Interaktom
 soubor všech interakcí v dané buňce nebo organismu
 meziproteinových – protein.protein
 protein–DNA interaktom
 ……
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Interactionsbetween molecules are centralto how cells
detect and respond to signalsand affect:
Gene expression (transcription & translation)
DNA replication,repair and recombination
Signalling
And many other processes....
Interactionsare (mainly) mediatedby many weak chemical
bonds(van der Waals forces, hydrogenbonds, hydrophobic
interactions)
Accumulationof many bonds influences affinity and specificity
of interactions
Protein-protien
 Dvouhybridní systém
 Ko-imunoprecipitace
 Knihovna „Phage display“
 „Pull-down assay“
 „Far-western blotting“
 Proteinová „microarray“
TEST
MB-2019-analýza proteinůainterakcí
Protein-Nucleic Acid Interactions
• A wide range of Biophysical Chemistry methods have been
used to study interactions between proteins and nucleic
acids
• Particularly good for determining the strength (affinity) of
the interactions
High affinity, μM – nM: tend to involve sequence-specific
interactions, e.g. restriction enzymes
Low affinity, mM – μM: proteins tend to recognise aspects
of “overall” structure i.e. not sequence-dependent
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Biophysical Chemistry Approaches for Studies of Molecular Interactions
• Wide range of Biophysical Chemistry approaches are useful for
studying molecular interactions:
NMR
X-ray crystallography
SPR (Surface plasmon resonance)
ITC (Isothermal titration calorimetry)
CD spectroscopy (Circular dichroism)
Gel electrophoresis
EPR (Electron paramagnetic resonance spectroscopy)
Mass spectrometry
Fluorescence
Footprinting
MB-2019-analýza proteinů a interakcí
Many of these techniques
are particularly useful for
determining the strength
(affinity) of interactions
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EMSA (“Gel Shift” Assay)
• Electrophoretic Mobility Shift Assay (EMSA) or “gel shift”
can provide information about protein-NA interactions
MB-2019-analýza proteinů a interakcí
[Protein]
DNA +
protein
+Ab
DNA +
protein
+ Ab
M
DNA
DNA alone
A fairly straightforward technique,
but only provides convincing data
for high affinity interactions
(typically <μM)
A fairly straightforward technique,
but only provides convincing data
for high affinity interactions
(typically <μM)
TEST
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“Footprinting” is a Technique to
Identify a DNA-binding site
Premise: DNA bound by protein will be protected from
chemical cleavage at its binding site
1) Isolate a DNA fragment thought to contain a binding
site and “label” it
2) Bind protein to DNA in one tube; keep another as a
“naked DNA” control
3) Treat both samples with chemical or enzymatic agent
to cleave the DNA
4) Separate the fragments by gel electrophoresis and
visualize bands on X-ray film or imager plate
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Protein-DNA
Footprinting
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Footprinting Results
of RNA Polymerase
Bound to Promoter
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Binding of Proteins to DNA Often
Involves Hydrogen Bonding
 Gln/Asn can form specific
H-bond with Adenine’s N-6
and H-7 H’s
 Arg can form specific Hbonds
with CytosineGuanine
base pair
MB-2019-analýza proteinů a interakcí
• Major groove is right size for -helix and has exposed
H-bonding groups
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DNA-binding domains
•Proteins generally recognise aspects of nucleic acid
sequence, or variations in structure and/or flexibility
•High-resolution structures of many protein-DNA
complexes have now been solved
•Similar structural domains occur in different
proteins:
Helix-turn-helix
Zinc-finger
Zinc-binding domain
Basic region-leucine zipper (bZIP)
β-sheet recognitionMB-2019-analýza proteinů a interakcí
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The Helix-turn-helix Motif is Common in
DNA-binding Proteins
 Each “helix-turn-helix” covers
~ 20 aa
One -helix for DNA
recognition, then -turn, then
another -helix
Sequence-specificbinding due
to contactsbetween the
recognition helix and the major
groove
MB-2019-analýza proteinů a interakcí
• Four DNA-binding helix-turnhelix
motifs in the Lac
repressor
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Helix-turn-helix
 Helix-turn-helix is most common observed DNAbinding
unit in prokaryotes
Berg, Tymoczko & Stryer, “Biochemistry”,
5th edn, 2002, p. 874
 Note that 34 Å corresponds to 1 turn of DNA
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Zinc-finger
 One of best-studied examples of DNA binding domain,
but also binds RNA
 Each covers ~30 aa
 Binding is relatively weak, so typically there are a series
of zinc fingers
MB-2019-analýza proteinů a interakcí
 “Finger” portion is a peptide loop cross-linked by Zn2+,
which is usually coordinated by 4 Cys, or 2 Cys + 2 His
 One type of consensus sequences is:
“Cys2/His2”: Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His
 Zinc is held in tetrahedral structure by conserved Cys
and His
Zn++ Zn++
= Cys
= His
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MB-2019-analýza proteinů a interakcí
 Regulatory protein
Zif268, complexed
with DNA
Zinc Finger Motif is Common in
Eukaryotic Transcription Factors
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β-recognition motif
 In some prokaryotic regulatory proteins, this is an
alternative DNA-binding motif
 E. coli methionine
repressor binds DNA
through insertion of
pair of β-strands into
major groove
Berg, Tymoczko & Stryer, “Biochemistry”, 5th edn, 2002, p.
874
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Studium interakcíprotein-protein
 Dvouhybridní systém
 Ko-imunoprecipitace
 Knihovna „Phage display“
 „Pull-down assay“
 „Far-western blotting“
 Proteinová „microarray“
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TEST
MB-2019-analýza proteinůainterakcí