understanding biology through structurescourse work 2009 proteins structures: introduction and...

Understanding Biology through Structures Course Work 2009

Proteins Structures:Introduction

and General Overview


Sequencing the Human Genome:A Landmark in the History of Mankind


…..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


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 andCrick

Perutz andKendrew

Hodgkin Pauling

Great ideas have always faced violent opposition from mediocre minds-Albert Einstein


Some Landmarks in Macromolecular Structure Determination……..contd.

Photosynthetic reaction centre

Potassium channelVirus


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


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)


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


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


DNA : Diffraction pattern


Model of DNA


Protein Structures • Primary structure

– Un-branched polymer– 20 side chains (residues or amino acids)


How does a protein adopt a unique 3D

conformation?


Peptide torsion angles


Phi-Psi map (Ramachandran map)


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%


Chicken Human Leishmania

Pyrococcus Thermotoga Vibrio

Three-dimensional structures of homologous proteins are very similar


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


•Annotated proteins in the databank: ~ 100,000

Limitations of Experimental Methods: Consequences

•Proteins with known structure: ~5,000 !

•Total number including ORFs: ~ 700,000


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KQFTKCELSQNLYDIDGYGRIALPELICTMFHTSGYDTQAIVENDESTEYGLFQISNALWCKSSQSPQSRNICDITCDKFLDDDITDDIMCAKKILDIKGIDYWIAHKALCTEKLEQWLCEKE

Predicting Protein Structure:Comparative (Homology) Modeling

Use as template & model

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KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRLShare

Similar Sequence

Homologous


How have the protein structures enhanced our

understanding of Biology?


Structure of antibody


•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


Antibody & Enzymes : ABZYMES

Diels-Alderase Catalytic Antibody 1E9 Complex With Its Hapten


Cholera Toxin: Recognition via sugar moiety


“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


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 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


•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


Mechanism of Influenza virus entry into cells

Understanding Influenza: A Success Story

• Flu different from common cold

• Cold characterized by fever or headache


Understanding Influenza : A Success Story

Crystal structure of Zanamivir: neuraminidase structure


Cryo-microscopic image of Dengue virus

18 ÅCarbohydrate recognition domain (CRD) of DC-SIGN


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 !

understanding biology through structurescourse work 2009 proteins structures: introduction and...

Documents

understanding biology

structurescourse work

understanding function

problems of biology

general overview slide

history of mankind slide

molecule nmr spectroscopy

diffraction pattern