Proteins Structures:

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Proteins Structures Introduction and General
Overview
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Sequencing the Human Genome A Landmark in the
History of Mankind
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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..
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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
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Some Landmarks in Macromolecular Structure
Determination
Watson and Crick
Perutz and Kendrew
Hodgkin and vitamin B12
Pauling
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Some Landmarks in Macromolecular Structure
Determination..contd.
Photosynthetic reaction centre
Virus
Potassium channel
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Structure determination made easy!

• 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

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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 Solubiliz
ation of the over-expressed protein Structure
determination of a molecule as it exists in
solution Size-limit is a major factor
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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

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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)

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X-ray versus NMR
X-ray 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 NOEs is slow and error prone

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DNA Diffraction pattern
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Model of DNA
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Landmarks in Macromolecular Structure
Determination
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Limitations of Experimental Methods Consequences

• Annotated proteins in the databank 100,000

• Total number including ORFs 700,000

• Proteins with known structure 5,000 !

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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 gt50
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Three-dimensional structures of homologous
proteins are very similar
Human
Leishmania
Chicken
Thermotoga
Vibrio
Pyrococcus
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The sequence- structure relationship
The relation between the divergence of sequence
and structure in proteins. Chothia C, Lesk AM.
EMBO J. 1986 Apr5(4)823-6.
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Predicting Protein Structure 1. Comparative
(Homology) Modeling
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2. Threading Basic Strategy
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Query
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Some landmark protein structures
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student
postdoc
Geneticist?
professor
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Structure of antibody
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AntigenAntibody 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.

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MHC molecules
Three-dimensional structures of MHC molecules
explain how these can bind large number of
peptides without any sequence specificity
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Recognition of MHC-I peptide complex by TCR
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Cholera Toxin Recognition via sugar moiety
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Antibody Enzymes ABZYMES
Diels-Alderase Catalytic Antibody 1E9 Complex
With Its Hapten
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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
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Structure of the ribosome
50S
30S
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Understanding Influenza A Success Story

• Flu different from common cold
• Cold characterized by fever or headache

Mechanism of Influenza virus entry into cells
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Understanding Influenza A Success Story
In 1999, the Food and Drug Administration (FDA)
approved two new drugs to fight the flu
zanamivir (Relenza) and oseltamivir (Tamiflu),
the first of a new class of antiviral drugs
called neuraminidase inhibitors. How do they
work? The surfaces of influenza viruses are
dotted with neuraminidase proteins.
Neuraminidase, an enzyme, breaks the bonds that
hold new virus particles to the outside of an
infected cell. Once the enzyme breaks these
bonds, this sets free new viruses that can infect
other cells and spread infection. Neuraminidase
inhibitors block the enzyme's activity and
prevent new virus particles from being released,
thereby limiting the spread of infection.
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Understanding Influenza A Success Story
Crystal structure of Zanamivir neuraminidase
structure
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Mutations and Their Effect on Protein Structures

• Mutations responsible for numerous diseases
• Sickle cell anemia (point mutation)
• Cystic fibrosis (point mutation)
• Huntingtons 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

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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

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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
?
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Inhibition of HIV protease