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Physics 303/607 Biology 303/607 Regular class times: MWF 10-10:50 AM http://www.wfu.edu/~shapiro/biophysics10/ Instructors: (1) Professor Kim-Shapiro, Phone: 758-4993, Office: 208 Olin, e-mail: [email protected], http://www.wfu.edu/~shapiro/ (2) Professor Macosko, Phone: 758-4981, Office: 315 Olin, e-mail:[email protected], http://www.wfu.edu/~macoskjc/ Office hours: Mondays and Wednesdays 2-4 pm; By appointment. Texts: 1. Principles of Physical Biochemistry, by K.E. van Holde, W. C. Johnson, and P.S. Ho 2. Neurodynamix, by W.O. Friesen and J.A. Friesen. 3. Supplementary texts on reserve: 1. Biophysical Chemistry Part II, Techniques for the study of biological structure and function, by Charles Cantor and Paul Schimmel (1980). 2. Biochemistry by Lupert Stryer (1988). 3. Additional reading will be assigned in the form of journal articles and handouts Physics 303/607 Biology 303/607 Grading: Undergraduate Students: 2 Midterm exams...........................40% Project………………………………10% Final Exam.....................................30 % Problem Sets..................................20% Graduate Students: 2 Midterm exams........................... 30% Project……………………………….20% Presentation of Journal Article.......10% ** Final Exam.....................................30 % Problem Sets.................................20% Emphasis in grading will be placed on how each problem is solved. All work showing how the solution was obtained must be shown. An answer with the correct answer but poor method is inferior to one with the wrong answer but good method. Problem sets will generally be assigned for each chapter and the students will have one week to complete them. Students may help each other on problem sets but each student must write their own solution to each problem. The project that all students do will be a 5-10 page paper focusing on a pparticular topic in biophysics. The project could be a service learning project (see Dr. Kim-Shapiro for more information on that). ** Graduate students will present one of the journal articles that are part of the reading assignments (also see reading list). Physics 303/607 Biology 303/607 Exam Schedule: Midterm 1: Friday, Feb. 29 (in-class) Midterm 2: Friday, April 18 (in-class) Final Exam: Friday, April 30, (2-5 PM) Miscellaneous: We will, at times, look at structures that are deposited in the protein data bank (http://www.rcsb.org/pdb/home/home.do). The data bank contains the coordinates of all solved protein, DNA, RNA and other bio-molecular structures, usually to atomic resolution. Physics 303/607 Biology 303/607 Tentative Syllabus: Part I Biophysical Methods 1. Introduction (Macosko) (~6 lectures) Biological Macromolecules, Molecular interactions, overview of thermodynamics Reading: van Holde, chapters 1-4 (partial). 2. X-ray diffraction, DNA Structure (Macosko) (~5 lectures) Fourier Transforms, Scattering, r(x) F(q), A helix , History of Watson and Cricks' discovery and its implications Reading: van Holde chapter 6, Watson and Crick Papers 3. Light Scattering, Sedimenation, Gel Electrophoresis, Higher Order DNA Structure (Kim-Shapiro) (~4 lectures) Sedimenation, mass spectrometry, Gel electrophoresis (Fick's Law), Light Scattering (Classical, Dynamic, Polarized) DNA Topology (Length, Twist, and Writhe), Chromosome Structure Reading: van Holde, chapters 5 and 7, Polarized Light Scattering 4. Absorption Spectroscopy, Protein Structure (Kim-Shapiro) (~4 lectures) UV, VIS spectroscopy, linear and circular dichroism Protein primary, secondary, tertiary, quaternary structure Reading: van Holde chapters 8-10 Physics 303/607 Biology 303/607 Tentative Syllabus (cont.) 5. Emission Spectroscopy (Macosko) (~4 