Presentation1 Biochem
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Transcript of Presentation1 Biochem
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1) Draw the general structure of an amino acid at pH 7.0.
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1) Draw the general structure of an amino acid at pH 7.0.
H+
R CH
CO O
NH3
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- Form depends on the pH:
pH 6-7 pH < 2pH > 9
they are amphoteric (react with acid and base)
H+R CH
COOH
NH3
R CH
CO O
NH3
OH
R CH
CO O
NH2
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2) Draw the structure of glutamic acid at pH 2, 7, 11
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2) Draw the structure of glutamic acid at pH 2, 7, 11
In an acid solution there are many protons H + so: COO- gains H+ and is
deionized (no charge)
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pH 7
COOH is ionized ie. Loses
H+ so now charged
pH 11
In a basic solution there are many
OH- (hydroxide ions), H3N is
deionized ie. loses H+ so now no
charge
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3) Give the name & one letter symbol for each of the following;
(a) an amino acid which has H as its R group.
(b) an aromatic amino acid
(c) a hydrophobic amino acid
(d) a polar amino acid
(e) a cyclical amino acid
(f) a positively charged amino acid
(g) an amino acid that can covalently bond to another identical amino acid.
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3) Give the name & one letter symbol for each of the following;
(a) an amino acid which has H as its R group.
Glycine (Gly) G
(b) an aromatic amino acid
Phenylalanine (Phe) F Tyrosine (Tyr) Y Tryptophan (Trp) W
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(c) a hydrophobic amino acid
Valine (Val) V Alanine (Ala) A Isoleucine (Ile) I Leucine (Leu) L
Are non-polar and dont interact with water (which is polar). Are aliphaticie. Have open chain hydrocarbons
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(d) a polar amino acid Glycine (Gly) G
Serine (Ser) S
Methionine (Met) MThreonine (Thr) T
Glutamine (Gln) Q
Cysteine (Cys) C
Asparagine (Asn) N
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(e) a cyclical amino acid
Proline (Pro) P
(f) a positively charged amino acid
These will be the basic amino acids
Histidine (His) H Lysine (Lys) L Arginine (Arg) R
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(g) an amino acid that can covalently bond to another identical amino acid.
Cysteine Cysteine (Cys) C with a disulphide bond
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4. What useful property of aromatic amino acids is utilised when studying
proteins in solution ?
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4. What useful property of aromatic amino acids is utilised when studying
proteins in solution ?
Aromatic amino acids can absorb UV light at 280nm. This is also how we
can detect and quantitate aromatic amino acids.
Absorption Spectrum for Cytochrome "C"
0
0.5
1
1.5
2
2.5
3
360 385 410 435 460 485 510 535 560 585 610 635 660
Wavelength (nm)
Absorbance
Cytochrome :"C" After sodium ascorbate added Baseline
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Sample requirements:sample to be analysed must contain onlyone absorbing component for which the calibration has beenperformed. If sample is a solution, a pure sample ofthe solvent should be used as a blank.
concentration
A
bsorbance
Linear relationship
between absorbance, A,
and concentration
A = -log(T)
A = ecl
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5. What two pieces of experimental evidence prove that polypeptide
conformation is dependent on primary structure?
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5. What two pieces of experimental evidence prove that polypeptide
conformation is dependent on primary structure?
1. Proteins will reform back into native confirmation if secondary,
tertiary and quartenary structure unfolded (denatured) but not if
primary sequence disrupted.
Denaturation- the unraveling of a proteins structure. Break down all
secondary, tertiary and quaternary structure. The disorganized protein willno longer act as intended.
When this occurs, protein strands will clump together - coagulate.
Examples - frying an egg
- Low pH in stomach
Temperature or pH outside the normal range can both cause denaturation.
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Denaturing a protein
heat
or
acid
heat
or
acid
denatured coagulated
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Renaturation
Providing the
1ostructure
remains in
place, then
protein will
often refold
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5. What two pieces of experimental evidence prove that polypeptide
conformation is dependent on primary structure?
2. Site specific mutagenesis: mutation in just one residue can change
structure and function of protein. Widely used to determine role of
individual residues in proteins. Conservative change eg. Hydrophobic
aa replaced with other hydrophobic aa usually results in similar
structure.
Eg. Sickle cell disease results due to Valine for Glutamine at residue 6 in
the Hbb chain.
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6. Discuss the 3-D conformation of a protein. In particular how is this related to activity.
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Sequence of amino acid residues in
polypeptide, e.g.
