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
Hierarchy in Understanding Function
…..AATGCCGCGTAGTCGGGTAAGGGTCTGAAGCTGAAATCTTTTCACACCGAGTCGATGGG…
…..GCCGCGTAGTCGGGTAAGGGTCACACCGAGTCGATGG…
….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
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 and
Crick
Hodgkin
Perutz and
Kendrew
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
Virus
Understanding Biology through Structures
Potassium channel
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
X-ray
• Producing enough protein for trials
• Crystallization time and effort
• Crystal quality, stability and size
control
• Finding isomorphous derivatives
• Chain tracing & checking
Understanding Biology through Structures
NMR
• 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
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 threedimensional 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
Three-dimensional structures of homologous
proteins are very similar
Chicken
Human
Leishmania
Pyrococcus
Thermotoga
Vibrio
Understanding Biology through Structures
Course Work 2009
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.
Understanding Biology through Structures
Course Work 2009
Limitations of Experimental Methods:
Consequences
•Annotated proteins in the databank: ~ 100,000
•Total number including ORFs: ~ 700,000
•Proteins with known structure: ~5,000 !
Understanding Biology through Structures
Course Work 2009
Predicting Protein Structure:
Comparative (Homology) Modeling
KQFTKCELSQNLYDIDGYGRIALPELICTMF
HTSGYDTQAIVENDESTEYGLFQISNALWCK
SSQSPQSRNICDITCDKFLDDDITDDIMCAK
KILDIKGIDYWIAHKALCTEKLEQWLCEKE
?
1alc
Homologous
Share
Similar
Sequence
Use as template
& model
Understanding Biology through Structures
KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAK
FESNFNTQATNRNTDGSTDYGILQINSRWWCND
GRTPGSRNLCNIPCSALLSSDITASVNCAKKIV
SDGNGMNAWVAWRNRCKGTDVQAWIRGCRL
8lyz
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
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.
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
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
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 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
valine side chain
-CH2-CH2-COO– acidic 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
Sickle Cell Anemia caused by
One Mutation
b
•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
Understanding Biology through Structures
Course Work 2009
Understanding Influenza:
A Success Story
• Flu different from common cold
• Cold characterized by fever or headache
Mechanism of Influenza
virus entry into cells
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 Å
Understanding Biology through Structures
Carbohydrate
recognition domain
(CRD) of DC-SIGN
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 !
Understanding Biology through Structures
Course Work 2009