lectures) Reading: van Holde, Chapter 11 6. Single Molecule biophysics (Macosko) (~3 lectures) Reading: Chapter 16 7. Electron Paramagnetic Resonance, Protein Function - Hemoglobin (Kim-Shapiro) (~4 lectures) Electron Paramagnetic Resonance, Hemoglobin cooperativity Studies using EPR and time-resolved absorption spectroscopy Reading: Handout Part II Membrane Biophysics 8. Biological membranes and Transport (Kim-Shapiro) (~4 lectures) Description of membranes, Diffusion, Facilitated transport, Nernst Equation, Donnan Equilibrium Reading: van Holde chapters 13-14 9. Nerve Excitation (Kim-Shapiro) (~3 lectures) Neurons, Action Potential, Propagation of action potential, measurements in membrane biophysics, Synaptic transmission Reading: Frisens Sections 1 and 2 Introduction-1 Structures of biological Macromolecules Homework (due Friday, Jan. 29): 1. 2. 3. 4. 5. What is the Central Dogma of Molecular Biology? Van Holde 1.2 (amino acid structure) Van Holde 1.4 (amino acid structure) Van Holde 1.7 (DNA structure) Protein data bank exercises (extra handout, protein, DNA structure) Reading: Van Holde, Chapter 1 Van Holde Chapter 3.1 to 3.3 Van Holde Chapter 2 (we’ll go through Chapters 1 and 3 first.) Paper list (for presentations) is posted on web site Introduction-1 Structures of biological Macromolecules Outline: 1) What will we study? 2) Central Dogma (movie) 3) Structure of proteins & Genetic Code Introduction-1 Structures of biological Macromolecules • We will mainly deal with: proteins, nucleic acids, (e.g. DNA, RNA) and . membranes From Voet & Voet Biochemistry (e.g cell walls) • Physical methods to examine the structure and function of these biological molecules Central dogma of Molecular Biology (As spoken in the language of biology, i.e. narratives not equations) Transcription (RNA polymerase) Genomic DNA Reverse Transcription mRNA (reverse transcriptase) Protein (Enzymes catalyze reactions in organism) (Proteins – building blocks of organism) Biological Macromolecules – General Prinicples - Well-defined stoichiometry & geometry. Not readily broken into tiny pieces - Monomer is the building block (amino acid→proteins, nucleic acid→DNA/RNA) (Macro = large. Up to ~ 25 residues = oligomer; >25 polymer) • 1° structure: one-dimensional sequence • 2° structure: local arrangement (a-helices, b-sheets, turns) →super secondary structures: hairpins, corners, a-b-a motifs, etc. • 3° structure: 3-D structure (e.g. folded protein), stabilized by H-bond, hydrophobic forces, van-der-Waals, charge-charge, etc • 4° structure: Arrangement of subunits (e.g. hemoglobin) - Configuration vs. Conformation: • Configuration – Defined by chemical (covalent bonds), must break bond to change configuration (e.g. L-amino acid, D-amino acid) • Conformation – Spatial arrangement (e.g. an amino acid polymer can have a huge number of different conformations, one of which is the natively folded protein). Important Molecular interactions in Biomolecules The structure of proteins 1° structure: Amino acid sequence – Twenty amino acids common to all organisms. – Each has amino group, carboxyl group, R group and a hydrogen in tetrahedral symmetry. Almost all organisms have “L” chirality, but some virus have the mirror-image “D” chirality. (see board) – Linked together by peptide bond. Peptide bond can be trans or cis. – Proteins have prosthetic groups (e.g. heme) and amino acids can get modified (sugars, phosphates, etc). – Two important angles: Φ: N-Ca bond, Ψ: C-Ca bond Ramachandran plot of allowed angles (dis-allowed due to steric hindrance). The structure of proteins 1° structure: Amino acid sequence – Given N amino acids, there are 20N different sequences. Sequence determines structure. If >20% homologous, probably similar