1.  -helix right-handed 3.6 aa per turn hydrogen bond N-H···O=C Collagen triple helix: three polypeptide intertwined A stereo, space-filling representation B2Protein structure － Secondary

2.  -sheet: hydrogen bonding of the peptide bond N-H and C=O groups to the complementary groups of another section of the polypeptide chainx A stereo, space-filling representation of the six- stranded antiparallel  sheet. Parallel  sheet: sections run in the same direction Antiparallel  sheet: sections run in the opposite direction fibroin

3. A two-stranded  sheet B2 Protein structure and function

4. B2Protein structure － Tertiary The different sections of  -helix,  -sheet, other minor secondary structure and connecting loops of a polypeptide fold in three dimensions

5. Noncovalent interaction between side chains that hold the tertiary structure together: van der Waals forces, hydrogen bonds, electrostatic salt bridges, hydrophobic interactions Covalent interaction: disulfide bonds Denaturation of protein by disruption of its 2 o and 3 o structure will lead to a random coil conformation B2 Protein structure and function

6. Many proteins are composed of two or more polypeptide chains (subunits). These subunits may be identical or different. The same forces which stabilize tertiary structure hold these subunits together. This level of organization called quaternary structure. A stereo, space-filling drawing showing the quaternary structure of hemoglobin  1-yellow;  1-light blue;  2-green;  -dark blue; heme-red back B2Protein structure － Quaternary

7. Advantages of the quaternary structure: 1.It allows very large protein molecules to be made, such as tubulin 。 2.It can provide greater functionality to a protein by combining different activities into a single entity. 3.The interactions between the subunits can often be modified by binding of small molecules and lead to the allosteric effects seen in enzyme regulation

8. Protein Folding: chaperones are involved in vivo (1)The rapidly reversible formation of local secondary structure (2)Formation of domains through the cooperative aggregation of folding nuclei (3)Assembly of domains into a “molten” globule (4)Conformational adjustment of the monomer (5)Final conformational adjustment of the dimeric protein to form the native structure. back B2 Protein structure and function

9. Prosthetic groups ( 辅基 ): covalently or noncovalently attached to many conjugated proteins, and give the proteins chemical functionality. Many are co-factors in enzyme reactions. Apoprotein ( 脱辅基蛋白 ) Examples : heme groups in hemogobin (Figure)Figure B2 Protein structure and function

10. Biological functions of proteins Enzymes: substrate binding, side chain in catalysis Signaling: cell membrane Transport and storage: hemoglobin transports oxygen Structure and movement: collagen, keratin, tubulin in cytoskeleton, actin and myosin for muscle contraction Nutrition: casein ( 酪蛋白 ) and ovalbumin( 卵清蛋白 ) Immunity: antibodies Regulation: transcription factors B2 Protein structure and function

11. B2Protein structure － Domains, motifs and families Domains: structurally independent units of many proteins, connected by sections with limited higher order structure within the same polypeptide. (Figure)(Figure) They can also have specific function such as substrate binding

12. Structural motifs: Groupings of secondary structural elements that frequently occur in globular proteins Often have functional significance and represent the essential parts of binding or catalytic sites conserved among a protein family  motif

13. Protein families: structurally and functionally related proteins from different sources The primary structures of c-type cytochromes from different organisms Motif

14. Protein purification An experimental step to purify the interested protein from other proteins and nonprotein molecules existing in the cells An essential experimental step when study any individual protein

15. The principal properties of proteins used for purification 1.Size: gel filtration chromatography 2. Charge: ion-exchange chromatography, isoelectric focusing electrophoresis 3. Hydrophobicity: hydrophobic interaction chromatography 4. Affinity: affinity chromatography 5. Recombinant techniques: involving DNA manipulation and making protein purification so easy

16. 1.gel filtration chromatography

17. 2. Charge: ion-exchange chromatography, isoelectric focusing, electrophoresis Isoelectric point (pI): the pH at which the net surface charge of a protein is zero - - - - - - - - + + + + + + + + pH=pI pH>pI pH<pI

18. Ion-exchange chromatography Column + anions + + + Sample mixture Protein binding Column + proteins Column + anions Ion displacing Purified protein

19. + Electrophoresis Protein migrate at different position depending on their net charge

20. Isoelectric focusing A protein will stop moving at position corresponding to its isoelectric point (pI) in a pH gradient gel.

