Download Amino Acids, Peptides and Proteins Dr AN Boa Section 1

Amino Acids, Peptides and Proteins
Dr AN Boa
Section 1 - Introduction
1.
Use alanine, CH3CH(NH2)CO2H, as an example amino acid to explain what is meant
by the following terms:
(i)
(ii)
(iii)
amphoteric;
zwitterion;
isoelectric point.
Comment on alanine’s solubility in water and in toluene.
2.
Draw the structure for the following amino acid / peptide derivatives described in
shorthand notation:
(i)
(ii)
(iii)
(iv)
3.
Ac-Tyr-OEt
Tyr(tBu)-OEt
Glu(OtBu)OEt
Pro.Lys(Ac).Gly
Draw the structure of diaminopimelic acid when in solution at i) pH 1, and ii) pH 12.
Sketch the acid-base titration curve for this amino acid and mark on this curve where
you would expect to find the isoelectric point.
HO2C
CO2H
NH2
4.
NH2
Draw the structure of histidine when in solution at (i) pH 1, and (ii) pH 12. Sketch a
fully labelled acid-base titration curve for this amino acid and mark on this curve
where you would expect to find the isoelectric point.
HO2C
NH
NH2
N
Amino Acids, Peptides and Proteins
Dr AN Boa
Section 2 – Separation and purification of amino acids and protein
1.
2.
3.
The following peptides 1-4 are subjected to electrophoretic analysis at pH 6.0.
1
Ala.Arg
Ala is CH3CH(NH2)CO2H
2
Phe.Ala.Arg
Arg is H2N(C=NH)NH(CH2)4CH(NH2)CO2H
3
Phe.Arg.Glu
Glu is HO2C(CH2)2CH(NH2)CO2H
4
Phe.Glu.Glu
Phe is PhCH2CH(NH2)CO2H
(i)
State the overall charge of each peptide at pH 6.0.
(ii)
Predict, with a brief explanation, which electrode they will move towards
and the relative rate of migration of each peptide.
The following peptides are subjected to electrophoretic analysis at pH 1.0, pH 6.0
and pH 12.0. At which pH will the separation be best and why?
1
Lys.Gly.Ala
Ala is CH3CH(NH2)CO2H
2
Gly.Asp.Lys
Asp is HO2CCH2CH(NH2)CO2H
3
Asp.Gly.Asp
Gly is CH2(NH2)CO2H
Lys is H2N(CH2)4CH(NH2)CO2H
Describe briefly the technique of X, explaining why it is particularly useful for the
separation of peptides and proteins.
(i)
X = SDS-PAGE
(ii)
X = anionic and cationic ion-exchange chromatography
(iii)
X = size exclusion (gel permeation) chromatography
(iv)
X = isoelectric focussing electrophoresis
Amino Acids, Peptides and Proteins
Dr AN Boa
Section 3 – Chemical and enzymatic reactions of amino acids and peptide
1.
What fragments are formed when the following peptide is treated with the
enzymes/reagents (i)-(v) indicated.
Asp.Arg.Val.Tyr.Met.His.Pro.Phe.Lys.Leu.Leu.Val.Tyr.Ser
2.
(i)
trypsin;
(ii)
chymotrypsin;
(iii)
cyanogen bromide;
(iv)
Edman’s reagent;
(v)
carboxypeptidase.
What products are obtained if the tripeptide Gly.Lys.Ala is subjected to end group
analysis using Sanger’s reagent?
Alanine, Ala, is CH3CH(NH2)CO2H,
Glycine, Gly, is CH2(NH2)CO2H, and
Lysine, Lys, is H2NCH2CH2CH2CH2CH(NH2)CO2H.
What is the major advantage of Edman’s approach to end group analysis over that of
Sanger?
3.
The peptide Asp.Gly.Ala.Asp is cleaved cleanly into two fragments when treated
with 6M hydrochloric acid at room temperature over several hours. Deduce the
structure of the two fragments and explain their formation.
Ala is CH3CH(NH2)CO2H;
Asp is HO2CCH2CH(NH2)CO2H and
Gly is CH2(NH2)CO2H
Amino Acids, Peptides and Proteins
Dr AN Boa
Section 4 – Polypeptide sequencing
1.
The pentapeptide A has the amino acid composition:
(Gly1, Ile2, Tyr2)
Use this and the following information to establish the structure of A.
A
A
A
2.
(i) FDNB
DNP-Ile
(ii) total acid hydrolysis
carboxypeptidase
partial acid hydrolysis
Tyr (the first amino acid released)
Products include only three dipeptides
Explaining fully your workings, determine the sequence of the polypeptide A from the
following experimental results:
(i)
Total acid hydrolysis of A gives:
A = (Ala, Arg, Gly, 2×Lys, Met, Phe, Pro, 2×Ser, Tyr, Val)
(ii)
Treatment of A with Edman’s reagent gives the thiohydantoin of Val.
(iii)
Treatment of A with carboxypeptidase first releases Ala.
(iv) Treatment of A with cyanogen bromide gives two peptides B and C with
composition:
B = (Ala, 2×Lys, Phe, Pro, Ser, Tyr)
C = (Arg, Gly, Met, Ser, Val)
(v)
Treatment of A with chymotrypsin gives three peptides D, E and F with
composition:
D = (2×Lys, Phe, Pro)
E = (Arg, Gly, Met, Ser, Tyr, Val)
F = (Ala, Ser)
(vi) Treatment of A with trypsin gives three peptides G, H and I with composition:
G = (Gly, Lys, Met, Tyr)
H = (Ala, Lys, Phe, Pro, Ser)
I = (Arg, Ser, Val)
Amino Acids, Peptides and Proteins
Dr AN Boa
Section 5 - Peptide synthesis
1.
Devise a synthesis of the peptide Lys.Ala.Asp (shown below) starting from its
constituent suitably protected amino acids (mechanisms are not required). State key
reagents needed for your synthesis.
O
H
N
H2N
H2N
N
H
4
CO2H
O
CO2H
2.
Draw the structure of peptide Ala.Pro.Asp.Gly and devise a synthesis for it starting
from suitably protected amino acids.
3.
Draw the structure of peptide Lys.Lys(ε→α)Asp and devise a synthesis for it starting
from suitably protected amino acids.
Note: (ε→α) here is to indicate that the side chain amine of the middle lysine is linked
to the α-acid of the aspartic acid.
4.
Why is racemisation an issue in peptide synthesis? What tactics/strategies can be
used to minimise this problem?
Section 6 – Protein structure
1.
Place the following inter-strand bond/interactions in order of increasing strength:
(i)
Disulfide bond
(ii)
Hydrogen bond
(iii)
van der Waals
(iv) Salt bridge
Draw an example to illustrate each type of interaction.
2.
Describe the difference between a parallel and antiparallel β-sheet.
3.
Explaining what is meant by “3.613 helix” discuss the structure of the α-helix.
4.
Explain what is meant when an α-helix is described as amphipathic and describe how
such helices are important in determining the tertiary and quaternary structures in
proteins.