structure. Converse not true: very different sequences can have similar structures. – Hydrophobicity/hydrophilicity values [or “hydropathy” values, i.e. “strong feeling about”] determines protein folding. In aqueous environment, the core is hydrophobic, the surface is hydrophilic; in the membrane, both are hydrophobic. – Kyte-Doolittle Scale – measure of hydrophobicity. Hydrophobicity is determined by measuring the energy DGtrans of transfering an amino acid from water to an organic solvent. DG trasnfer RT ln P , where P aq nonaq , mole fraction • If DGtrans is positive – hydrophobic; if negative hydrophilic. – There are charged and uncharged side chains. Proteins have net charge and pockets of positive and negative charges, salt bridges. Isoelectric point: pH where net charge of protein is 0. Central dogma revisited Transcription (RNA polymerase) Genomic DNA mRNA Genetic Code Protein Genetic Code (these tables are just a piece of the “Genetic Code”) The G-ball: A new way to explore the Genetic Code Key: Starred residues use class-II (3’-OH charging, dimeric) aminoacyl synthetases. Residues in italics are charged (white: positive, black: negative). Size of font corresponds to residue size (and using all lowercase for smaller than average, all uppercase for larger than average, first letter uppercase for average). The structure of proteins • • Negatively charged 1° structure: A polymer with a unique amino acid sequence. There are twenty different amino acids -3 -2.6 Ala Arg 4 Asn Asp 2 Positively charged Cys Gln 0 1 2 3 4 5 -4.6 His Ile -2 Charged amino acids -4 6 Glu Gly Leu Lys Met -7.5 Phe Pro -6 -8 Ser Thr Trp Tyr Val -1.7 Nonpolar (hydrophobic) amino acids, aromatic The structure of proteins 1.0 2.5 • 1° structure: A polymer with a unique amino acid sequence. • There are twenty different amino acids Nonpolar (hydrophobic) amino acids, alkyl 1.0 2.3 2.2 Hydrophobic amino acids 3.1 -0.29 Nonpolar (hydrophobic) amino acids 1.1 Polar amino acids The structure of proteins 0.67 -0.75 • 1° structure: A polymer with a unique amino acid sequence. • There are twenty different amino acids -1.1 Polar amino acids, disulfide with adjacent Cys 0.17 Polar amino acids, amines Uncharged, polar amino acids -2.7 -2.9 Polar amino acids, aromatic 0.08 a-helix (© by Irvine Geis) The structure of proteins Biochemistry Voet & Voet 2° structure: alpha helix Alpha helix: - right-handed helix - 0.15 nm translation (rise) - 100° rotation (twist) - 3.6 residues/turn - Pitch: 0.54 nm - stabilized by H-bonds between NH and CO group (four residues up). Red – oxygen Black – carbon Blue – nitrogen Purple – R-group White – Ca Hydrogen-bonds between C-O of nth and N-H group of n+4th residue. The structure of proteins 2° structure: beta strand Beta sheet: - Can have parallel and anti-parallel - Distance between residues: 0.35 nm - H-bonds between NH and CO groups of adjacent strands stabilized structure. Note: Color-in atoms for practice The structure of proteins Higher Order Structure: Super secondary (+2°) structure: b turns, b-Hairpin, Greek Key, a-a, bab, b barrel H-bonding disfavored in aqueous environment b-sheets inside globular proteins (prions: a-helix b sheet) Domains (are to 3° structure as sheets and helices are to +2° structure): Structurally or functionally defined, eg calmodulin, DNA binding domain 3° Structure: Overall 3-D structure Next time: pictures of peptide chains in fibrinogen molecule Use sphere, ball and stick, ribbon representation 4° Structure Non-covalently linked 3° Structures (eg Hemoglobin ) Homodimer vs hetero dimer, Hemoglobin is a heterotetramer Dany’s lecture starts here Outline for Friday January 15, 2010 • Introductions – How much Biology, Physics, Chemistry have you had •Web Page