understanding biology through structurescourse work 2009 proteins structures: introduction and...
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Understanding Biology through Structures Course Work 2009
Proteins Structures:Introduction
and General Overview
Understanding Biology through Structures Course Work 2009
Sequencing the Human Genome:A Landmark in the History of Mankind
Understanding Biology through Structures Course 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
Understanding Biology through Structures Course 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…..
Understanding Biology through Structures Course Work 2009
Some Landmarks in Macromolecular Structure Determination
Watson andCrick
Perutz andKendrew
Hodgkin Pauling
Great ideas have always faced violent opposition from mediocre minds-Albert Einstein
Understanding Biology through Structures Course Work 2009
Some Landmarks in Macromolecular Structure Determination……..contd.
Photosynthetic reaction centre
Potassium channelVirus
Understanding Biology through Structures Course 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
Understanding Biology through Structures Course 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
Understanding Biology through Structures Course 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)
Understanding Biology through Structures Course 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-ray NMR
Understanding Biology through Structures Course 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
Understanding Biology through Structures Course Work 2009
DNA : Diffraction pattern
Understanding Biology through Structures Course Work 2009
Understanding Biology through Structures Course Work 2009
Model of DNA
Understanding Biology through Structures Course Work 2009
Protein Structures • Primary structure
– Un-branched polymer– 20 side chains (residues or amino acids)
Understanding Biology through Structures Course Work 2009
How does a protein adopt a unique 3D
conformation?
Understanding Biology through Structures Course Work 2009
Peptide torsion angles
Understanding Biology through Structures Course Work 2009
Phi-Psi map (Ramachandran map)
Understanding Biology through Structures Course Work 2009
The Protein Folding Problem
Amino acid sequence of a polypeptide has all the information required to determine its three-dimensional topology
Understanding Biology through Structures Course 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?
Understanding Biology through Structures Course 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%
Understanding Biology through Structures Course Work 2009
Chicken Human Leishmania
Pyrococcus Thermotoga Vibrio
Three-dimensional structures of homologous proteins are very similar
Understanding Biology through Structures Course 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
Understanding Biology through Structures Course 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
Understanding Biology through Structures Course Work 2009
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KQFTKCELSQNLYDIDGYGRIALPELICTMFHTSGYDTQAIVENDESTEYGLFQISNALWCKSSQSPQSRNICDITCDKFLDDDITDDIMCAKKILDIKGIDYWIAHKALCTEKLEQWLCEKE
Predicting Protein Structure:Comparative (Homology) Modeling
Use as template & model
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KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRLShare
Similar Sequence
Homologous
Understanding Biology through Structures Course Work 2009
How have the protein structures enhanced our
understanding of Biology?
Understanding Biology through Structures Course Work 2009
Structure of antibody
Understanding Biology through Structures Course 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
Understanding Biology through Structures Course Work 2009
Antibody & Enzymes : ABZYMES
Diels-Alderase Catalytic Antibody 1E9 Complex With Its Hapten
Understanding Biology through Structures Course Work 2009
Cholera Toxin: Recognition via sugar moiety
Understanding Biology through Structures Course Work 2009
“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
Mechanism of F0F1 ATP Synthase
Understanding Biology through Structures Course 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
Understanding Biology through Structures Course 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 individualval-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
Understanding Biology through Structures Course 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
Understanding Biology through Structures Course 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
Understanding Biology through Structures Course Work 2009
Understanding Influenza : A Success Story
Crystal structure of Zanamivir: neuraminidase structure
Understanding Biology through Structures Course Work 2009
Cryo-microscopic image of Dengue virus
18 ÅCarbohydrate recognition domain (CRD) of DC-SIGN
Understanding Biology through Structures Course 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 !
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