Protein Classification and Structure Prediction Amino acid ...

Protein Classification and
Structure Prediction
Amino acid composition
• Basic Amino Acid
Structure:
• The side chain, R,
varies for each of
the 20 amino acids
Side chain
H
H
Amino
group
N
R
C α
H
C
O
OH
Carboxyl
group
Proteins are chains of amino acids
The Peptide Bond
• Dehydration synthesis
• Repeating backbone: N–CN
α –C –N–C α –C
Peptidyl polymers
• A few amino acids in a chain are called a polypeptide. . A
protein is usually composed of 50 to 400+ amino acids.
• Since part of the amino acid is lost during dehydration
synthesis, we call the units of a protein amino acid
residues.
carbonyl
carbon
amide
nitrogen
• Convention – start at amino terminus and proceed to
carboxy terminus
Krane & Raymer
Side chain properties
• Carbon does not make hydrogen bonds with
water easily – hydrophobic
• O and N are generally more likely than C to h-h
bond to water – hydrophilic
• The amino acids forms three general groups:
• Hydrophobic
• Charged (positive/basic & negative/acidic)
• Polar
The Hydrophobic Amino Acids
Proline severely
limits allowable
conformations!
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The Charged Amino Acids
The Polar Amino Acids
More Polar Amino Acids
Planarity of the peptide bond
And then there’s…
Psi (ψ) – the
angle of
rotation about
the Cα-C bond.
Phi (φ) – the
angle of
rotation about
the N-Cα bond.
The planar bond angles and bond
lengths are fixed.
Krane & Raymer
Primary & Secondary Structure
• Primary structure = the linear sequence of amino
acids comprising a protein:
AGVGTVPMTAYGNDIQYYGQVT…
• Secondary structure
• Regular patterns of hydrogen bonding in proteins
result in two patterns that emerge in nearly every
protein structure known: the α-helix
and the
β-sheet
• The location of direction of these periodic, repeating
structures is known as the secondary structure of the
protein
The alpha helix
φ ≈ ψ
≈ −60°
Krane & Raymer
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Properties of the alpha helix
• φ ≈ ψ ≈ −60°
• Hydrogen bonds
between C=O of
residue n, , and
NH of residue
n+4
• 3.6 residues/turn
• 1.5 Å/residue rise
• 100°/residue turn
Properties of a-helices
• 4 – 40+ residues in length
• Often “dual-natured”
• Half hydrophobic and half hydrophilic
• Mostly when surface-exposed
exposed
• For many α-helices
• Helix formers: Ala, Glu, Leu,
Met
• Helix breakers: Pro, Gly, Tyr,
Ser
Krane & Raymer
Krane & Raymer
The beta strand (& sheet)
φ ≈ − 135°
ψ ≈ +135°
Properties of beta sheets
• Formed of stretches of 5-105
residues in extended
conformation
• Pleated – each C α a bit above or
below the previous
• Parallel/aniparallel
aniparallel,
contiguous/non-contiguous
Krane & Raymer
Parallel and anti-parallel
b-sheets
Anti-parallel is slightly energetically favored
Anti-parallel
Parallel
Turns and Loops
• Secondary structure elements are connected by regions
of turns and loops
• Turns – short regions
of non-α, , non-β
conformation
• Loops – larger stretches with no secondary structure.
Often disordered.
• “Random coil”
• Sequences vary much more than secondary structure regions
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Levels of Protein
Structure
• Secondary structure
elements combine to
form tertiary structure
• Quaternary structure
occurs in multi-enzyme
complexes
• Many proteins are active
only as homodimers,
homotetramers, , etc.
Protein Structure Examples
Krane & Raymer
• Two cysteines in
close proximity
will form a
covalent bond
• Disulfide bond,
disulfide bridge,
or dicysteine
bond.
• Significantly
stabilizes tertiary
structure.
Disulfide Bonds
Determining Protein Structure
• There are O(100,000) distinct proteins in human
proteome.
