Regular class times: MWF 10-10:50 AM Physics 307/607Biology 307/607 Instructors: (1) Professor Martin Guthold, Phone: , Office: 302 Olin,(2) Professor Kim-Shapiro, Phone: , Office: 208 Olin,Office hours: Guthold: M, W, F, 2:00 pm 3:00 pm, and by appointment. Kim-Shapiro: W, T, 2:15 pm 4:00 pm, and by appointment Texts: 1.Principles of Physical Biochemistry, 2 nd ed. 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 Syllabus 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. Homework: 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. TA: Jiajie, Olin 311, The project that all students do will be a 5-10 page paper focusing on a particular topic in biophysics. The project could be a service learning project (see instructors for more information on that). Project topic is due in two weeks (Wednesday, Jan. 27) Project outline is due before spring break (Friday, March 4) Complete project due last day of class (Wednesday, April 27) ** Graduate students need to do a 5-10 minutes presentation on one of the journal articles that are part of the reading assignments (see reading list); or another article relevant to a lecture topic. Graduate Students: 2 Midterm exams % Project.10% Presentation of Journal Article % ** Final Exam % Problem Sets % Grading: Undergraduate Students: 2 Midterm exams % Project10% Final Exam % Problem Sets % Exam Schedule: Midterm 1: Friday, Feb. 26 (in-class) Midterm 2: Wednesday, April 20 (in-class) Final Exam: Friday, April 29, (9:00 am 12:00 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. Over 114,000 structures (Jan. 2016).http://www.rcsb.org/pdb/home/home.do Syllabus Tentative Syllabus: 1.Introduction (Guthold) (~9 lectures) 1.1 Biological Macromolecules; 1.2 Molecular interactions; 1.3 Overview of Thermodynamics Reading: van Holde, chapters 1-4 (partial). 2.Sedimenation, Gel Electrophoresis, Higher Order DNA Structure, Light Scattering, (Kim-Shapiro) (~8 lectures) Sedimenation, mass spectrometry, Gel electrophoresis (Fick's Law), Light DNA Topology (Length, Twist, and Writhe), Chromosome Structure Scattering (Classical, Dynamic, Polarized) Reading: van Holde, chapters 5 and 7, Polarized Light Scattering 3.X-ray diffraction, DNA Structure (Guthold) (~5 lectures) Braggs law, von Laue condition, Fourier Transforms, Scattering, (x) F(q), A helix, History of Watson and Cricks' discovery and its implications Reading: van Holde chapter 6, Watson and Crick Papers 2.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 Syllabus Tentative Syllabus (cont.) 5.Biological membranes and Transport (Kim-Shapiro) (~3 lectures) Description of membranes, Diffusion, Facilitated transport, Nernst Equation, Donnan Equilibrium Reading: van Holde, chapters 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 7.Emission Spectroscopy (Guthold) (~3 lectures) Reading: van Holde, Chapter 11 8.Single Molecule biophysics (Guthold) (~3 lectures) Reading: van Holde, Chapter 16 Syllabus Reading: Van Holde, Chapter 1 Van Holde Chapter 3.1 to 3.3 Van Holde Chapter 2 (well go through Chapters 1 and 3 first.) Homework (due Wednesday, Jan. 27): 1.What is the Central Dogma of Molecular Biology? Describe, sketch in your own words. 2.Van Holde 1.2(amino acid structure) 3.Van Holde 1.7(DNA structure) 4.Protein data bank exercises (see extra handout) 5.Protein & DNA structure exercises (see extra handout) Paper list (for presentations) is posted on web site:Introduction-1 Structures of Biological Macromolecules In this course we will mainly study three of the four main types of Biomacromolecules found in all living organisms*: nucleic acids (DNA, RNA), amino acids (proteins),lipids (membranes) Look at physical methods to examine the structure and function of these biological molecules * A fourth class is carbohydrates, but we will not talk much about those From: Voet & Voet Biochemistry Introduction-1 Structures of Biological Macromolecules AFM image of -DNA (Guthold group) DNA mismatch repair protein MutS (image: Salsbury group) Bovine pulmonary artery endothelial cells Image: Justin Sigley, WFU Physics) Triple stain: DNA (nucleus ) blue Actin fibers red Microtubules green Scale bar: 10 m Introduction-1 Structures of Biological Macromolecules Outline Nucleic acids, DNA, RNA DNA structure, twist, rise, linking number Amino acids, proteins Protein structure, 1 o, 2 o, 3 o, 4 o structure Properties of amino acids, (small, large, neutral, charged, hydrophobic, hydrophilic, etc.) Protein data bank (PDB) Central Dogma, Replication, Transcription, Translation Genetic code, DNA/RNA codons Biological Macromolecules General Prinicples - Well-defined stoichiometry & geometry. Not readily broken into tiny pieces - Monomer is the building block (nucleic acid DNA/RNA; amino acid proteins) (Macro = large. Up to ~ 25 residues = oligomer; >25 = polymer) 1 structure: one-dimensional sequence 2 structure: local arrangement ( -helices, -sheets, turns); sometimes super- secondary structures: hairpins, corners, 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). Nucleosome