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Protein Folding & Biospectroscopy F14PFB Dr David Robinson Lecture 2 Principles of protein structure and function • Function is derived from structure • Structure is derived from amino acid sequence • Different activities and shapes of proteins due to different amino acid sequences A reminder… • Basic Amino Acid Structure: – The side chain, R, varies for each of the 20 amino acids Side chain R H O N C C H Amino group H OH Carboxyl group The Peptide Bond • Dehydration synthesis • Repeating backbone: N–C –C –N–C –C O O – Convention – start at amino terminus and proceed to carboxy terminus Levels of Protein Structure The folded protein structure is stabilized by a variety of weak chemical interaction, and in some cases covalent (disulfide) bonds between cysteine residues Disulfide bond: R– CH2–S–S–CH2–R Cys Cys - helix Myoglobin Hemoglobin Protein structure: overview Structural element Description primary structure amino acid sequence of protein secondary structure helices, sheets, turns/loops super-secondary structure association of secondary structures domain self-contained structural unit tertiary structure folded structure of whole protein • includes disulfide bonds quaternary structure assembled complex (oligomer) • homo-oligomeric (1 protein type) • hetero-oligomeric (>1 type) Primary & Secondary Structure Primary structure = the linear sequence of amino acids comprising a protein: AGVGTVPMTAYGNDIQYYGQVT… Secondary structure • Regular patterns of hydrogen bonding in proteins result in two patterns that emerge in nearly every protein structure known: the -helix and the -sheet • The location of direction of these periodic, repeating structures is known as the secondary structure of the protein The alpha helix 60° Properties of the alpha helix 60° Hydrogen bonds between C=O of residue n, and NH of residue n+4 3.6 residues/turn 1.5 Å/residue rise 100°/residue turn Properties of -helices 4 – 40+ residues in length Often amphipathic or “dual-natured” • Half hydrophobic and half hydrophilic • Mostly when surface-exposed If we examine many -helices, we find trends… • Helix formers: Ala, Glu, Leu, Met • Helix breakers: Pro, Gly, Tyr, Ser The beta strand (& sheet) 135° +135° Properties of beta sheets Formed of stretches of 5-10 residues in extended conformation Pleated – each C a bit above or below the previous Parallel/antiparallel, contiguous/non-contiguous Parallel and anti-parallel -sheets Anti-parallel is slightly energetically favoured Anti-parallel Parallel Turns and Loops Secondary structure elements are connected by regions of turns and loops Turns – short regions of non-, non- conformation Loops – larger stretches with no secondary structure. Often disordered. • “Random coil” • Sequences vary much more than secondary structure regions Levels of Protein Structure Secondary structure elements combine to form tertiary structure Quaternary structure occurs in multienzyme complexes • Many proteins are active only as homodimers, homotetramers, etc. Protein Folding • Forming polypeptide chain requires energy and information (template) – ie translation from RNA protein SEQUENCE Protein Folding • Forming polypeptide sequence requires energy and information (template) • Forming native conformation requires NO ADDITIONAL energy or information (SELF ASSEMBLY) Protein folding Amino acid sequence contains all information necessary for folding into a specific threedimensional structure Protein Folding Proteins, in general, do NOT fold as they are synthesized on the ribosome Folding of RNAse A in the test tube denaturation renaturation Incubate protein in guanidine hydrochloride (GuHCl) or urea 100-fold dilution of protein into physiological buffer - the amino acid sequence of a polypeptide is sufficient to specify its three-dimensional conformation Thus: “protein folding is a spontaneous process that does not require the assistance of extraneous factors” Anfinsen, CB (1973) Principles that govern the folding of protein chains. Science 181, 223-230. Protein Folding Many proteins fold by Assisted Self Assembly Correct assembly (native conformation) requires assistance by CHAPERONES Protein unfolding = Denaturation Loss of structure and function – – – – Heat Extreme pH Detergents Urea Protein unfolding = Denaturation Why do these conditions cause loss of structure and function? – – – – Heat Extreme pH Detergents Urea Lysozyme Lysozyme Tertiary: complete three-dimensional structure Quaternary: arrangement of subunits (in multisubunit protein) Hemoglobin Quaternary structure • Held together by weak interactions between side (R/functional) groups as well as covalent disulfide bonds Structure-function relationship • Function is derived from structure • Structure is derived from sequence Sickle-cell disease Normal red blood cells Sickle shaped red blood cells Due to single amino acid change in haemoglobin Sickle-cell disease Sickle-cell disease • Single specific amino acid change causes change in protein structure and solubility • Results in change in cell shape • Causes cells to clog blood vessels Amino acids