• Two methods for revealing positions of atoms in 3-D: 3
• X-Ray Crystallography
• X-ray diffraction pattern + mathematical construction
• Good protein crystal needed, good resolution of diffraction needed
ed
• Nuclear Magnetic Resonance
• Small proteins only (< 250 residues)
• Inter-proton distances + geometric constraints
• 24,000+ 3D structures have been determined so far
(including duplicates with different ligand bound, etc.)
Krane & Raymer
PDB Holdings List: 23-Mar
Mar-2004
PDB: Growth (Mar 2004)
X-ray
Diffraction
and other
NMR
Proteins,
Peptides,
& Viruses
19427
2998
Protein/Nucleic
Acid Complexes
944
97
Nucleic
Acids
726
575
Carbohydrates
14
4
total
21111
3674
Total
22425
1041
1301
18
24785
Please note that theoretical models have been removed.
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Experimentation to grow
protein crystals
X-Ray Crystallography
Trial-and-error
Experimentation
Observables
Partial Success
~0.5mm
Trial 1
Control
Parameters
Failure
Success
Trial 3
Trial 2
• The crystal is a mosaic of millions of copies
of the protein.
• As much as 70% is solvent (water)!
• May take months (and a “green” thumb) to
grow.
Krane & Raymer
Krane & Raymer
X-Ray diffraction
• Image is averaged over:
• Space (many copies)
• Time (of the diffraction experiment)
Protein Crystal Growth in Space
• Protein crystal growth experiments on over 20 shuttle
missions since 1984
• Larger Crystals in 45.4% of the cases
• New Crystal Structures in 18% of the cases
• ≥ 10% increase in X-Ray X
Crystallography Brightness in 58%
of the cases
• Less thermal motion in 27.2% of the cases
• An X-Ray X
Crystallography resolution improvement of ~0.3 Å
in 42.4% of the cases
• An X-Ray X
Crystallography resolution improvement of 0.3 to
0.5 Å in 9.9% of the cases
• An X-Ray X
Crystallography resolution improvement of 0.5 to
1.0 Å in 9.9% of the cases
Krane & Raymer
The Protein Folding Problem
• Proteins self-assemble in solution. Almost all of the
information necessary to determine the complex 3-D 3
structure is in the amino acid sequences
• Central dogma:
Sequence specifies structure
• Central question:
“Given a particular sequence of
amino acid residues (primary structure), what will the
tertiary/quaternary structure of the resulting protein be?”
Levinthal’s paradox
• Consider a 100 residue protein. If each residue
can take only 3 positions, there are 3 100 = 5 ×
10 47 possible conformations.
• If it takes 10 -13
s to convert from 1 structure to
another, exhaustive search would take 1.6 × 10 27
years!
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Ideas on protein folding
• It is believed that hydrophobic collapse is a key
driving force for protein folding
• Hydrophobic core!
• Proteins are, in fact, only marginally stable
• Native state is typically only 5 to 10 kcal/mole more
stable than the unfolded form
• Many proteins help in folding
• Protein disulfide isomerase – catalyzes shuffling of
disulfide bonds
• Chaperones – break up aggregates and (in theory)
unfold misfolded proteins
What determines fold?
• Anfinsen’s experiments in 1957 demonstrated
that proteins can fold spontaneously into their
native conformations under physiological
conditions. This implies that primary structure
does indeed determine folding or 3-D 3 D structure.
• Some exceptions exist
• Chaperone proteins assist folding
• Abnormally folded Prion proteins can catalyze
misfolding of normal prion proteins that then
aggregate
Other factors
• Physical properties of protein that influence
stability & therefore, determine its fold:
• Rigidity of backbone
• Amino acid interaction with water
• Hydropathy index for side chains
• Interactions among amino acids
• Electrostatic interactions
• Hydrogen, disulphide bonds
• Volume constraints
CASP changed the
landscape
• Critical Assessment of Structure Prediction competition.
Even numbered years since 1994
• Solved, but unpublished structures are posted in May,
predictions due in September
• Various categories
• Relation to existing structures, ab initio, , homology, fold, etc.
• Partial vs. Fully automated approaches
• Produces lots of information about what aspects of the
problems are hard, and ends arguments about test sets.
• Results showing steady improvement, and the value of
integrative approaches.
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