1. Understanding Biology through StructuresCourse Work 2009 Proteins Structures: Introduction and General Overview

2. Understanding Biology through StructuresCourse Work 2009 Sequencing the Human Genome: A Landmark in the History of Mankind

3. Understanding Biology through StructuresCourse Work 2009 …..AATGCCGCGTAGTCGGGTAAGGGTCTGAAGCTGAAATCTTTTCACACCGAGTCGATGGG… …..GCCGCGTAGTCGGGTAAGGGTCACACCGAGTCGATGG… ….APTCHYLDELAKGGRLDATIKRDGLGVLVWAQND…. Hierarchy in Understanding Function “We may, I believe, anticipate that the chemist of the future who is interested in the structures of proteins, nucleic acids, polysaccharides, and other complex substances with higher molecular weights will come to rely upon a new structural chemistry, involving precise geometrical relationships among the atoms in the molecules and the rigorous application of the new structural principles, and that great progress will be made, through this technique, in the attack, by chemical methods, on the problems of biology and medicine.” -Linus Pauling, Nobel Lecture, 1954

4. Understanding Biology through StructuresCourse Work 2009 Paradigm: Function of biological macromolecules is intricately related to their three-dimensional shape and structure. Structural knowledge is therefore an important step in understanding function. Techniques available: X-ray crystallography, NMR, CD, Fluorescence spectroscopy, Mass spectrometry…..

5. Understanding Biology through StructuresCourse Work 2009 Some Landmarks in Macromolecular Structure Determination Watson and Crick Perutz and Kendrew Hodgkin Pauling Great ideas have always faced violent opposition from mediocre minds -Albert Einstein

6. Understanding Biology through StructuresCourse Work 2009 Some Landmarks in Macromolecular Structure Determination……..contd. Photosynthetic reaction centre Potassium channel Virus

7. Understanding Biology through StructuresCourse Work 2009 Experimental Methods of Structure Determination X-ray crystallography Solubilization of the over-expressed protein Obtaining crystals that diffract Structure determination by diffraction of protein crystals Size of a molecule: no theoretical limit Nuclear Magnetic Resonance spectroscopy Solubilization of the over-expressed protein Structure determination of a molecule as it exists in solution Size-limit is a major factor

8. Understanding Biology through StructuresCourse Work 2009 Principles of X-ray crystallography Crystals act as a three-dimensional grating and produce diffraction The diffraction pattern contains complete information on the placement of scatterers (electrons in atoms) By fourier transforming the diffraction pattern, we can obtain information on the structure of the molecule in the crystals

9. Understanding Biology through StructuresCourse Work 2009 Principles of NMR Measures nuclear magnetism or changes in nuclear magnetism in a molecule NMR spectroscopy measures the absorption of light (radio waves) due to changes in nuclear spin orientation NMR only occurs when a sample is in a strong magnetic field Different nuclei absorb at different energies (frequencies)

10. Understanding Biology through StructuresCourse Work 2009 X-ray versus NMR Producing enough protein for trials Crystallization time and effort Crystal quality, stability and size control Finding isomorphous derivatives Chain tracing & checking Producing enough labeled protein for collection Sample “conditioning” Size of protein Assignment process is slow and error prone Measuring NOE’s is slow and error prone X-rayNMR

11. Understanding Biology through StructuresCourse Work 2009 Structure Determination Made Easy (Modern Crystallographers Three Rings) Advances in molecular biology –Ability to produce and modify proteins in large quantities at will Advances in instrumentation –Synchrotron radiation sources, detectors, NMR machines Advances in computational techniques –Improved hardware and novel algorithms of structure determination

12. Understanding Biology through StructuresCourse Work 2009 DNA : Diffraction pattern

13. Understanding Biology through StructuresCourse Work 2009

14. Model of DNA

15. Understanding Biology through StructuresCourse Work 2009 Protein Structures Primary structure –Un-branched polymer –20 side chains (residues or amino acids)

16. Understanding Biology through StructuresCourse Work 2009 How does a protein adopt a unique 3D conformation?

17. Understanding Biology through StructuresCourse Work 2009 Peptide torsion angles

18. Understanding Biology through StructuresCourse Work 2009 Phi-Psi map (Ramachandran map)

19. Understanding Biology through StructuresCourse Work 2009 The Protein Folding Problem Amino acid sequence of a polypeptide has all the information required to determine its three- dimensional topology

20. Understanding Biology through StructuresCourse Work 2009 If a polypeptide sequence corresponds to a unique conformation of the protein, how does nature take care of point mutations in the primary sequences?

