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Proteins Structures: Introduction and General Overview. Sequencing the Human Genome: A Landmark in the History of Mankind. …..GCCGCGTAGTCGGGTAAGGGTCACACCGAGTCGATGG…. Hierarchy in Understanding Function. …..AATGCCGCGTAGTCGGGTAAGGGTCTGAAGCTGAAATCTTTTCACACCGAGTCGATGGG….
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Proteins Structures: Introduction and General Overview
Sequencing the Human Genome: A Landmark in the History of Mankind
…..GCCGCGTAGTCGGGTAAGGGTCACACCGAGTCGATGG… Hierarchy in Understanding Function …..AATGCCGCGTAGTCGGGTAAGGGTCTGAAGCTGAAATCTTTTCACACCGAGTCGATGGG… ….APTCHYLDELAKGGRLDATIKRDGLGVLVWAQND…. “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
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…..
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
Some Landmarks in Macromolecular Structure Determination……..contd. Photosynthetic reaction centre Virus Potassium channel
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
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
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) Principles of 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-ray versus NMR X-ray NMR
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 Structure Determination Made Easy(Modern Crystallographers Three Rings)
Primary structure Un-branched polymer 20 side chains (residues or amino acids) Protein Structures
The Protein Folding Problem Amino acid sequence of a polypeptide has all the information required to determine its three-dimensional topology
If a polypeptide sequence corresponds to a unique conformation of the protein, how does nature take care of point mutations in the primary sequences?
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%
Three-dimensional structures of homologous proteins are very similar Human Leishmania Chicken Thermotoga Vibrio Pyrococcus
The sequence- structure relationship The relation between the divergence of sequence and structure in proteins. Chothia C, Lesk AM. EMBO J. 1986 Apr;5(4):823-6.
Limitations of Experimental Methods: Consequences • Annotated proteins in the databank: ~ 100,000 • Total number including ORFs: ~ 700,000 • Proteins with known structure: ~5,000 !
Homologous KQFTKCELSQNLYDIDGYGRIALPELICTMFHTSGYDTQAIVENDESTEYGLFQISNALWCKSSQSPQSRNICDITCDKFLDDDITDDIMCAKKILDIKGIDYWIAHKALCTEKLEQWLCEKE KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL Share Similar Sequence 1alc ? Use as template & model 8lyz Predicting Protein Structure: Comparative (Homology) Modeling
Antigen:Antibody complex • Antibodies bind to antigens by recognizing a large surface, and through surface complementarity. • Thus, these complexes have a very high affinity for each other.
Antibody & Enzymes : ABZYMES Diels-Alderase Catalytic Antibody 1E9 Complex With Its Hapten
Mechanism of F0F1 ATP Synthase “Throughout our endeavors we have been motivated by the expectation that the detailed knowledge of its (F0F1 ATP synthase) structure would lead to a better understanding of how ATP is made.” -John Walker
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
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 -CH2-CH2-COO– acidic side chain valine side chain -CH-(CH3)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
Sickle Cell Anemia caused by One Mutation • •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 b
Understanding Influenza: A Success Story • Flu different from common cold • Cold characterized by fever or headache Mechanism of Influenza virus entry into cells
Understanding Influenza : A Success Story Crystal structure of Zanamivir: neuraminidase structure
Cryo-microscopic image of Dengue virus Carbohydrate recognition domain (CRD) of DC-SIGN 18 Å
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 !