Transcript Lecture_12_2005

• Protein domains vs. structure domains - an
example.
Genome of the week
• Nanoarchaeum equitans - archaea
– Hyperthermophile
– Diverged early in evolution from other archaea
– New kingdom of archaea?
• Obligate symbiont with Ignicoccus
• Smallest completely sequenced genome
– <500kB
– Genome reduction observed in symbionts (Schmidt)
– Is N. equitans a “primitive” archaea or is the genome
undergoing reductive evolution?
• N. equitans lacks genes necessary for many
aspects of central metabolism.
– Can’t make lipids, vitamins, amino acids, etc.
– Parasite, not symbiont? First archaea
• Genome is quite compact.
– 95% of genome codes for genes. 552.
• Not primitive.
– Has complete set of information pathway and cell cycle
genes found in archaea.
• No longer undergoing reductive evolution.
– Normally would find pseudogenes - not found.
Protein complexes - why?
• Proteins often function as large, multisubunit complexes.
– ribosomes
• Can get clues about the function of a protein
by knowing what other proteins it contacts.
Protein:protein interactions
• Genetic approach
– Yeast 2-hybrid
• Biochemical approach
– Co-immunoprecipitation
– Fusion protein affinity chromatography
• Cell-biology
– FRET - fluorescence resonance energy transfer
• Computational
– Rosetta Stone
– Co-regulation
– Phylogenetic analysis
Yeast 2-hybrid approach
• Based on the fact some transcriptional activators
have separable DNA binding (BD) and
transcriptional activation domains (AD).
– GAL4, LexA
• Protein you are studying = Bait
– Fused to the DNA binding domain of GAL4
• Protein(s) you are screening = Fish or Prey
– Fused to the activation domain of GAL4
• Transform Bait and Fish plasmids into yeast,
measure the expression of a reporter gene.
– Usually a gene can be selected for when expressed.
Image from: http://www.bioteach.ubc.ca/MolecularBiology/AYeastTwoHybridAssay/
Yeast 2-hybrid on a genome wide
scale
• Clone every gene in your genome into both
the “bait” and “fish” vectors.
• Systematically screen each gene for
interactions.
– Mate individual yeast strains.
• Many false positives.
Interactome
Term to define all of the protein interactions
that take place in the cell.
Book example - predicting human
interactions.
Based on data that only 10% of the
measured interactions are physiological
Yeast 2-hybrid
• False-positives
– Some baits are “sticky” leading to non-functional
interactions
• False negatives
– Binding not tight enough to detect interaction
– Fusion proteins often do not fold correctly
• Works best when comparing two proteins
suspected of interacting
• Bacterial 2-hybrid systems
Co-immunoprecipitation
• Using an antibody to isolate and purify a
protein from a whole cell lysate.
• Normally you will only purify the protein
the antibody recognizes.
• Any additional proteins that co-purify are
candidates for interacting proteins.
Hirano et al, 1997 Cell, Vol 89, 511-521, 16 May 1997
Fusion protein affinity
chromatography
• Express the protein of interest as a fusion
protein.
– 6-8X His residues
– Glutathione S-transferase (GST)
– Other “tags”
• Bind and purify the protein of interest
– Poly His residues will bind Ni2+
– GST will bind glutathione
Image from: Sigma-Aldrich
Fusion proteins - identifying
interactions.
• In vivo - express fusion protein in vivo
– Purify complexes from the cell
• In vitro - overexpress protein in vitro
– Bind fusion protein to a column and run whole
cell lysate through the column. Identify
proteins that “stick” to the fusion protein.
Difficulties when using
biochemical approaches
• Stability of protein:protein interactions.
– Many are not stable enough to survive purification.
• Is the fusion protein functional?
– Many times fusions will not be functional.
• Quality of the antibody.
– Is it good enough to precipitate enough protein for
analysis?
Computational methods
• Rosetta stone analysis
– Search for proteins that are separate in one
organism but are fused into one protein in
another organism.
Computational methods
• Co-expression
– Genes that are in operons are often functionally
linked. (not always true).
– Determine if the structure of an operon is
conserved, indicating co-expression.
– Candidates for interaction.
– Not a great method.
Phylogenetic analysis
• Search for the presence of a protein in all
organisms.
• Determine the distribution.
• Identify other proteins that also show this
distribution.
• Functionally interact? Physically?
PLEX
• Protein Link EXplorer.
• Uses phylogenetic profiles to predict
possible associations.