Question

5. Draw the tripeptide leucine, cysteine, phenylalanine, and name it.

6. How can you identify the primary, secondary, tertiary, and quaternary structures of proteins? Explain.

7. which amino acids are particularly prone to forming -sheet structure? why?

Answer 1

#1.

Ans. #2. Tertiary and quaternary structures can be differentiated using native PAGE and SDS-PAGE or SDS-PAGE in presence of 2-mercapthanol.   

# 2- mercaptoethanol (b- mercaptoethanol, a commonly used reducing agent used in SDS-PAGE) breaks disulfide bridges (-S-S-) in protein resulting two cysteine residues with free thiol groups. Because of breaking disulfide bonds, the two or more subunits linked by disulfide bond in a multimeric protein are separated and migrate through the gel as independent subunits.

# Following effects can be observed on rate of movement of protein through the gel under these conditions-

Case I: Native PAGE: A multimeric protein gives a single band in native page.

Case II. SDS-PAGE in presence of 2-mercapthanol: Because of breaking of disulfide bonds in presence of 2-mercapthanl, the two or more subunits linked by disulfide bond in a multimeric protein are separated and migrate through the gel as independent subunits.

If two or more bands are observed, the protein is exists in a quaternary structure. And, the subunits are linked together by disulfide bonds.

If only one band is observed, the protein exists in tertiary structure.

Case III. SDS-PAGE: SDS disrupts non-covalent interactions (H-bonds, ionic interactions, hydrophobic interactions, etc.) in proteins. So, all the subunits held together non-covalent interactions would be separated in presence of SDS as denaturing agent.

If two or more bands are observed, the protein is exists in a quaternary structure. And, the subunits are linked together by non-covalent interactions.

If only one band is observed, the protein exists in tertiary structure.

# However, the primary, secondary and tertiary structures can no longer be always distinguished using electrophoresis. In such cases, the evaluation of structure would be required as follow-

Primary structure: Linear polymer of amino acids of a polypeptide chain is called its primary structure. It gives the amino acid sequence of the peptide chain.

            N-ter- YTRSPMLKHIDNIMHITA///EARTH-C-ter

Secondary structure: The localized folding of small segments (≈ 3- 30 residues) of a polypeptide into ordered geometrical units is called secondary structure. A single polypeptide may have several of these localized geometric units, thus are sometimes also called the ‘repeating unit/ structure’. Secondary structures are stabilized purely by non-covalent interactions like van der Waal’s forces, hydrophobic interactions and H-bonds. Example- α -helix and β- sheet.

Tertiary Structure: The overall 3D conformation of a polypeptide chain is called its tertiary structure. Tertiary structure may also be stabilized by intra-peptide chain (intramolecular) disulfide bridge (-S-S-) between cysteine residues brought together in close proximity during folding. The non-covalent interactions includes H-bonds, ionic interactions, hydrophobic interactions, etc. among the residues of the same polypeptide chain.

Quaternary Structure: The quaternary structure of protein is an association of two or more polypeptides into a distinct structural/functional unit. Two polypeptides in quaternary structure may be held together by inter-peptide chain (intermolecular) disulfide bonds and/or non-covalent interactions. The non-covalent interactions includes H-bonds, ionic interactions, hydrophobic interactions, etc. among the residues of the same polypeptide chain, and among residues of two or more polypeptides.  

#3. A beta sheet consists of two or more-beta strands held together by hydrogen bonds.

Amino acids with large aromatic side chains (ex- Phenylalanine, tryptophan, tyrosine) and those with large, branched side chain (ex- Isoleucine, Valine, threonine) favor the formation of beta strands. Presence of amino acids with bulky side chains facilitates the trans-configuration of peptide bonds which in turn favors the formation of beta-strands. Moreover, proline is abundant at the edges (bends) of beta-strand because it produces kinks in the peptide bonds. Formation of kinks is crucial for bend (zig-zag or pleated) pattern of the beta-strands.