21. 3. Hydrophobicity: hydrophobic interaction chromatography Similar to ion-exchange chromatography except that column material contains aromatic or aliphatic alkyl groups

22. 4. Affinity chromatography ding d Enzyme-substrate binding Receptor-ligand binding Antibody-antigen binding Substrate analogs: competitive inhibitors

23. 5. Recombinant techniques: Clone the protein encoding gene of interest in an expression vector with a purification tag added at the 5’- or 3’ end of the gene Protein overexpression in a cell Protein purification with affinity chromatography.

24. Mass Determination  Gel filtration chromatography and SDS-PAGE Comparing of the unknown protein with a proper standard Popular SDS-PAGE: cheap and easy with a 5-10% error SDS: sodium dodecyl sulfate, makes the proteins negatively charged and the overall charge of a protein is dependent on its mass.

25. Mass Determination Mass spectrometry: Molecules are vaporized and ionized (by Xe/Ar beam), and the degree of deflection (mass-dependent) of the ions in an electromagnetic field is measured Extremely accurate (0.01% error), but expensive ESI (electrospray ionization) and MALDI (matrix- assisted laser desorption/ionization) can measure the mass of proteins smaller than 100 KDa Helpful to detect post-translational modification Protein sequencing: relying on the protein data base

26. Determine the primary structure of a protein: p Amino acid composition: 1.Acid treatment to hydrolyze peptide bonds: 6M HCl, 110°C for 24 hrs. 2.Chromatographic analysis However, you cannot get the sequence!

27. Specific enzyme/chemical cleavage: Pepsin cleaves after lysine(K) or arginine (R) V8 protease cleaves after glutamic acid (E) Cyanogen bromide cleaves after methionine (M) Edman degradation: Performed in an automated protein sequencer Determine the sequence of a polypeptide from N- terminal amino acid one by one. Phenylisothiocyanate 苯 异硫氰酸 reaction) Expensive and laborious Determine the primary structure of a protein: protein sequencing

28. Protein sequence analysis (1) Sequence: HLMGSHLVDALELVMGDRGFEYTPKAWLV Pepsin T1 HLMGSHLVDALELVMGDR T2 GFEYTPK T3 AWLV V8 V1 HLMGSHLVDALE V2 LVMGDRGFE V3 YTPKAWLV

29. Protein sequence analysis (2) Computer analysis: T1 HLMGSHLVDALELVMGDR V1 HLMGSHLVDALE V2 LVMGDRGFE T2 GFEYTPK V3 YTPKAWLV T3 AWLV Sequence: HLMGSHLVDALELVMGDRGFEYTPKAWLV

30. Most protein sequences are deduced from the DNA/cDNA sequence Direct sequencing: determine the N-terminal sequences or some limited internal sequence  construction of an oligonulceotide or antibody probe  fishing the gene or cDNA

31. X-ray crystallography and NMR Determing the 3-D structure of a protein X-ray crystallography: Measuring the pattern of diffraction of a beam of X- rays as it pass through a crystal. The first hand data obtained is electron density map, the crystal structure is then deduced. A very powerful tool in understanding protein 3-D structure Many proteins have been crystallized and analyzed

32. X-ray crystallography and NMR Determing the 3-D structure of a protein Measuring the relaxation of protons after they have been excited by radio frequencies in a strong magnetic field Measure protein structure in liquid but not in crystal Protein measured can not be larger than 30 KDa NMR: Nuclear magnetic resonance (NMR) spectroscopy

33. Summary 1.20 Amino acids: D & E (-), H, K, R (+), S, T, C, N, Q (polar), G,A,V,L, I, M, P (nonpolar), F, Y,W (aromatic) 2.Protein structure: primary, secondary, tertiary, quaternary 3.Protein analysis: purification, sequencing, mass determination, X-ray crystallography and NMR