3.4 Protein Analysis

Question 1

What is the most common method for identifying protein structure?

Answer 1

X-ray crystallography is the most reliable and common method; 75 percent of the protein structures known today were analyzed through this method.

Question 2

X-ray crystallography

Answer 2

• measures electron density on an extremely high-resolution
  scale and can also be used for nucleic acids.
• An X-ray diffraction pattern is
  generated in this method
• The small dots in the diffraction pattern can then be interpreted to determine the protein’s structure.

Question 3

How can the AA’s that compose a protein be determined?

Answer 3

• by complete protein hydrolysis and subsequent chromatographic analysis
• However, the
  random nature of hydrolysis prevents AA sequencing
• Instead sequential digestion of the protein with specific cleavage enzymes is used

Question 4

Edman degradation

Answer 4

• uses cleavage to sequence proteins of up to 50 to 70 amino acids.
• The Edman degradation selectively and sequentially
  removes the N-terminal amino acid of the protein, which can be analyzed via mass spectroscopy.
• For larger proteins, digestion with chymotrypsin, trypsin, and cyanogen bromide, a synthetic reagent, may be used

Question 5

Limits of Edman degradation

Answer 5

Because disulfide links and salt bridges are broken to reduce the protein to its primary structure, their positions cannot be determined by these methods.

Question 6

How is protein activity monitered?

Answer 6

• Protein activity is generally determined by monitoring a known reaction with a given concentration of substrate and comparing it to a standard.

Question 7

How is concentration determined?

Answer 7

• almost exclusively through spectroscopy

Question 8

Why can proteins be analyzed with UV spectroscopy?

Answer 8

• Because proteins contain aromatic side chains
• However, this type of analysis is particularly sensitive to sample contaminants
• Bradford method is most common for colorimetric changes because of its reliability and simplicity in
  basic analyses

Question 9

Bradford Protein Assay

Answer 9

• mixes a protein in solution with Coomassie Brilliant Blue dye
• The dye is protonated and green-brown in color prior to
  mixing with proteins
• dye gives up protons upon binding to amino acid groups, turning blue in the process
• Ionic attractions between the dye and the protein then stabilize this blue form of
  the dye; thus, increased protein concentrations correspond to a larger concentration of blue dye in solution.
• Samples of known protein
  concentrations are reacted with the Bradford reagent and then absorbance is measured to create a standard curve. The unknown sample is then exposed to the same conditions, and the concentration is determined based on the standard curve.
• This is a very accurate method when only one type of
  protein is present in solution, but because of variable binding of the Coomassie dye with different amino acids, it is less accurate when more than one protein is present.