NH of either residue i+2, i+3, i+5, or i+4 β-turn i→i+3 H-bond (most common) γ-turn: i→i+2 H-bond α-turn i→i+4 H-bond π-turn i→i+5 H-bond Secondary (2o) structure Helices • -helix • 310-helix • p-helix b Sheets • Antiparallel • Parallel Turns • b,g,,and p Unstructured Tertiary (3o) structure • Proteins fold into compact tertiary structures with hydrophobic cores. (spacefill representation) 43 Structural classification: http://www.cathdb.info/cgi-bin/cath/GotoCath.pl?link=class.html Tertiary (3o) structure • It is a bit difficult to see and understand anything here (sticks representation) 44 Tertiary (3o) structure • To simplify representation, secondary structure diagrams are used 45 Quaternary (4o) structure Dimer of identical subunits (Cro protein of bacteriophage lambda) Quaternary structure • • • • • • Coat of rhinovirus (common cold) 60 copies of 4 different subunits, 3 outside, red, blue, green 47 Protein structure databases • Primary: UniProt http://www.uniprot.org/ • Secondary: DSSP http://rcsb.org • Tertiary: PDB http://rcsb.org • Quaternary: PQS http://www.ebi.ac.uk/pdbe/pqs/ DNA and RNA Structure NOTE: • • • • • • Components • Sugar • Base • Phosphate 5’ to 3’ direction T->U in RNA RNA - extra –OH at 2’ of pentose sugar DNA - deoxyribose Numbering • Single vs double strands • DNA more stable Voet, Donald and Judith G. Biochemistry. John Wiley & Sons, 1990, p. 792. 49 The 5 Bases of DNA and RNA Purines NOTE: • • • • • • • Pyrimadines and Purines T->U in RNA Names Numbering Bonding character Position of hydrogen Tautomers Pyrimadines 50 Tautomeric Structures • Keto vs enol (OH) • Different hydrogen bonding patterns 51 Geometry of Watson Crick Base Pairs • A:T and G:C pairs are spatially similar • 3 H-bonds vs 2 (GC rich?) • Sugar groups are attached asymmetrically on the same side of the pair • Leads to a major and minor grove • Bases are flat but the hydrogen bonding leads to considerable flexibility • Base stacking is flexible Voet, Donald and Judith G. Biochemistry. John Wiley & Sons, 1990, p. 797. Pharm201 Lecture 2 2010 52 Definition of Major and Minor Groove Hydrogen bonding of WC base pair Mechanisms of recognition The canonical Watson-Crick base pair, shown as the G-C pair. Positions of the minor and major grooves are indicated. The glycosidic sugar-base bond is shown by the bold line; hydrogen bonding between the two bases is shown in dashed lines. Pharm201 Lecture 2 2010 53 Base Morphology The base-pair reference frame is constructed such that the x-axis points away from the (shaded) minor groove edge. Images illustrate positive values of the designated parameters. Reprinted with permission from Adenine Press from (Lu, et al., 1999). Pharm201 Lecture 2 2010 54 Backbone Conformation Voet, Donald and Judith G. Biochemistry. John Wiley & Sons, 1990, p. 807. Pharm201 Lecture 2 2010 55 A Beta-nucleoside • Ring is never flat – has 5 internal torsional angles • The pucker is determined by what is bound • A variety of puckers have been observed • Pucker has a strong influence on the overall conformation Pharm201 Lecture 2 2010 56 The Glycosidic Bond Anti Syn • Connects ribose sugar to the base 57 Change in sugar conformation affects the backbone C3’ C2’ C3’-Endo C3’ C2’ C2’-Endo 58 ..and the position of A DNA the bases relative to the helix axis B DNA Pharm201 Lecture 2 2010 59 Canonical B DNA Neidle, Stephen. Nucleic Acid Structure and Recognition. Oxford University Press, 2002, p. 34. Pharm201 Lecture 2 2010 60 Canonical B DNA • First determined experimentally by fiber diffraction (Arnott) • C2’-endo sugar puckers • High anti glycosidic angles • Right handed – 10 base pairs per turn • Bases perpendicular to the helix axis and stacked over the axis • Overall bending as much as 15 degrees (result of base morphologies – twist and roll) – {machine learning – sequence vs overall conformation?} • Over 230 structures 25 with base mis-pairing – only cause local perturbations • Strong influence of hydration along spine http://ndbserver.rutgers.edu/index.html Pharm201 Lecture 2 2010 61 A DNA Neidle, Stephen. Nucleic Acid Structure and Recognition. Oxford University Press, 2002, p. 36. Pharm201 Lecture 2 2010 62 Canonical A DNA • C3’-endo sugar puckers – brings consecutive phosphates closer together 5.9A rather than 7.0 • Glycosidic angle from high anti to anti • Base pairs twisted and nearly 5A from helix axis • Helix rise 2.56A rather than 3.4A • Helix wider and 11 base pairs per repeat • Major groove now deep and narrow • Minor grove wide and very shallow Pharm201 Lecture 2 2010 63 Z-DNA Pharm201 Lecture 2 2010 64 Z-DNA • • • • • • • • • Helix has left-handed sense Can be formed in vivo, given proper sequence and superhelical tension, but function remains obscure. Narrower, more elongated helix than A or B. Major "groove" not really groove Narrow minor groove Conformation favored by high salt concentrations, some base substitutions, but requires alternating purine-pyrimidine sequence. N2-amino of G H-bonds to 5' PO: explains slow exchange of proton, need for G purine. Base pairs nearly perpendicular to helix axis GpC repeat, not single base-pair – P-P distances: vary for GpC and CpG – GpC stack: good base overlap – CpG: less overlap. • • Zigzag backbone due to C sugar conformation compensating for G glycosidic bond conformation Conformations: – G; syn, C2'-endo – C; anti, C3'-endo Pharm201 Lecture 2 2010 65 Z-DNA • • • • Convex major groove Deep minor groove Alternate C then G Spine of hydration Pharm201 Lecture 2 2010 66 Quadruplex DNA 1NP9 Jmol Pharm201 Lecture 2 2010 67 tRNA Invariant L-shape 1EVV jmol Saenger, Wolfram. Principles of Nucleic Acid Structure. Springer-Verlag New York Inc., 1984, p. 333. Pharm201 Lecture 2 2010 68 tRNA H bonds between distant regions Neidle, Stephen. Nucleic Acid Structure and Recognition. Oxford University Press, 2002, p. 148. Pharm201 Lecture 2 2010 69 The Ribosome • Complex of protein and RNA • Small 30S subunit – controls interactions between mRNA and tRNA • Large 50S subunit – peptide transfer and formation of the peptide bond Pharm201 Lecture 2 2010 70 Nucleic acid structure databases • Primary: UniProt http://www.uniprot.org/ • Tertiary: PDB http://rcsb.org contains all of http://ndbserver.rutgers.edu/ Molecular viewers • Pymol • Rasmol • Jmol • Molmol • VMD • Swiss PDB etc… (please note, only freeware are listed) Transcription and translation (DNA RNA Protein) Transcription and translation (DNA Protein)