Oct 25
This is only a first attempt at the `fleshing out' process; any contribution or comment is very welcome, constructive or otherwise. - Alan Mills 13/10/94 ........................................and subsequently on 24th.October 1994 ______________________________________________________________________________ see also URLs http://www.cryst.bbk.ac.uk/education/PPS.html & http://seqnet.dl.ac.uk:8000/vsns-pps/consult/sept30.html ______________________________________________________________________________ THE PRINCIPLES OF PROTEIN STRUCTURE INTRODUCTION -Prerequisites as PMR has previously listed, plus any amendments. We may cite references for those who need a little revision. Such stuff will be sent to all students pre-registering -Objectives & Assessment The overall course objective is to provide the student with a broad understanding of the main fundamentals of the structure of proteins. Each course section will have it's own individual objectives, which may be the subject of continuous self-assessment using forms provided by us. -Course Map, or Contents Page (this page HTMLised?) Important to prevent students from `wandering off' through any external hlinks that we offer, and to enable them to get back to where they want to be easily. Possibly using click buttons as in the O manual - see http://kaktus.kemi.aau.dk/oman/junk.html OVERVIEW OF PROTEIN SYNTHESIS -Cellular Organisation distinguish prokaryotes and eukaryotes, nucleus, DNA, chromosomes, membrane(s), cytosol, ER, lumen of ER, Golgi, organelles, ribosomes, mention viruses(?) -Transcription DNA, Watson-Crick A:T, G:C, unwinding of double helix, RNA polymerase, U not T, template directed synthesis of ssRNA ->rRNA, tRNA, mRNA, introns/exons in eukaryotes, splicing (regulation, operons and promoters?) -Translation ribosome, attachment to Shine-Dalgarno sequence, genetic code, start (Met) and stop codons, coding sequence = gene, tRNA-AA, growth of nascent AA-chain and folding up, mention chaperones -Post-translational processing distinguish ER-directed vs. cytoplasmic, signal peptide, peptidase, foldases, glycosylation, myristoylation, oligomerisation, binding of co-factors, etc -Transport into ER, to Golgi, lysosomes & export, to and across membrane, exocytosis, nuclear targeting, pores and channels, binding proteins, periplasm, circulatory systems in higher organisms PRIMARY STRUCTURE OF PROTEINS -Nomenclature (Genetic & Single-Letter Codes) Bases, reading frames, genetic code, 20 AAs, 3 and 1 letter codes, self-assessment form to test rote learning. Note sulphur in M, C. -Databases Advances in sequencing, HUGO, automatic recognition and translation of coding ORFs, protein and DNA databases, NBRF-PIR, EMBL, GENBANK, SwissProt, Japan DB, GDB, non-redundant -The Amino Acids & Their Properties Re-emphasise single letter code (more sensitive alignment) 0.Chemical Structure Diagrams, note hydrogens, acid + base, RASMOL samples, peculiarity of Glycine and Proline, rote learning 1.Size smallest to largest, relationship to acceptance of mutations, influence on flexibility for Glycine 2.Charge D, E negative; H, K, R positive. Effect of pH, i.e.pKa (esp. H). H-bonding & solvent. Salt-bridges. 3.Hydrophobic A, V, L, I, M definitely. Simple meaning of term, Also F, P, W, C, Y, T, G, and R to some extent; each needs discussing. 4.Aromatic F, Y, W - chemical stability of benzene ring, electron delocalisation, ring currents(?), interactions to pi clouds. 5.Polar N, Q, S, T, also Y, W - hydrogen bonding donor/acceptor interactions to solvent, and to m/c & s/c. 6.Conformationally unusual G - flexible due to lack of steric hindrance P - inflexible due to s/c bonding back phi~-60 degrees -Chirality Point up difference between L- and D- amino acids and also that several carbon atoms within side chains are chiral. Tetrahedral geometry -The Disulphide Bond Explain that nearby cysteines can be oxidised to form disulphides, which may improve stability. Bridges are common in extracellular proteins (and ER lumen) but rare in cytosolic proteins. Converse is true for isolated thiols. Summarise geometry. Refer to thiol-redox mechanisms & proteins. ?canonical disulphide in Igs. Cross-linking. PROTEIN GEOMETRY -Nomenclature Greek letters; N- & C-termini (chain direction); single-letter code. Different methods of representation (ball&stick, points&lines; space-filling; atomic radii. Colour conventions. -The Peptide Bond Condensation reaction. Planar bond; charge delocalisation; semi-rigidity. Bond lengths and angles. H-bonding potential. -Torsion Angles 1.Main Chain phi/psi Definition of torsion angle. Explanation of phi `before' and psi `after' C-alpha. Steric hindrance leads to preferred values. Refer forward to Rama plot & secondary structures Refer back to Glycine and Proline. -Torsion Angles 2.Side Chain Rotamers Steric hindrance leads to preferred values. gauche + and -, & trans, more populated in known structures. Packing in core. -Ramachandran Plot Who was he? What is the plot, and what does it show. Refer to secondary structures, and to turns (alpha-left). Maybe polyproline and bridge regions. Why is it useful? OVERVIEW OF MOLECULAR FORCES -Covalent Bonds; Lengths and Angles (& distortions thereof) Bond concept. Geometric data. Strong forces involved in bond stretching or breaking. Lesser in angle distortion. Functional form(?) and approx.values(?) Lennard-Jones (6-12). Aromatic rings(?) -Van