Biol2012 Exploring Proteins PDF

Biol2012 Exploring Proteins Jörn Werner  Last time 2: Investigating matter 10-9m 10-6m 10-3m 1+1m 0.1nm 1nm 10nm 100nm 1mm 10mm 100mm 1mm 1cm 10cm 1.0m Atom Molecule Protein Bacteriu...

Biol2012 Exploring Proteins Jörn Werner  Last time 2: Investigating matter 10-9m 10-6m 10-3m 1+1m 0.1nm 1nm 10nm 100nm 1mm 10mm 100mm 1mm 1cm 10cm 1.0m Atom Molecule Protein BacteriumEukaryotic Cell C.elegans Organ Human Light microscopy electron microscopy x/n ray scattering x-ray diffraction NMR  Nuclear Magnetic Resonance Nucleus gives rise to spectrum  Proteins have 100-1000s of nuclei: Most abundant are protons (1H) Also used Carbon (13C) and nitrogen (15N) Every peak corresponds to one specific proton in the protein  Chemical shift dispersion indicates presence of absence of 3D structure Example: prion protein Native Prion unfolded protein PrPC Solubilised fibrillar form of the prion folded protein ppm Use correlations between two protons (i.e. spins) E.g. if two protons are close in space ( 200Kda – EM diffraction (also in combination with cryo EM) Requires (only) 2D crystals (e.g. good for membrane proteins) Resolution ~ 3-4Å possible  Strengths and limitations of different techniques: scattering X-ray and neutron scattering possible – Strengths: applicable to large range of macromolecules – Proteins, DNA/RNA, small to large complexes, and assemblies measurement in solution ~mg of pure protein required – Limitations: Resolution ~20Å, Only shape information, best suited in combination with other techniques Neutron scattering requires more material and best suited with some level of deuteration (i.e. recombinant protein production) Strengths and limitations of different techniques: x-ray – crystallography Strengths: highest resolution (less then 1Å) enormous information content: do chemistry by structure applicable to large range of macromolecules – Proteins, DNA/RNA, small to large complexes, and assemblies – Limitations: Molecule needs to form crystals – may prove impossible – Crystal environment may influence the result of dynamic parts of a protein or weakly interacting systems Static structure Several mg of pure protein required Strengths and limitations of different techniques: NMR – Strengths: Versatile tool to investigate any macromolecule – Each atom a potential “spy” to investigate a protein Experiments in solution or in solids (membranes, aggregates) Structure determination folding, Protein ligand interactions with local resolution dynamics (site specific with out perturbation of protein) highest resolution (less then 1Å) enormous information content: do chemistry by structure applicable to large range of macromolecules – Limitations: Several mg of pure protein required (isotope labelling , recombinant protein production Solutions structures limited to small (