How to compact 2 meters of DNA into 2 m-sized nucleus? (like folding a 1000 km long long fishing line (1 mm diameter) into 1m sized ball) 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 Later: This is also how DNA and RNA match up (hybridize) in the binding pocket of RNA polymerase during transcription! Normal Watson-Crick base pairing BaseBase plus ribose sugar Nucleoside (RNA) Base plus deoxy ribose sugar Deoxy-nucleoside (DNA) Base plus ribose sugar plus phospate (nucleotide)* Adenine (A)Adenosine (A)Deoxy-adenosine (dA)Adenosine monophospate (AMP) Cytosine (C) Cytidine (C)Deoxy-cytidine (dC)Cytidine monophospate (CMP) Guanine (G) Guanosine (G)Deoxy-guanosine (dG)Guanosine monophospate (GMP) Thymine (T)(Methyluridine, m 5 U)Thymidine (dT)m 5 UMP Uracil (U)Uridine (U)Deoxy-urdine (dU)Uridine monophosphate (UMP) A bit of nucleic acid nomenclature * Can also have two or three phosphates, and de-oxy variety, too B-DNA (most common): - right-handed nm rise bp per turn nm pitch - adopted in aqueous - 2 nm diameter A-DNA: - right-handed - broader than B nm rise - 11 bp per turn nm pitch - adopted in non-aqueous - most common form for RNA nm diameter - 19 o inclination of base pairs Z-DNA: - left-handed - zig-zaggy - ~12 bp per turn - adopted sometimes by (CG) n repeats nm diameter cruciform Triple-strand The structure of DNA and RNA RNA molecules are more variable and can adopt structures that resemble proteins (e.g. t-RNA below). Twist, rise and linking number in DNA L = T + W L, linking number: Number of times one edge of ribbon linked around other topological property cannot change w/o cutting. (calculate by L = T + W) T, twist = winding of Watson around Crick integrated angle of twist/2 along length, not an integer, necessarily (calculate by T = (number of base pairs/(base pairs/turn)) W, writhe = wrapping of ribbon axis around itself noninteger, geometric property Supercoiling (Writhe) important in vivo (most DNA is slightly negatively supercoiled). = superhelical density Note: There are topoisomerases to convert topoisomers. They can remove a knot by breaking double-stranded DNA and re-ligating DNA. Mutated topoisomerases cause cancer. = W/T Sample problem A circular, plectonemic (braided) helix of DNA is in the B form and has a total of 1155 base pairs. 1.What is the twist of the DNA? 2.The DNA has a superhelical density of The DNA is put into an alcohol solution and it takes the A form. What is the W, T, L, and ? 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: : N-C bond, : C-C bond Ramachandran plot of allowed angles (dis-allowed due to steric hindrance). Trans and cis peptide bond The structure of proteins Given N amino acids, there are 20 N 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 G trans of transferring an amino acid from organic solvent (or vapor) to water (more in introduction-3*). If G trans 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. 1 structure (primary structure): Amino acid sequence Kyte-Doolittle scale also uses structural data, in addition to G transfer The structure of proteins 1 structure: A polymer with a unique amino acid sequence. There are twenty different amino acids Charged amino acids Positively charged Negatively charged Source: Kyte J & Doolittle, RF; J. Mol. Biol. 157, 110 (1982) The structure of proteins 1 structure: A polymer with a unique amino acid sequence. There are twenty different amino acids Hydrophobic amino acids Nonpolar (hydrophobic) amino acids, aromatic Nonpolar (hydrophobic) amino acids, alkyl Nonpolar (hydrophobic) amino acids Source: Kyte J & Doolittle, RF; J. Mol. Biol. 157, 110 (1982) The structure of proteins 1 structure: A polymer with a unique amino acid sequence. There are twenty different amino acids Uncharged, polar amino acids Polar amino acids, aromatic Polar amino acids, amines Polar amino acids, disulfide with adjacent Cys Polar amino acids Source: Kyte J & Doolittle, RF; J. Mol. Biol. 157, 110 (1982) The structure of proteins Alpha helix: - right-handed helix nm translation (rise) - 100 rotation (twist) residues/turn - Pitch: 0.54 nm - stabilized by H-bonds between NH and CO group (four residues up). 2 structure (secondary structure): alpha helix -helix ( by Irvine Geis) Biochemistry Voet & Voet Red oxygen Black carbon Blue nitrogen Purple R-group White C Hydrogen-bonds between C-O of n th and N-H group of n+4 th residue. The structure of proteins 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. 2 structure: beta strand Note: Color-in atoms for practice The structure of proteins 3 10 helix: - right-handed helix nm translation (rise) - 120 rotation (twist) residues/turn - Pitch: 0.60 nm - stabilized by H-bonds between NH and CO group (three residues up). 