Transcript Protein Folding Problem

The Protein Folding Problem
Bionanotechnology
Protein folding is
“one of the great unsolved problems of science”
Alan Fersht
protein folding can be seen as a connection between
the genome (sequence) and what the proteins
actually do (their function).
Protein folding problem
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Prediction of three dimensional structure from its
amino acid sequence
Translate “Linear” DNA Sequence data to spatial
information
Why solve the folding problem?
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Acquisition of sequence data relatively quick
Acquisition of experimental structural information
slow
Limited to proteins that crystallize or stable in
solution for NMR
Protein folding dynamics
Electrostatics, hydrogen bonds and van der Waals forces hold a
protein together.
Hydrophobic effects force global protein conformation.
Peptide chains can be cross-linked by disulfides, Zinc, heme or
other liganding compounds. Zinc has a complete d orbital , one
stable oxidation state and forms ligands with sulfur, nitrogen and
oxygen.
Proteins refold very rapidly and generally in only one stable
conformation.
Random search and the
Levinthal paradox
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The initial stages of folding must be nearly random, but if the entire process was
a random search it would require too much time. Consider a 100 residue
protein. If each residue is considered to have just 3 possible conformations the
total number of conformations of the protein is 3100. Conformational changes
occur on a time scale of 10-13 seconds i.e. the time required to sample all
possible conformations would be 3100 x 10-13 seconds which is about 1027 years.
Even if a significant proportion of these conformations are sterically disallowed
the folding time would still be astronomical. Proteins are known to fold on a
time scale of seconds to minutes and hence energy barriers probably cause the
protein to fold along a definite pathway.
Physical nature of protein folding
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Denatured protein makes many interactions with
the solvent water
During folding transition exchanges these noncovalent interactions with others it makes with
itself
What happens if proteins don't fold correctly?
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Diseases such as Alzheimer's disease, cystic
fibrosis, Mad Cow disease, an inherited form of
emphysema, and even many cancers are believed to
result from protein misfolding
Protein folding is a balance of forces
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Proteins are only marginally stable
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Free energies of unfolding ~5-15 kcal/mol
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The protein fold depends on the summation of all
interaction energies between any two individual
atoms in the native state
Also depends on interactions that individual atoms
make with water in the denatured state
Protein denaturation
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Can be denatured depending on chemical
environment
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Heat
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Chemical denaturant
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pH
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High pressure
The
Protein Folding
Folding Problem
The
Protein
Problem
A major hurdle must be crossed before bionanotechnology
will have general applicability:
We must be able to predict the folded structure of a protein
starting only with its chemical sequence.
Without this ability, we will merely shadow evolution,
poking and prodding existing proteins until they are changed
into something that we want.
The Protein Folding Problem
The protein folding problem poses grave difficulties for two
reasons.
1. The first is the sheer magnitude of the problem. Typical
proteins have several hundred amino acids. Each is connected to
its neighbors through two flexible linkages that may adopt a
range of stable conformations. In addition, each amino acid has a
flexible side chain that can adopt a number of stable local
conformations. Together, these many levels of torsional freedom
define a staggeringly large conformational space that is beyond
all current computational prediction methods.
The Protein Folding Problem
The protein folding problem poses grave difficulties for two
reasons.
2. The second problem lies in the method used to estimate the
stability of each trial conformation during a prediction
experiment. Folded proteins have thousands of internal contacts,
each of which adds a tiny increment of stabilization to the entire
structure.
The Protein Folding Problem
The protein folding problem poses grave difficulties for two
reasons.
2. Many water molecules are freed as proteins
fold, as the protein chains shelter their carbon-rich portions
inside.
This freeing of water is a strong force pushing proteins toward a
folded structure. Entropy, on the other hand, works against the
favorable energies of internal contacts and water release.
Protein Structure Prediction
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Why ?
Type of protein structure
predictions
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Sec Str. Pred
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Homology Modelling
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Fold Recognition
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Ab Initio
Secondary structure prediction
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Why
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History
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Performance
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Usefullness
Why do we need structure prediction?
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3D structure give clues to function:
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active sites, binding sites, conformational changes...
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structure and function conserved more than sequence
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3D structure determination is difficult, slow and
expensive
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Intellectual challenge, Nobel prizes etc...
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Engineering new proteins
The Use of Structure
The Use of Structure
The Use of Structure
It's not that simple...
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Amino acid sequence contains all
the information for 3D structure
(experiments of Anfinsen, 1970's)
But, there are thousands of atoms,
rotatable bonds, solvent and other
molecules to deal with...
Levinthal's paradox
Structure prediction
Summary of the four main approaches to structure prediction. Note
that there are overlaps between nearly all categories.
Approac h
Difficulty
Usefulness
Compar ative Proteins of
modelling
known
(Homolog y
structure
modelling)
Identify related structure with
sequence methods, copy 3D
coords and modi fy where
necessary
Relatively easy
Very, if sequence identity
drug design
Fold
recognition
Proteins of
known
structure
Same as above, but use more
sophisticated methods to find
related structure
Medium
Limited due to poor models
Secondary
structure
prediction
Sequencestructure
statistics
Forget 3D arrangeme nt and
Medium
predict where the helices/strands
are
Can improve align ments,
fold recognition, ab initio
ab initio
tertiary
structure
prediction
Energy
functions,
statistics
Simulat e folding, or generate lots Very hard
of structures and try to pick the
correct one
Not really
Method
Knowledge
Secondary structure predictions
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Ignore 3D, it's too hard!
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Usually concentrate on helix, strand and ``coil''.
Pattern recognition, but which patterns?
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some amino acids have preferences for helix or strand; due to
geometry and hydrogen bonding
spatial (along sequence) patterns, alternating hydrophobics (helical
wheel)
conservation (down alignment) in different members of protein
family; insertions and deletions
Three main generations/stages in SSP method development since
1970's.
What is ``known secondary
structure''?
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Of critical importance in training/assessment of
SSP methods
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Can be defined:
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visually by structural biologist
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by geometric and chemical criteria (, angles,
distances between atoms, hydrogen bonds...) by
programs like DSSP and STRIDE
Secondary structures -Helix
Secondary Structure - Sheet
Secondary structure - turns
Other secondary structure prediction
methods
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turn prediction
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transmembrane helix prediction
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coiled coil
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Dissorder predictions
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contact prediction, disulphides
What use is it?
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No 3D means no clues to detailed function, so...
Accurate secondary structure predictions help
sequence analysis: finding homologues, aligning
homologues, identifying domain boundaries.
Can help true 3D prediction
Future improvements to SSP
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Long range information
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Baker
Folding pathway and/or 3D-information