Question

You are trying to determine the structure of a protein that you know is pure. You carry out a series of experiments on this protein. The results are listed below.

Describe what you can determine about the protein’s structure. (For example, what can you determine about the protein’s molecular weight, primary, secondary, tertiary, quaternary structure?)

· Size exclusion chromatography of the native protein indicates an apparent molecular weight of 160,000.

· SDS polyacrylamide gel electrophoresis of the protein carried out WITHOUT the addition of a disulfide-bond reducing agent shows the protein as having an apparent molecular weight of 80,000. After treatment with performic acid, the same technique reveals two bands of molecular weights 35,000 and 45,000.

·The Sanger reagent (fluorodinitrobenzene) identifies Ala and Leu as amino terminal residues, in roughly equal amounts.

Answer 1

Size exclusion chromatography separates the native protein based on charge. The molecular weight was determined to be 160,00. Native protein is in its properly folded and assembled state and has all four levels of structure.

Proteins have four levels of organization: primary, secondary, tertiary, and quaternary structure. Primary structure is the amino acid sequence of the polypeptide chain. The amino acids in the polypeptide chain are joined together by peptide bonds. Peptide bond is formed between the alpha amino group of one amino acid and the carboxyl group of another amino acid with loss of water. Secondary structure determines the folding pattern of the polypeptide chain. This structure is stabilized by hydrogen bonds between N-H and C=O groups. There are two types of secondary structure- alpha helix and beta pleated sheet. Alpha helix is the arranged of polypeptide chain like a coiled spring. The carbonyl and NH groups are placed parallel to each other. The structure is rigid as all amino acids are involved in hydrogen bond formation. There are 3.6 amino acid residues per turn. In beta-pleated structure , the polypeptide chain has strands side by side due to self-folding. Bonds are formed between neighboring polypeptide chain. Tertiary structure is the 3-dimensional arrangement of the polypeptide and results due to interactions between side chains of the amino acids or between side chains and polypeptide backbone distant in sequence. Weak forces such as Van Der Vaal’s forces, ionic interactions, disulphide bonds, hydrophobic interaction and hydrogen bonds are involved in tertiary structure. Quaternary structure occurs in proteins that have many polypeptide subunits. These subunits are joined together by noncovalent bonds, salt bridges or disulphide bond interactions as tertiary structure.

SDS-Polyacrylamide Gel electrophoresis separates proteins based on molecular weight. SDS or sodium dodecyl sulfate gives the protein a net negative charge by binding to it, after it is denatured by heating. SDS only reduces non-covalent bonds and helps in reduction of tertiary and quaternary structure. It does not break disulphide bonds. However, since the first SDS PAGE is carried out without disulphide bonds reducing agents, disulphide bonds will not be affected. SDS cannot reduce disulphide bonds. Since this PAGE gave only one fragment of 80000, the quaternary structure was affected. The protein was a homodimer of two subunits, each of 80000. The quaternary structure lacked disulphide bonds.

The SDS PAGE with perfomic acid gave two fragments of 35000 and 45000. Performic acid is a disulphide reducing agent that breaks down disulphide bonds. Hence, the tertiary structure of the protein was affected. The protein was composed of two subunits of 80000, each with two polypeptide chains of 35000 and 45000 respectively.

The Sanger’s reagent (fluorodinitrobenzene) reacts with the N terminal amino acid of the protein, which are identified by chromatography. Hence, it is used to identify N-terminal amino acid in the protein. Hence, two polypeptides has Ala and Leu as their N Terminal amino acid.

Results can be summarized as follows:

The native protein with a molecular weight of 160,000. The protein is composed of two subunits of 80000 each. Both the subunits are individually made up two polypeptide chains of 35000 and 45000 linked by one or more disulphide bonds. The two polypeptidesof 35000 and 45000 have Ala and Leu as their N-terminal amino acid.