Macromolecules structure and interactions Sergiev P.V. 1755 - - PowerPoint PPT Presentation

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MSU & SkolTech

Macromolecules structure and interactions

Sergiev P.V.

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Structure of macromolecules

X-ray structure analysis 1. 2. 3. 4. Purification of macromolecule Crystallization Diffraction Calculation The basement of the method is X-ray beam diffraction ~ Å wavelength 1

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Structure of macromolecules

X-ray structure analysis beam crystal detector reflections coordinates and intensities are measurable, but phases are required

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Structure of macromolecules

X-ray structure analysis Method of isomorphous replacement Max Perutz Nobel Prize 1962 heavy atom Inclusion of a heavy atom, e.g. Hg to the molecule Modified molecule should have the same structure and crystal lattice two cristals are needed ( )

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Structure of macromolecules

X-ray structure analysis

Method of multiwavelength anomalous dispersion MAD ( ) selenomethionine Se Utilization of several waivelengths to solve phase problem. No necessity for two crystals

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Structure of macromolecules

X-ray structure analysis Molecular replacement If similar structure is known

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Structure of macromolecules

X-ray structure analysis R-factor shows quality of structure calculation

R |F (h)-F (h)| ______________ F (h) = S

S

h h

• bs

calc

• bs

Good R-factor 0.1-0.15 resolution Å R 2.1 , = 0.08 Resolution - ability to distinguish objects located at certain minimal distance

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Structure of macromolecules

crystallization

solvent evaporation temperature drop change addition of precipitant inorganic salts organic solvents рН ( , ) CrystalFarm - 400 plates well screening of the crystallization conditions 96

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Structure of macromolecules

Diffraction Usually not in the wet lab, but in the specialized synchrotron facility

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Structure of macromolecules

X-ray structure analysis PDB - protein data bank nucleic acids +complexes >10 structures (+ ) 0 000 Eukaryotic ribosome 75 chains >3 MDa : ,

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Structure of macromolecules

Nuclear magnetic resonance (NMR) magnetic field spin For certain nuclei (spin N+ ) projections

• f spin are quanted

P = hm (m = I, I-1, ...., -I) for proton In external magnetic field two projections have different energy +1/2, -1/2

z I I

½ D n E=h Electromagnetic energy could be absorbed

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Structure of macromolecules

NMR

For available magnetic field intensities corresponding frequencies are hundreds of MHz (radiowaves) Difference in energy is very small in comparison with thermal movement lower level is populated only more 0.001-0.07% E

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Structure of macromolecules

NMR

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Structure of macromolecules

NMR

factors influencing NMR signal Atoms connected by chemical bonds (chemical shift) Number of atoms, connected through 2-4 bonds (multiplex) Proximal atoms in 3D 1. 2. 3.

CH3 CH3 CH2

CH3 O O CH2 CH3

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Structure of macromolecules

2D NMR (COSY)

CH3 CH3 CH2

CH3 O O CH2 CH3 CH2 CH3 CH2 CH3

COSY spectra detects proximity between atoms in a chemical structure

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Structure of macromolecules

2D NMR (NOESY) NOESY spectrum proximity of atoms in 3D

p53 1H-1H NOESY spectrum

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Structure of macromolecules

NMR HSQC Different types of atoms, requires stable isotope labeling

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Structure of macromolecules

X-ray structure analysis vs. Nuclear magnetic resonance X-ray NMR

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Interaction of macromolecules

Affinity chromatography: co-purification

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Interaction of macromolecules

Affinity chromatography: co-purification Requires tagging of one component leads to purification of its partners

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Interaction of macromolecules

Immobilized phase is glutathione, glutathione S-transferase as a tag glutathione is used for elution

Affinity chromatography: tags for purification GST

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Interaction of macromolecules

Affinity chromatography: tags TAP (tandem affinity purification) tag

TAP tag immobilized IgG protein CBP protein А TEV TEV protein protein

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Interaction of macromolecules

Affinity chromatography: tags TAP (tandem affinity purification) tag

immobilized calmodulin Ca

2+

EGTA protein protein protein

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Interaction of macromolecules

Affinity chromatography: other tags SPA tag CBP 3X FLAG TEV myc ? X myc HA HA protein protein protein

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Interaction of macromolecules

Affinity chromatography: co- immunopurification by specific antibodies

protein G (A)

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Interaction of macromolecules

Affinity purification of RNA and RNA-protein complexes

MS2 coat protein binding site MS2 coat protein ZZ TEV IgG

Streptomycin (tobramycin) aptamer streptomycin (tobramycin) Streptavidin binding aptamer Streptavidin

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Interaction of macromolecules

Yeast two-hybrid system reporter lacZ ( ) Gal4 DNA binding domain Gal4 activation domain protein 1 protein 2

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Identification of interacting parts

• f macromolecules

Deletion analysis

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Identification of interacting parts

• f macromolecules

Binding site mutation (scanning mutagenesis)

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Identification of interacting parts

• f macromolecules

Cross-linking

bifunctional reagent X cleavage X fragment separation and detection of a cross-link X

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Identification of interacting parts

• f macromolecules

RNA (DNA) – protein interaction sites Footprinting

Modification reagent

• r nuclease

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Identification of interacting parts

• f macromolecules

Binding site mutation (scanning mutagenesis)

RNases G T2 A C,U V1 DMS A,C,G(N7) kethoxal G CMCT U T1 all nucleotides dsRNA chemical reagents

A C G U K1 K2 K3

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Identification of interacting parts

• f macromolecules

Limited proteolysis

protease X

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Identification of interacting parts

• f macromolecules

toeprinting

DNA polymerase or Reverse transcriptase 1 2 3 4 1 2 3 4

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Study of macromolecule interactions

Surface plasmon resonance

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Study of macromolecule interactions

Isothermal titration calorimetry