Protein Structures: Data Representation

1)1) Primary StructurePrimary Structure: character string.

2)2) Secondary StructureSecondary Structure:

3)3) Tertiary StructureTertiary Structure:

4)4) Quaternary StructureQuaternary Structure:

Identifying sub-structures in a large protein based on sequence.

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

2) Alpha Helix

3) Beta Sheet

A Hydropathy plot identifies domains within a protein that are soluble (region of “charged” amino acids) or insoluble (region of “uncharged” amino acids).

An alpha helix is a group of amino acids within a proteins that arrange themselves in a helical structure.

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 PlotCommonly 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 acidc) repeat calculationd) continue this though the entire protein sequence.

ELRLRYCAPAGFALLKCNDADYDGFKTNCSNVSVVHCTNLMNTTVTTGLLLNGSYSENRT

Transmembrane domains are 20 amino acids, but any size window can be used.

1) Calculate average using amino acids-specific constants.

2) Move window 1 amino acid.

3) Calculate average.

Solubility Constants(Kyte & Doolittle)A Alanine 1.8 R Arginine -4.5 N Asparagine -3.5D Aspartic acid -3.5C Cysteine 2.5Z Glutamine -3.5E Glutamic acid -3.5G Glycine -0.4H Histidine -3.2I Isoleucine 4.5L Leucine 3.8K Lysine -3.9M Methionine 1.9F Phenylalanine 2.8P Proline -1.6S Serine -0.8T Threonine -0.7W Tryptophan -0.9Y Tyrosine -1.3V Valine 4.2

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

X = 30.05 / 20

X = 1.503

Secondary Structure Prediction: Hydropathy Plot

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 temperature-dependent 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 StructureQuaternary 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 beta-sheet 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 futurewhen 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 Disease40-90 (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)

Senile Plaque

Neuronal cell nuclei (blue circles)

Amyloid Precursor Protein

Beta Amyloid Protein

Self Aggregation

Alzheimer’s Disease

42 amino acids long

500 nm

Beta-Amyloid Aggregated in Water

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 repeats6-34 Normal Gene36-120 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 involvesCAG repeats >60.

Huntingtin Gene

Huntingtin Protein

Normal

Abnormal

> 40 CAG codons

10-30 CAG codons

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 wild-type (b) mice at 27 months of age. Only the striatum (Str) in the HD-repeat mutant mouse brain was immunoreactive with EM48. Ctx, cortex. High-magnification 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).

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

Prion Protein Diseases

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 amino-acid 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.

Normal FormDisease Form

(self aggregating)

TransmissibleSpongiform

Encephalopathy