Structural Techniques

Chromatography

Gel Filtration

• Also called size exclusion. Separates proteins by molecular weight
• Resin is made of porous beads
• Elute largest to smallest

Affinity Chromatography

• The resin contains a covalently bound ligand that binds selectively to the protein tag
• This only works if there is a tag on the protein
• Contaminating protein and cellular debris is washed off
• Protein is eluted with a high concentration of ligand which out competes resin, releasing the protein

High Pressure Liquid Chromatography (HPLC)

• Reverse phase: hydrophobic resin, gradient of mobile phase from polar -> non-polar
• Measure time through gradient and determine when protein elutes (retention time)
• Hydrophobic molecules bind stronger (and longer) to the resin

Ion Exchange

• Separates proteins by charge
• Anion exchange: binds negatively charges proteins to a positive resin
• Cation exchange: binds positively charges proteins to a negative resin

Mass Spectrometry (MS)

• Mass spec analyzers contain:
• 1) An ion source to ionize proteins (MALDI or electrospray);
• 2) Mass analyzer which separates proteins based on their m/z ratio (TOF or ion traps);
• 3) Strike detector which tells the relative abundance of each ion;
• 4) Computerized data system to acquire, store and process data.
• Mass spec is highly sensitive (can detect down to 1x10-15 mol of certain proteins) and can easily distinguish between two very similar proteins.
• Mass spec gives researchers a RELATIVE amount of protein, not an absolute amount.

X-ray Crystallography

• Some proteins can form crystal structures of repeating units
• These are then placed in a X-ray diffractometer where they are exposed to X-rays
• The crystals scatter the X-rays into diffraction patterns
• These patterns can be interpreted into 3D structures
• The x,y,z coordinates of each atom can be identified to create a high resolution structure

Electron Microscopy

• Accelerated electrons are used as a source of illumination
• Significantly higher resolving power than light microscopes
• Magnification up to 10,000,000x compared to 2,000x
• Cryo-EM allows samples to be images in cryogenic temperatures

Nuclear Magnetic Resonance (NMR)

• Protons in a nuclei exhibit either up or down spin states
• Under an external magnetic field, these nuclei will either align or not
• The difference between these alignments are detected through the absorption of energy
• Therefore you need to measure nuclei with a odd number of protons: H-1, C-13, N-15
• NMR spectra allow us to determine protein structure

Infrared Spectroscopy (IR)

• Protein's contain many C-H, N-H, O-H, and C=O bonds
• These possess bending, stretching and vibrating properties
• These motions absorb IR light wavelengths
• IR is able to detect H-bond interactions (secondary structure)

Circular Dichroism

• Measures the absorption of UV light
• This technique utilizes the asymmetry of proteins
• Secondary structures absorb at specific wavelengths:
• α-helix: 222, 208nm (negative), 195nm (positive)
• β-sheet: 217nm (negative)
• Disordered: 198nm (negative)