21. Understanding Biology through StructuresCourse Work 2009 Triosephosphate Isomerase Structures of E. coli, B. stearothermophilus, P. falciparum, T. brucei, S. cerevesiae, chicken, human TIMS are identical though amino acid sequences differ by >50%

22. Understanding Biology through StructuresCourse Work 2009 Chicken HumanLeishmania Pyrococcus ThermotogaVibrio Three-dimensional structures of homologous proteins are very similar

23. Understanding Biology through StructuresCourse Work 2009 The relation between the divergence of sequence and structure in proteins. Chothia C, Lesk AM. EMBO J. 1986 Apr;5(4):823-6. The sequence- structure relationship

24. Understanding Biology through StructuresCourse Work 2009 Annotated proteins in the databank: ~ 100,000 Limitations of Experimental Methods: Consequences Proteins with known structure: ~5,000 ! Total number including ORFs: ~ 700,000

25. Understanding Biology through StructuresCourse Work 2009 ? KQFTKCELSQNLYDIDGYGRIALPELICTMF HTSGYDTQAIVENDESTEYGLFQISNALWCK SSQSPQSRNICDITCDKFLDDDITDDIMCAK KILDIKGIDYWIAHKALCTEKLEQWLCEKE Predicting Protein Structure: Comparative (Homology) Modeling Use as template & model 8lyz 1alc KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAK FESNFNTQATNRNTDGSTDYGILQINSRWWCND GRTPGSRNLCNIPCSALLSSDITASVNCAKKIV SDGNGMNAWVAWRNRCKGTDVQAWIRGCRL Share Similar Sequence Homologous

26. Understanding Biology through StructuresCourse Work 2009 How have the protein structures enhanced our understanding of Biology?

27. Understanding Biology through StructuresCourse Work 2009 Structure of antibody

28. Understanding Biology through StructuresCourse Work 2009 Antibodies bind to antigens by recognizing a large surface, and through surface complementarity. Thus, these complexes have a very high affinity for each other. Antigen:Antibody complex

29. Understanding Biology through StructuresCourse Work 2009 Antibody & Enzymes : ABZYMES Diels-Alderase Catalytic Antibody 1E9 Complex With Its Hapten

30. Understanding Biology through StructuresCourse Work 2009 Cholera Toxin: Recognition via sugar moiety

31. Understanding Biology through StructuresCourse Work 2009 “Throughout our endeavors we have been motivated by the expectation that the detailed knowledge of its (F 0 F 1 ATP synthase) structure would lead to a better understanding of how ATP is made.” -John Walker Mechanism of F 0 F 1 ATP Synthase

32. Understanding Biology through StructuresCourse Work 2009 Mutations and Their Effect on Protein Structures Mutations responsible for numerous diseases Sickle cell anemia (point mutation) Cystic fibrosis (point mutation) Huntington’s disease (insertion of extra amino acids) HIV uses mutations to its advantage a drug that binds to an HIV protein may not bind very well only a few viral generations later

33. Understanding Biology through StructuresCourse Work 2009 Sickle Cell Anemia caused by One Mutation Sickle cell anemia is caused by a point mutation in hemoglobin b chain (a is unaffected) val-his-leu-thr-pro-glu-glu … normal individual val-his-leu-thr-pro-val-glu … affected individual Only one amino acid is change in the entire sequence of the protein glutamic acid side chain -CH 2 -CH 2 -COO – acidic side chain valine side chain -CH-(CH 3 ) 2 nonpolar side chain The hemoglobin molecule folds up and functions (binds oxygen) The mutation caused the protein to clump up in the cells The clumping up distorts the cell shape and makes them architecturally weaker

34. Understanding Biology through StructuresCourse Work 2009 The surface of the protein has side chains sticking out. Polar and charged side chains help the protein stay dissolved in water The glutamic acid to valine mutation is a surface mutation   Sickle Cell Anemia caused by One Mutation

35. Understanding Biology through StructuresCourse Work 2009 Mechanism of Influenza virus entry into cells Understanding Influenza: A Success Story Flu different from common cold Cold characterized by fever or headache

36. Understanding Biology through StructuresCourse Work 2009 Understanding Influenza : A Success Story Crystal structure of Zanamivir: neuraminidase structure

37. Understanding Biology through StructuresCourse Work 2009 Cryo-microscopic image of Dengue virus 18 Å Carbohydrate recognition domain (CRD) of DC-SIGN

38. Understanding Biology through StructuresCourse Work 2009 Challenges for Structural Biology How can the process of structure determination be expedited? Can we predict the structures of proteins accurately? How can we use the structures in designing novel therapies? Thank You !