incl. Service learning projects, HW etc •Review •Motivating question •Nucleic acids Introduction-1 Review. • Central Dogma • Primary, secondary, tertiary, quaternary structure – PDB and VMD/Rasmol • Molecular Interactions • Amino acids, peptide bonds, angles aq D G RT ln P , where P , mole fraction • Kyte Dolittle trasnfer nonaq • Genetic Code More detail on Kyte-Doolittle DG trasnfer RT ln P , where P • • • • aq nonaq , mole fraction They used water to vapor, others have used water to ethanol. If aq > nonaq then DG is positive – hydrophilic If nonaq > aq then DG is negative – hydrophobic e.g. DGtransfer for val is –2.78, DGtransfer for glu is +8.59 (in Kcal/mole) • Kyte and Doolittle actually used combnation of (1) –0.69 DGtransfer + 2.32 (2) (48.1)(fraction 100% buried) – 4.5 (3) (16.45)(fraction 95% buried). They combined these three things to get a hydropathy index. • Now hydrophobic is positive and hydrophilic is negative. Questions Consider the DNA from a single white blood cell of yours. a) If you were to stretch it all out, how long would it be? b) Is the DNA different from that cell than that from one of your endothelial cells? c) Is the DNA from your white blood cell different from that from the person sitting next to you? The structure of DNA and RNA • • • Four monomer building blocks RNA has ribose instead of 2’deoxyribose RNA has Uridine instead of Thymidine Stabilizing factors in double-stranded (ds)-DNA cruciform Triple-strand B-DNA: A-DNA: Z-DNA: - right-handed - right-handed - left-handed - most common form - broader than B - zig-zaggy - 0.34 nm rise - 0.26 nm rise - ~12 bp per turn -10.5 bp per turn - ~11 bp per turn - 3.4 nm pitch - 2.8 nm pitch - adopted sometimes by (CG)n repeats. - adopted in aqueous - adopted in non-aqueous - most common form for RNA - has “hole” down the center RNA molecules are more variable and can adopt structures that resemble proteins (e.g. t-RNA below). Aptamers are DNA and RNA molecules that fold into a 3D structure and bind substrates (much like proteins) What are aptamers? Aptamers (from apt: fitted, suited; Latin aptus: fastened) • Oligonucleotides which have a demonstrated capability to specifically bind molecular targets with high affinity (KD = 10-6 to 10-9 M). • First described by Joyce1 (1989), Tuerk & Gold2 and Ellington & Szostak2 (1990). • Binding properties depend on 3D structure and thus on sequence. 1 G. F. Joyce Gene 82: 83-87 (1989) 2C. Tuerk & L. Gold, Science 249, 505 (1990). 3A. D. Ellington & J. W. Szostak, Nature, vol. 346, pp. 818-822, 1990 Three-dimensional solution structure of the thrombinbinding DNA aptamer d(GGTTGGTGTGGTTGG) that we are working with (initially). Twist, rise and linking number in DNA Lk = Tw + Wr s = Wr/Tw Lk = linking number: Number of times one edge of ribbon linked around other – topological property cannot change w/o cutting. (calculate by Lk = Tw+Wr) Twist = winding of Watson around Crick – integrated angle of twist/2p along length, not an integer, necessarily (calculate by Tw = (number of base pairs/(base pairs/turn)) Writhe = wrapping of ribbon axis around itself – noninteger, geometric property Supercoiling (Writhe) important in vivo (most DNA is slightly negatively supercoiled). There are topoisomerases to convert topoisomers Problem A plectonemic helix of DNA is in the B form and has a total of 1155 basepairs. • What is the twist of the DNA? • The DNA has a superhelical density of -0.273. The DNA is put into an alcohol solution and it takes the A form. What is the DWr, DTw, DLk, and Ds? Compact 2 meters of DNA into mm-sized nucleus? (like folding a 1000 km long long fishing line (1 mm diameter) into 1m sized ball) Nucleosome http://www.rit.edu/~gtfsbi/IntroBiol/images/CH09/figure-09-07.jpg