Protein Analysis Methods

X-ray Crystallography

• Protein analysis method that determines the full 3D atomic structure of proteins.

• Already purified protein is made into a crystal, the x-rays are shined through the crystal, the x-rays scatter/diffract through the crystal in a pattern, scientists use the pattern to determine a protein’s entire 3D structure/character.

• X-rays used because visible light is too long in wavelength

• Can detect structural changes that occur after substrate binding

NMR Spectroscopy

• Protein analysis method that is used to figure out a protein’s 3D structure and how it moves in solution based on hydrogen atoms.

• Put purified protein in a strong magnetic field, detects spin interactions of hydrogen nuclei, absorb and emit radio waves, signals tell scientists where atoms are located relative to each other, then can be used to determine interactions within proteins such as two alpha helixes.

• Detects interactions between hydrogen atoms to determine spatial relationships

Mass Spectroscopy

• Protein analysis method that is used to identify proteins and determine their mass

• Purified protein is broken into smaller pieces, they are ionized and vaporized, sent through mass spectrometer where the time-of-flight data is measured and that determines how heavy the mass is and masses are used to figure out which protein is present.

• Can detect covalently bound organic cofactors in enzymes

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