E XPERIMENTAL METHODS TO STUDY PROTEIN STRUCTURE AND FOLDING

Determination of protein structure Complete 3D structure –X-Ray diffraction spectroscopy –Nuclear Magnetic Resonance spectroscopy Low-resolution structure –Cryomicroscopy Elements of structure –Electron Spin Resonance (ESR) spectroscopy –UV/VIS spectroscopy –Circular Dichroism spectroscopy –Fluorescence/Fluorescence Resonance Energy Transfer spectroscopy –IR/Raman spectroscopy –Mass spectroscopy (cross-linking experiments)

X-Ray crystallography Bragg equation

Intensity image (structure factors) Electron-density map with atom positions fitted The phase problem

Top row: azurin, flavodoxin, rubredoxin Bottom row: azidomethemerytrin, lamprey hemoglobin, bacteriochlorophyl The „hanging drop” method of protein crystallization. The dialysis method of protein crystallization. Protein crystallization

Nuclear magnetic resonance spectroscopy Resonance absorption at  E=h =const (600 MHz, 900 MHz), B 0 varies to achieve resonance.

Chemical shift/coupling constants  2,2-dimethyl-2-silapentane-5-sulfonate (DSS) as reference

The 1 H NMR spectrum of ubiquitin

2D NMR spectroscopy

Comparison of the NMR and X-ray structure of tendamistat (a  -sheet protein) NMR determination of protein structure

Cryoelectron microscopy

Small X-Ray scattering (SAXS) Raw SAXS data (RAR1-GST-Tag fusion protein) The resulting distance distribution Kozak and coworkers, Acta Physica Polonica, 2010, 117, 307

Schematic representation of the protein Actual shape determined by SAXS

Protein folding – types of experiments Thermal denaturation Chemical denaturation Mechanical unfolding Kinetic experiments Mutational studies

Protein folding - measurements Differential scanning calorimetry (DSC) Spectroscopy –Circular dichroism (CD) –Infrared/Raman –Fluorescence/Fluorescence energy transfer –Nuclear magnetic resonance (NMR) –Small angle X-ray (SAXS) and small angle neutron scattering (SANS) Atomic force microscopy (AFM)

Wild type Acid-denaturated wild type L16A mutant C-terminal peptide Religa et al., J. Mol. Biol., 333, (2003)

Millet et al.. Biochemistry 41, (2002) SANS studies of protein unfolding

Structure of closed and open form of the DnaK (Hsp70) chaperone

Fluorescence studies of closing and opening of Hsp70 Mapa et al., Molecular Cell 38, 89, 2010.

CD spectroscopy

Gruebele and coworkers, Proc. Natl. Acad. Sci., USA, 2003, 100, Investigation of the folding of the FBP28 WW domain and its mutants

Atomic force microscopy (AFM)

 -values Mutation affects the folded state but not the transition state Mutation affects both the folded state and the transition state Matouschek A, Kellis JT, Serrano L, Fersht AR. (1989). Mapping the transition state and pathway of protein folding by protein engineering. Nature 340:122

Blind men and a elephant (a story from ancient China)

Gruebele and coworkers, Proc. Natl. Acad. Sci., USA, 2003, 100,

Lewandowska et al., Biophysical Chemistry, 2010, 151, 1-9