Transcript Slide 1

Protein Structures: Data Representation
1) Primary Structure: character string.
2) Secondary Structure:
Identifying sub-structures in a large protein
based on sequence.
3) Tertiary Structure:
4) Quaternary Structure:
3-Dimensional Representation
Protein Database Bank (PDB)
This is a complicated file format structure that
support numerous programs, and contains
information regarding the primary structure
(sequence), 3-D structures (x, y, z
coordinates), size and linking of specific atoms
in structures, etc.
Secondary Structure Prediction:
1) Hydropathy Plot
A Hydropathy plot identifies domains within a protein that are soluble (region of
“charged” amino acids) or insoluble (region of “uncharged” amino acids).
2) Alpha Helix
An alpha helix is a group of amino acids within a proteins
that arrange themselves in a helical structure.
3) Beta Sheet
A beta sheet is a group of amino acids within a protein
that arrange themselves in a stable aligned (parallel)
configuration.
Secondary Structure Prediction: Hydropathy Plot
Commonly used to identify alpha helices that span a membrane (i.e. anchor protein to cell wall).
1) Choose a “moving window” that travels along the protein sequence;
a) calculates the overall “solubility” of the amino acids in the window.
b) moves in amino acid
c) repeat calculation
d) continue this though the entire protein sequence.
Transmembrane
domains are 20
amino acids, but
any size window
can be used.
ELRLRYCAPAGFALLKCNDADYDGFKTNCSNVSVVHCTNLMNTTVTTGLLLNGSYSENRT
1) Calculate
average using
amino acidsspecific constants.
2) Move window
1 amino acid.
3) Calculate
average.
Secondary Structure Prediction: Hydropathy Plot
ELRLRYCAPAGFALLKCNDADYDGFKTNCSNVSVVHCTNLMNTTVTTGLLLNGSYSENRT
X = (-3.5)+(3.8)+(-4.5)+(3.8)+(-4.5)+(-1.3)+(2.5)+(1.8)+(-1.6)+(1.8)+(-0.4)+(2.8)+(1.8)+(3.8)+(3.8)+(-3.9)+(2.5)+(-3.5)+(-3.5)+(1.8)
WINDOW SIZE: 20
Solubility Constants
(Kyte & Doolittle)
A Alanine
1.8
R Arginine -4.5
N Asparagine -3.5
D Aspartic acid -3.5
C Cysteine 2.5
Z Glutamine -3.5
E Glutamic acid -3.5
G Glycine
-0.4
H Histidine -3.2
I Isoleucine 4.5
L Leucine
3.8
K Lysine
-3.9
M Methionine 1.9
F Phenylalanine 2.8
P Proline
-1.6
S Serine
-0.8
T Threonine -0.7
W Tryptophan -0.9
Y Tyrosine -1.3
V Valine
4.2
X = 30.05 / 20
X = 1.503
Positive numbers are hydrophobic (insoluble)
Negative numbers are hydrophilic (soluble)
Highly insoluble regions represent positions for protein insertion into the membrane.
Protein Folding: Computationally Modeling Biochemistry
OBJECTIVE:
Utilize the sequence information, along with temperaturedependent biomolecular interaction constants, to computationally
“predict” a protein’s tertiary structure.
CHALLENGES:
A) It is NOT known how proteins fold in nature.
B) More detailed or mathematically-intensive methods can’t be
completed in a reasonable time (given current computer
capabilities).
C) There are essentially no experimental methods to verify or validate
that a predicted protein is “correct” – or “how correct”.
Monte Carlo simulation of a folding event. Each frame displays the average position of
a 48-mer chain during a 10^4 iteration time window. The color of each bead represents
the variance of the position of the bead during this time interval, with yellow/green
indicating large fluctuations and blue indicating small fluctuations. The entire folding
event takes 8 x 10^5 iterations.
Evolution of Protein Folding Methods:
1) Lattice Methods: 3D lattice of residue or atomic positions.
2) Off-Lattice Methods: Not reliant on predetermined 3D positions.
Can include solvent effects.
3) All Atoms Methods/Modeling: EXTREMELY computationally
intensive.
Tactics
A) Initially calculate secondary structures minimums (fold sheets and
helices), then calculate minima for remaining sequence.
B) Emulate Protein synthesis process, starting from amino-terminus.
C) Utilize existing NMR and X-ray crystal structures that match
sequence under investigation.
Protein Self-Assembly: Good AND Bad
Quaternary Structure: the interaction of
multiple proteins to form larger
functional structures.
Many proteins bind to themselves to
form homodimers and
homopolymers. Many proteins bind
to other proteins to form
heterodimers and heteropolymers.
Many diseases involve self-aggregating proteins (especially
neurodegenerative diseases).