-Asp-Leu-Met-Thr-Ser-Tyr-
or
DLMTSY
-Hydrolysed to amino acids
-Polypeptide sequencing
e.g. Edman degradation
N-terminus
C-terminus
Primary Structure
6. Discuss the 3-D conformation of a protein. In particular how is this related to activity.
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Secondary Structure
Long chains of amino acids will commonlyfold into a regular repeating structure,
such as - helix or - pleated sheet
Structureis a result of:
- peptide bond angles
- R group interactions
- hydrogen bonding between amino acidswithin the protein.
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RGroup Interactions
R-Groups interact in a variety of ways:
- Hydrogen bonding- Ionic linkages
- Metal ion coordination
- Hydrophobic interactions- Covalent bonds
- Steric Hindrance
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-Helix
H
|
N
C
||
O
H
|
N
C
||
O
H
|
N
C||
O
C
||OH
|
N
C
||
O
H|
N
C
||
O
H
|N
C||
O
C
||
O
C
||
O
H
|
N
H
|
N
H
|
NEvery amide hydrogen and
carbonyl oxygen is involved
in a hydrogen bond.
R-Groups are on the outside
of the helix
Properties of an -helixinclude strength and low
solubility in water.
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-Pleated sheets
Another secondary structure for protein.
Hydrogen bonding between adjacent sheets of protein.
C|
R
R|
C
R|
C
R|
C
R|
C
C|
RC|R
C|
R
C|
R
C|
R
N
|H
N|
H
N|
H
O||C
O||C
O||C
O||C
C
||O
C
||O
C||
O
C||
O
H|
N
H|
N
H|
N
H|
N
N|
H
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Turns and Loops
Turn- 4 residues.
- Reverse direction of the main polypeptide
chain (several subtypes).
- Connect regions of more regular secondary
structure (-helix, -sheet) Loop- 6-16 residues. (Random Coil)
- Continuous segment of a polypeptide chain.
Both have no extended secondary structure. They
serve to tie together other units.
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Protein Tertiary Structure
Interactions between amino acid residuesand environment results in a proteintaking on a stable, compact arrangement.
Central dogma of protein folding
The primary structure determines thetertiary structure
Proteins with a unique primarystructure tend to fold spontaneously into adistinct tertiary structure.
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Protein Tertiary Structure
Folding occurs asa stepwiseprocess.
Only the final formis biologicallyactive.
This final form isthe proteinsnativeconformation
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Quaternary structure
of proteins
Many proteins are not single peptide strands.
They are: - combinations of several proteins
- aggregate of smaller globular
proteins.
Conjugated protein - incorporate another type of group that performs a
specific function.
prosthetic group eg; Haem
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Multi Subunit Proteins
HomomultimersIdentical Subunits
eg Homodimer
Heteromultimers- Different Subunitseg Heterodimer
Subunit Association is usually non-covalent
Association and Stabilisation due to samefactors that hold tertiary structure
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Hydrophobic attractions
- Attractions between R groups of non-polar amino acids.
Hydrogen bonding
- Interaction between polar amino acid
- R groups.
Ionic bonding- Bonding between + and - charged
amino acid R groups.
Disulfide LinkagesCys S=S Cys
Subunit Stabilisation
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Summary of protein structure
primary secondary
tertiary quaternary
H O
| ||
H2N - C - C
|R
H
|
N - C - COOH
| |H R
H O
| ||
- NH - C - C -
|R
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7) Compare and contrast the structure and function of myoglobin and haemoglobin
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7) Compare and contrast the structure and function of myoglobin and haemoglobin
Both oxygen binding proteins, Myoglobin for O2storage, Haemoglobin for O2
transport. Myoglobin is a single polypeptide chain, haemoglobin is a tetramer of 2
different subunits ( & ). Both similar tertiary structure but only haemoglobinhas quarternary structure.
O2 binding to myoglobin is hyperbolic
O2 binding to hemoglobin is co-operative allosteric
When plenty of O2 (lungs),stronger binding capacity. Binding of an O2 results in
conformational change and promotes binding of next. When low O2
as in tissues, weak
binding capacity.
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8) What special features characterize transmembrane proteins?
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8) What special features characterize transmembrane proteins?
Transmembrane proteins, such as cell surface receptors or ion channels, have exposed
hydrophobic residues that allow them to embed in the hydrophobic core of lipid bilayer
membranes, such as the plasma membrane. They are often characterized by membrane
spanning alpha-helices, i.e. TM7 receptors.
Membrane receptors
transduce external
signal from large
signalling moleculesto cell interior,
without signalling
molecules entering
cells
e.g. 7
transmembrane helix
(7TM) receptors, such
as -adrenergic
receptor