Peptide, Proteins 2011
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Transcript of Peptide, Proteins 2011
Chapter 4Part II: Peptides, and
Proteins
MARIA CRISTINA R. RAMOS, Ph.D.
Professor of Chemistry
Department of Chemistry
Four Levels of Protein Structure
Levels of Protein Structures
• Primary (1o)- refers to the order of amino acids in a polypeptide chain
• Secondary (2o)- refers to the arrangement in space of the atoms in the backbone of the polypeptide chain
• Tertiary (3o)-refers to the arrangement of all the atoms, including those of the side chain, in the polypeptide chain
• Quarternary (4o)- refers to the arrangement of all the subunits of the protein
Primary Structure
Formation of Peptide Bond
Primary Structure Determination
Amino Acid Composition
• Total Hydrolysis followed by quantitative analysis of the liberated amino acids
1. Acid Hydrolysis
2. Base Hydrolysis
N and C-terminal Determination
N-terminal Determination
Edman Degradation
•Sequentially removes one residue at a time from the amino end.
•Phenyl isothiocyanate reacts with amino group to form a phenylthiocarbamoyl derivative.
•Mild acid conditions create cyclic derivative.
•Cyclic derivative is separated by chromatography to identify amino acid
C-terminal Determination
Cyanogen bromide: C-side of M
SAMPLE PROBLEMS
State the cleavage pattern of the following polypeptides by the indicated agents
• S-A-F-K-P by chymotrypsin
• T-C-G-M-N by CNBr
• L-R-G-D by hydrazine
• V-W-K-P-R-E by trypsin
An unknown decapeptide was isolated and characterized. Complete hydrolysis of this peptide gave : F(2), A,G,C,K,N,T, W and V. Treatment with carboxypeptidase releases A. Reaction with Edman’s reagent gave PTH-T and a nonapeptide. The nonapeptide was treated with trypsin and gave 2 peptides: (V-C-G-A) and (N-F-F-W-K). Give the sequence of amino acid in the decapeptide.
• You have isolated from a rare fungus an octapeptide that prevents baldness and you wish to determine its amino acid sequence. The amino acid composition is K2, D, Y, F, G, S, A. Reaction of the intact peptide with dansyl chloride yields dansyl-A. Cleavage with trypsin yields peptides whose compositions are: (K, A, S) and (G, F, K) plus a dipeptide. Reaction with chymotrypsin releases free D, a tetrapeptide with composition (k, S, F, A) and a tripeptide whose composition following acid hydrolysis is (G, K, Y). The enzymatic digests are each carried out on the whole, undansylated peptide.What is the sequence?
Determine the sequence of the following heptapeptide:• Amino acid analysis of the heptapeptide revealed that the
original peptide was composed of: R, V, Y, E, K, A and G. • Reaction of the heptapeptide with dansyl-Cl and acid
hydrolysis gave dansyl-A. • Digestion of the heptapeptide with: • carboxypeptidase gave G as the first detectable amino
acid. • trypsin gave free R, a dipeptide (A-K) and a tetrapeptide
containing E, G, Y and V • digestion of the tetrapeptide above (derived from the
trypsin digestion above) with chymotrypsin gave two dipeptides: V-Y and E-G.
• pepsin gave a tetrapeptide and a tripeptide (Y-E-G).
Determine the amino acid sequence of a heptapeptide (2 M, D, R, K, F, G) was isolated from the urine of a three-
toed sloth, given the following results:
• Reaction of the heptapeptide with FDNB gave DNP-M. • Limited proteolysis with carboxypeptidase indicated that M was
the first amino acid released • Cyanogen bromide (CNBr) reaction with the heptapeptide
released one equivalent of free homoserine lactone • Chymotrypsin digestion of the heptapeptide yielded a
pentapeptide and a dipeptide. Reaction of the pentapeptide with dansyl-Cl gave dansyl-M.
• Trypsin digestion yielded two M-containing tripeptides and free R.
• Digestion of the heptapeptide with pepsin gave a tetrapeptide (containing M, R, K and D) and a tripeptide (M, F, and G).
The peptide glucagon from the Nile tilapia was treated with chymotrypsin, and the resulting fragments were sequrnced.
A second sample of the polypeptide was treated with trypsin, and the fragments were sequenced. What is the
sequence of the polypeptide?
Chymotrypsin fragments
• LMNNKRSGAAE
• SNDY
• HSEGTF
• LEDRKAQDF
• VRW
• SKY
Trypsin Fragments
• AQDFVR
• WLMNNK
• HSEGTFSNDYSK
• RSGAAE
• YLEDRK
The sequence of crinia-angiotensin, an angiotensin II-like undecapeptide from the skin of the Australian frog, is
determined. A single round of Edman degradation releases DNP-Ala. A second round sample of the peptide is then
treated with chymotrypsin. Two fragments are released with the following amino acid composition: fragment 1: (H,P,F,V) and fragment 2 (A,D,R,G,P,I,Y). Next, a third sample of the
peptide was treated with trypsin, which results in two fragments with the following amino acid compositions:
fragment III (A,D,R,G,P) and fragment IV ( H,I,P,F,Y,V). Treatment of another sample with elastase yields three
fragments, two of which are sequenced: fragment V (H-P-F) and fragment VI (A-P-G). What is the sequencre of the
undecapeptide?