Question

A protein you are studying is believed to have a beta barrel secondary structure that consists of several duplicate hydrophobic amino acid sequence repeats. What potential analytical issues would you encounter in the purification and analytical assessment of this type of protein and would you anticipate using detergents to help solubilize this protein (explain)?

Answer 1

Proteins are biomolecules which plays an important role in biological system. They perform a variety of functions in organisms such as they serves as catalyst (enzymes) in metabolic reactions, transporter for molecule from one to another place, in replication process etc. To perform these functions proteins attain a biologically stable structure. Amino acids are building blocks of proteins which are linked together by peptide bond in a chain form. Amino acids are small molecules that is made up of a basic amino group (-NH2), a acidic group (-COOH) and side chain R group. There are 20 amino acids and they differed from each other at R group. Each protein amino acids sequence vary from each other which is encoded by genetic material which is responsible for their structure and their activity. In organisms, most of the proteins present in their unique biologically active 3-dimensional structure which is achieved by protein folding ( due to various kind of bond formation and interaction of amino acids of a protein). Depending upon the arrangement of polypeptide there are 4 type of structure of protein:

1. Primary structure: It refers to simple amino acid sequence where amino acids are linked to each other by peptide bonds.
2. Secondary Structure: It refers to structure which is formed by hydrogen bonding among neighboring amino acids of a protein sequence. Common example are α- Helix,  β- sheets and turns. α- Helix is formed by H-bonding of an amino acids with fourth amino acid down in the chain.  β- sheets as its name suggests are formed when two or more portion of chain arrange next to each other forming like sheet. The sheet structure is held together by H-bond formation among the participating amino acids.
3. Tertiary structure: It refers biologically active 3-dimensional structure of proteins which is formed by different kind of interaction and bonding such as disulfide bond, hydrophobic interactions, among the spatially separated secondary structure of protein.
4. Quaternary structure: It refers to structure which is formed by interaction between two or more polypeptide chain.

Beta barrel are part of many transmembrane proteins where they act as transpoter for molecules. Beta barrels are  β- sheets consisting of tandem repeats which form barrel like structure where first strand is hydrogen bonded with last one. Mostly strand have alternate hydrophilic and hydrophobic residue, so hydrophobic residue are oriented to inner side of protein structure forming a hydrophobic core and hydrophilic amino acids are positioned outward exposing themselves to local hydrophilic environment. In contrast to this membranes have just opposite orientation of amino acid residue where hydrophobic residues are exposed outward to the lipid bilayer which is hydrophobic in nature.

While purification of membrane proteins with several hydrophobic repeats, we will face difficulty to purify them because they are deeply imbedded in hydrophobic environment of lipid bilayer. Lipids are insoluble in water which makes it difficult to purify them. To purify membrane bound proteins, harsh detergent has to be used to solubilize the proteins. Detergents are amphiphilic compound with polar and non-polar domain. Sodium dodecyl sulfate (SDS), sodium cholate, N-lauryl sarcosine are some example of detergents used in membrane protein purification.These detergents solubilize the protein by disrupting the lipid bilayer. For example, SDS monomer solubilize membrane protein by disrupting cell membrane and form micelles.