der Waals Forces Radii; electron clouds. Non-bonded! Dipoles and induced dipoles. Lennard-Jones (6-12). Magnitudes. -Hydrogen Bonds Proton `sharing'. Donors and acceptors. Angle and distance limits. Importance to main-chain folds. Importance of H2O. Energy magnitudes. -Electrostatic Effects, Salt Bridges Coulomb attraction and repulsion. Screening effect of H2O and core atoms. Local fields, pKa changes. GRASP(?) Examples of salt bridges (mainly surface). -The Hydrophobic Effect Entropy of solvent (dG=dH+TdS), effects at surface, partition coeffts., `greasy' vs. hydrophilic (polar & charged). Magnitude of effect. Importance to folding (core vs.surface). Hydropathy plots and sliding window(?) -The Importance of Solvent Dry proteins denature! In folding and solubility. & in many reaction mechanisms. -Overall Energetics, Stability, & Energy Minimisation Add all terms together. Relate to folding (& pathway). Marginal stability. Thermophiles(?) Computational minimisation. SECONDARY STRUCTURE -General, including representations, CD, prediction etc Remind primary, secondary, super-secondary, tertiary, quaternary Folding produces strands and helices first. Such structures in most proteins - `architectural elements'. Arrows and cylinders. Strands lie together in sheets. Examples. (Cameo on circular dichroism?) Page on secondary structure prediction. -The Alpha Helix 1. The classic right-handed alpha-helix. Hydrogen bonding pattern 3.6 per turn. 100 degree turn. Carbonyls all pointing `down' - macrodipole. No prolines and few glycines. Side chains sticking out. Graphical examples. Linus Pauling. -The Alpha Helix 2. Variations on the theme (3-10, pi). Amphipathicity. Capping(?). AA propensities. Length variation. Trans-membrane helices. -Beta Sheets 1.Parallel The concept of the extended strand in detail (inc.direction). H-bonding between adjacent strands. Pleated(ness). Silks. -Beta Sheets 2.Anti-Parallel Different H-bonding pattern. Also pleated. Hairpins. -Beta Sheets - General Twist. Mixed sheets. 1, +2x, etc nomenclature. Refer forward to folds -Turns Predominance of Pro & Gly. Positive phi. Tight turns; hairpins, classification(s). (P-loop in nucleotide-binders?) -`Random' Coil All the loops in between, which are not hairpins. Connecting sec.struct.elements. Often at surface. Evolutionary variability. Indels. SUPER-SECONDARY STRUCTURE -Motifs comprised of combinations of elements -Beta hairpin -Helix hairpin -Helix-turn-helix (DNA binding vs EF-hand) -beta-alpha-beta as in wound alpha-beta proteins and the right-handed crossover -(?)beta corners -and others(?) TERTIARY STRUCTURE -Globular (mainly enzyme) vs Fibrous, Structural, and Membrane Proteins Most biochemistry mediated by (1000s of) enzymes; mostly soluble and cytosolic. Cover co-factors. Also eg hormones. Plus all the cellular machinery. Describe collagen, keratin, and some other ECM proteins. Then some plasma stuff(?). Distinguish membrane associated, and integral membrane proteins. Describe receptors, channels, pores and pumps in outline. Page on exptl(?) X-Ray & NMR methods. -Domains Distinct folding units. Give examples. Show difficulties of exact definition (compound domains). Use small disulphide domains as paradigm. Zinc fingers. Multi-domain structures. -Folds; classification and representation (point to SCOP?) 0.General AA propensities different for classes, and for environments. Drawings, nomenclatures, etc. 1.All alpha ROP, 4-helix bundle, globins, some cytokines Plenty of lovely graphical examples. 2.All beta Ig sandwich, up-down barrels, lectins, Greek keys, jelly rolls Plenty of lovely graphical examples. 3.Wound alpha/beta, and variations mainly enzymes, mainly cytosolic. Rossman fold, and variations on theme. A huge section!? nucleotide-binding proteins. Tim barrels. Plenty of lovely graphical examples. 4.Mixed alpha+beta Ribonuclease, lysozome, serpins, etc. Plenty of lovely graphical examples. 5.Other distinctive folds eg beta propellor eg alpha-beta horseshoe (RNase inhibitor) eg beta-trefoil fold eg . . . -Mosaic Proteins Mainly extra-cellular. Intron/exon boundaries. Exon-shuffling in evolution. Receptors and ECM proteins. Examples with diagrams (a la Campbell and Patthy) QUATERNARY STRUCTURE -Dimers, Trimers, Tetramers & + Mainly in cytosolic enzymes, but also eg hormones, cytokines Haemoglobin. Receptor dimerisation. Examples. -Multi-enzyme complexes, Chaperonins, Cellular `Machinery' tryptophan synthase(?), Reverse transcriptase polymerases, GroeL, - a mixed bag, help! -Families and Superfamilies see SCOP URL http://www.bio.cam.ac.uk/scop refer forward to next section of course -Functional Groupings EC enzyme classification. Hydrolases, transferases, isomerases, etc. Four major protease groupings. Cytokines. Kinases. PROTEIN INTERACTIONS -Cooperativity, Allosteric Effects Explanation of allostery and cooperativity in enzymes Haemoglobin, glycogen phosphorylase, and other examples -Conformational Changes (related to function, regulation?) Hinge motions. Serpins. F1 ATPase. Actin/myosin. -Interactions among Proteins (receptors, carriers, Igs, hydrolases, etc) Binding to receptors, their activation (dimerisation and activation). Proteolysis in processing and degradation. Carriers, eg transferrin, ferritin, retinol-b-pr., other means of transport. Alan Mills ........................ 24th.October 1994
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Sept 30 1994