Mad Cow Disease (Prion Proteins)
Alzheimer’s Disease (beta-Amyloid Peptide)
Huntington’s Disease
Why neuro-diseases?
1) Because the blood flow (nutrients) to the brain is highly regulated, and
proteins that aggregate tend to collect – and are NEUROTOXIC. Note that
these proteins ALSO aggregate in peripheral tissues, but are “cleared” and
do not appear to be sufficiently toxic.
2) Brain cells (neurons) do NOT regenerate in a manner equivalent to
peripheral tissues (particularly in older people).
3) Loss of neuronal cells leads to altered cognitive capabilities, which is not
the case in peripheral tissues (e.g. slight muscle atrophy).
Neurodegenerative Protein Diseases = Beta Sheet
Structures!!!
Beta-sheet structures are sometimes called
“amyloid” structures.
Hence the term: Amyloidopathy
NOTE: The molecular forces that assemble betasheet structures ALSO cause them to self-assemble!
2 key concepts regarding age-related diseases….
1) Increased human health & longevity “invents diseases”. Before the modern age, nature
had rarely seen a 60 year old human. Imagine the age-related diseases of the future
when the average human life span is >120 years.
2) Evolutionary pressures did not select for humans to live much longer than 35-40 years.
So inherited mutations that lead to age-related diseases were not “selected out” of the
human population. This fact has NOT changed in modern times.
Alzheimer’s Disease
40-90 (sporadic at 60+, familial at 40+), increases with age
Men more common under the age of 80 yrs
Women more common over the age of 80 yrs
(J Neurol Neurosurg Psychiatry 1999;66;177 in BMJ 1999 Feb 27;318(7183);614)
Alzheimer’s Disease
Amyloid Precursor Protein
Beta Amyloid Protein
42 amino acids long
Self Aggregation
Neuronal cell nuclei (blue circles)
Senile Plaque
Beta-Amyloid Aggregated in Water
500 nm
Huntington’s Disease
Incidence
2-8 persons per 100,000 worldwide with focal population clusters
Cause Known:
excess of trinucleotide (CAG) repeats (encode glutamine)
#CAG repeats
6-34
Normal Gene
36-120 HD Mutation (majority 40-50 CAG repeats, 33-40 yr onset)
Number of repeats inversely related to age of onset. Juvenile onset is rare and involves
CAG repeats >60.
Huntingtin Gene
10-30 CAG codons
Normal
Abnormal
> 40 CAG codons
Huntingtin Protein
Normal
Abnormal
Figure 1. Specific localization
of huntingtin aggregates in
HD-repeat mutant mouse
brain.
Low-magnification micrographs
are shown of brain sections from
HD-repeat mutant (a) and wildtype (b) mice at 27 months of
age. Only the striatum (Str) in
the HD-repeat mutant mouse
brain was immunoreactive with
EM48. Ctx, cortex. Highmagnification light micrograph
(c) and electron microscopy (d)
show EM48−immunoreactive
aggregates in the neuronal
nucleus (arrows). n, Nucleus.
Immunofluorescent double
labelling shows that striatal
neurons containing intranuclear
EM48−reactive aggregates are
labelled by antibodies to
calbindin-D (stars in e), but not
by antibodies to nitric oxide
synthase (NOS; f) or
parvalbumin (PARV; g). Scale
bars, 10 m (a−c,f− g) and
0.5 m (d).
Prion Protein Diseases
Creutzfeldt-Jakob Disease
CJD
humans
variant Creutzfeldt-Jakob Disease
vCJD
humans; acquired from cattle with BSE
Bovine Spongiform Encephalopathy
BSE
"mad cow disease"
Kuru
infectious; in humans who practiced cannibalism in Papua New Guinea
Gerstmann-Sträussler-Scheinker disease
GSS
inherited disease of humans
Fatal Familial Insomnia
FFI
inherited disease of humans
Scrapie
infectious disease of sheep and goats
other animal TSEs
cats, mink, elk, mule deer
1) Inter-species effect due to similarity between prion protein sequences.
2) The role of the normal prion protein in nature is not understood.
3) The disease involves a mis-folding of the prion protein to a beta-sheet
structure, which then self-aggregates.
The illustration below compares a normal prion protein (PrpC) to a
disease-causing form (PrpSc). The two structures exhibit two different,
classic protein motifs, called "alpha helices," and "beta sheets." Alpha
helices, seen here in the normal prion (left), consist of linked aminoacid building blocks that spiral around like a coiled spring. Beta sheets
form when amino acid chains line up in a flat plane within the protein,
as in the disease-causing protein shown here.
Transmissible
Spongiform
Encephalopathy
Normal Form
Disease Form
(self aggregating)