Exploring Protein Structure and Conformation

1. Polypeptide and protein structure Nafith Abu Tarboush DDS, MSc, PhD natarboush@ju.edu.jo www.facebook.com/natarboush

2. Protein conformation • Many conformations are possible for proteins due to flexibility of amino acids linked by peptide bonds • At least one major conformations has biological activity, and hence is considered the protein’s native conformation or the native protein

3. Levels of Protein Structure • 1°structure: sequence and number, from N to C • 2°structure: the ordered 3-dimensional arrangements (conformations) in localized regions of a polypeptide chain, backbone interactions through hydrogen bonding; • e. g., -helix and -pleated sheet • 3˚ structure: 3-D arrangement of all atoms • 4˚ structure: arrangement of monomer subunits with respect to each other

4. Primary Structure of Proteins • Zigzag arrangement • R-groups • Determination? • 1o Sequence & 3-D conformation; relation to functional properties (MW& genetic mutations) • Site-directed mutagenesis and structure function relationship

5. Shape-Determining Interactions in Proteins • Is it ordered or spaghetti? • Hydrogen, ionic, covalent, & hydrophobic

6. Secondary Structure of Proteins • What is the secondary structure of proteins? “folding of the backbone” • What are the bonds that have a free rotation? What is the implication? These angles repeat themselves in regular secondary structures • Two main kinds: α-helix and β-pleated sheet • They are periodic; their features repeat at regular intervals • Stability of secondary structure

7. The α-helix • H-bonds are parallel to the helix axis, same segment • C=O binds N—H four residues away • linear arrangement of H-bonds (maximum strength and stability) • Turns occur every 3.6 residues, right handed, clockwise • The pitch (linear distance between corresponding points on successive turns) is 5.4 Å • Proteins have varying amounts of α-helical structures • What factors affect the helix (specific amino acids, electrostatic repulsion, steric repulsion)

8. The β-sheets • Backbone is almost completely extended • R groups extending above and below the sheet • H-bonds are intra-chain or inter-chain bonds • Perpendicular to the direction of the protein chain • Parallel vs. anti-parallel • Zigzag structure

9. Others regular ones • Why do we have them? • Loops vs. turns • Also known as β-bends • Involve 4 amino acids (H-bond: C=O of 1 & N–H of 4) • A motif is a repetitive super-secondary structure. Regardless of function • Domains; protein conformations with similar functions (leucine zipper)

10. -Helices and -Sheets • Supersecondary structures: a combination of - and -sections • b unit: (parallel) •  unit: (helix-turn-helix), anti-parallel • -meander: an anti-parallel sheet formed by a series of tight reverse turns connecting stretches of a polypeptide chain • Greek key: a repetitive super-secondary structure formed when an anti-parallel sheet doubles back on itself • -barrel: created when -sheets are extensive enough to fold back on themselves

12. Fibroin, β-sheets, alternating glycine and alanine α-keratins, bundles of α-helices Fibrous Proteins • Contain polypeptide chains organized approximately parallel along a single axis: • Consist of long fibers or large sheets • Mechanically strong • Insoluble • play an important structural role • Examples are • Keratin • Collagen • fibroin

13. Globular Proteins • Folded to, a more or less, spherical shape • Soluble • Polar vs. non-polar, exterior vs. interior • Most of them have substantial sections of -helix and -sheet

14. 3˚ Structure • The 3-dimensional arrangement of all the atoms in the molecule • Simple vs. conjugated • Backbone hydrogen bonding, side chains hydrogen bonding, hydrophobic interactions, electrostatic attraction, electrostatic repulsion, metal coordination • Not every protein have all kinds of interactions (myoglobin & hemoglobin; no disulfides) (trypsin & chymotrypsin, no metal complexes) • Interactions between side chains also plays a role

15. Forces That Stabilize Protein Structure

16. 3°& 4°Structure • Tertiary (3°) structure: the arrangement in space of all atoms in a polypeptide chain • It is not always possible to draw a clear distinction between 2°and 3°structure • Quaternary (4°) structure: the association of polypeptide chains into aggregations (dimers, trimers, tetramers,…etc). Non-covalent interactions. Simple or conjugated

17. Determination of 3°Structure • X-ray crystallography • uses a perfect crystal; that is, one in which all individual protein molecules have the same 3D structure and orientation • exposure to a beam of x-rays gives a series diffraction patterns • information on molecular coordinates is extracted by a mathematical analysis called a Fourier series • 2-D Nuclear magnetic resonance • can be done on protein samples in aqueous solution

18. X-Ray and NMR Data • High resolution method to determine 3˚ structure of proteins (from crystal) • Diffraction pattern produced by electrons scattering X-rays • Series of patterns taken at different angles gives structural information • Determines solution structure • Structural info. Gained from determining distances between nuclei that aid in structure determination • Results are independent of X-ray crystallography

19. Complex Protein Structures • Covalent conjugation to carbohydrates (glyco-proteins): post-translational, function, N-linked (-N of Asn) & O-linked (-OH of Ser or Thr) & occasionally to the –OH of hydroxy-lysine • Lipoproteins: Non-covalent, store & transport lipids and cholesterol • Extremely important: ex. Erythrocytes & blood groups (glycoshpingolipids) • Phosphoproteins: contain phosphate groups esterified to Ser, Thr, or Tyr.; the phosphate group usually regulates protein function

20. Chemical Properties of Proteins • 1. Protein Hydrolysis • The reverse of protein synthesis • Digestion of proteins is hydrolyzing peptide bonds • Takes place in the stomach and small intestine

21. 2. Protein Denaturation • How the protein preserve its shape? • What is denaturation? It affects physical, chemical, and biological properties, such as enzymes • Solubility decreased, ex. eggs • Heat (≈≥50 οC) • Mechanical agitation • Detergents • Organic compounds: acetone, ethanol, bacterial proteins • pH change: disrupt salt bridges, milk turns sour because it has become acidic • Most denaturation is irreversible

22. Protein Folding & prediction • The integration of biochemistry and computing has led to bioinformatics • Predicting the 3˚ structure (limitations) • First step is to search for sequence homology

23. Hydrophobic Interactions & Chaperons • Hydrophobic interactions are spontaneous • Chaperones aid in correct & timely folding of many proteins • hsp70 were the first chaperone proteins discovered • Chaperones exist in organisms from prokaryotes to humans

24. Structure & diseases (prion, Alzheimer) • Mad-cow disease (bovine spongiform encephalopathy), and human spongiform encephalopathy (Creutzfeldt-Jakob disease) • The cause is a small (28-kDa) protein called a prion (Met129) • Alzheimer's Disease: buildups of plaques and fibers of hard insoluble material • Amyloid protein mis-folded and broken down (≈40 a.a, Ab protein) and aggregates