Treatment of a polypeptide with Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) yields two peptides: 1. I-Q-K-H-C-R-C-A-K-M-V-S 2. F-C-R-L-K-D-C-K-N-D Treatment...

Treatment of a polypeptide with Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) yields two peptides:

1. I-Q-K-H-C-R-C-A-K-M-V-S

2. F-C-R-L-K-D-C-K-N-D

Treatment of the intact polypeptide with Trypsin yields fragments with the following amino acid composition:
(Ser, Met, Val) (Lys, Gln, Ile) (Phe, Ala, Cys2, Arg, Lys) (Asp, Asn) (Leu, Lys)
(Lys, His, Asp, Cys2, Arg)

a) The intact (untouched) polypeptide is labeled with 1-Fluoro-2,4-dinitrobenzene (FDNB) labeling, followed by acid hydrolysis. What will be observed?

b) Name and briefly describe a method which may determine if the intact polypeptide was a monomer or a dimer

Solutions

Expert Solution

a) 1-Fluoro 2,4-dinitrobenzene (FDNB) is a protein sequencing molecule. Through this molecule, it can be confirmed that which amino acid residues are present in the polypeptide chain. Thus when it is used in this particular situation, due to the action of trypsin followed by acid hydrolysis the peptide bond will break after the Arg (Arginine) amino acid residue. This reaction will yield two polypeptides from the given sequence. It is specific for the trypsin to cut the sequence after Arg.

b) For determining the intact polypeptide was a monomer or a dimer the Fluorescence Correlation Spectroscopy (FCS) is used which is based on the principle of fluctuation of fluorescence intensity that it the photon counting histogram analysis. This fluorescence intensity is changing due to the Brownian movement of the molecules present in the solution. If the molecule is entangled with other chains or it is in the dimeric form then the intensity change will be low and can differ from the fluctuation created by the monomer. Set up for this experiment or biophysical technique is as follows:

gladiator answered 3 hours ago

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