2 structure: 3 10 helix The structure of proteins Domains: Structurally or functionally defined protein regions, e. g. DNA binding domain 3 Structure (tertiary structure): Overall three dimensional structure of whole protein 4 Structure (quaternary structure): Larger assembly of several proteins or subunits (non-covalently linked or linked by cystines (e. g., hemoglobin 2 ) Higher Order Structure: Super secondary (+2) structure: turns, -Hairpin, Greek Key, , , barrel (a) Stick model; (b) van der Waals surface model; (c) ribbon model; (d) solvent accessible surface model; (e) caricature of molecule. Example: Structure of Fibrinogen (look at this structure in Protein Data Bank) Six polypeptide chains: 2 A (610 a.a.), 2 B (461 a.a.), and 2 (411 a.a.) (human numbering). Trinodular: 2 external D nodules; central E nodule (N-termini) Parts not resolved: loopy -C region stretching back to E nodule (after residue 220), N-terminal of - and - chains (fibrinopeptides A and B) and N-terminal of -chain (2x96 residues), C- terminal of -chain(2x 16 residues). Dimensions: about 45 nm x 4.5 nm 17 disulfide bonds: within E nodule and braces at ends of the alpha helix coiled coils E noduleD nodule Crystal structure of Chicken Fibrinogen (2x 1364 a.a.). Z. Yang, J. M. Kollman, L. Pandi, R. F. Doolittle, Biochemistry 40, (2001) b-hole a-hole Formation of Fibrin Fibers (major structural component of blood clots) + thrombi n fibrinoge n fibrin B A Fibrinogen b a Fibrinopeptides A & B Thrombin Fibrin (protofibrils) Protofibril formation Further lateral aggregation Lateral aggregation and branching SEM image (Hantgan) of fibrin clot (plus platelets) 10 m AFM image (Guthold) of fibrin clot 10 m Image: M. Kaga, P. Arnold; Voet & Voet, Biochemisty, Wiley & Sons, NewYork, 1990 The protein data bank An Information Portal to Biological Macromolecular Structures nearly 100,000 structures (Jan 2014) Go to:Well do some exercises related to the homework. DNA is just a super-long string of four different bases. Proteins do all the work and action in an organism (structure, catalyze reactions, etc). How does the information (letter code) contained in DNA get translated into specific proteins? ? Central dogma of Molecular Biology Describes how the genetic information encoded (stored) in the letter sequence of DNA is first transcribed and then translated into an amino acid sequence, i.e. into proteins. (Crick, F.H.C. (1958): On Protein Synthesis. Symp. Soc. Exp. Biol. XII, ; Crick, F. H. C. (1970): Central Dogma of Molecular Biology. Nature 227, )On Protein Synthesis. (Enzymes catalyze reactions in organism) (Proteins building blocks of organism) The genome, or genomic DNA (deoxyribonucleic acids), of an organism consists of a very long sequence of four different nucleotides with bases A, C, G, T. Genomic DNA is a double-stranded helix comprised of two complementary strands, held together by A-T and C-G base pairs. The entire genome is replicated by DNA polymerases (a protein) and passed on to daughter cells during cell division. The genome consists of many (usually thousands) of genes. A gene is a specific, defined nucleic acid sequence that encodes one particular protein. The human genome consists of about 310 9 base pairs and only about 30,000 genes (in higher organisms, large parts the genome (80 98%) do not encode any known proteins). Genomic DNA Replication (DNA polymerase) mRNA Transcription (RNA polymerase) Transcription: RNA polymerase (a protein) binds to the beginning of one particular gene and synthesizes an exact mRNA copy of that gene. RNA (ribonucleic acid) consists of nucleotides with bases A, C, G, U. It is single-stranded. Transcription stops at the end of each gene and the RNA chain is released. A gene is on the order of a thousand bases. Protein Translation (Ribosome) Translation: The RNA is moved to the ribosome. The ribosome reads the RNA sequence (with the help of t-RNA) and synthesizes an amino acid chain (polypeptide). The polypeptide folds into a three-dimensional structure a protein (or part of a protein). There are 20 different amino acids, thus three RNA letters are needed to code for one amino acid. These triplets of RNA letters are called codons. Central dogma Picture in prokaryotic (bacterial) cell and eukaryotic (higher) cell Eukaryotic cell The human genome has about 30,000 genes (and lots of non-coding DNA) Simply speaking: one gene one polypeptide Prokaryotic cell (no nucleus) Transcription (making RNA from a DNA template): RNA polymerase binds at a promoter (beginning of a gene), unwinds DNA, and starts synthesis of an RNA copy of the gene The sequence of bases in DNA codes for the sequence of amino acids in proteins First real-time movies of a transcribing RNA polymerase 1,2 1.S. Kasas et al., Biochemistry 36, 461 (1997). (see Fig of book) 2.M. Guthold et al., Biophysical Journal 77, 2284 (Oct, 1999). Kasas moviemovie Credit: 8 minute movie of inner workings of a cell BioVisions, Harvard University Central dogma continued Translation: Ribosome is reading codons of mRNA, and with the help of tRNA, synthesizes a polypeptide. mRNA is translated into polypeptide chain mRNA messenger RNA tRNA transfer RNA mRNA Genetic Code (same in all organism) UAU, UAC = Tyrosine