Last Lecture: Protein 1 Structure

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Last Lecture Protein 1 Structure Today
Protein 2 Structure
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Problems to Consider 1. primary structure
determination (see handout) 2. Draw the
tetrapeptide Glu-Lys-Arg-Ser A titration of
this peptide reveals 5 pKas 2.10,4.07,9.21,
10.54,12.48 Calculate the pI for this
peptide. 3. Consider the following mixture of
proteins A MW 5000 Da, pI 9 B MW
10,000 Da pI 9.5 C MW 5500 Da pI
4 Describe a purification scheme to separate
these peptides using a combination of anion
exchange and size exclusion (gel filtration)
chromatography. What pH should the buffers used
be? 4. A GST fusion protein, GST-A is cleaved
to yield free GST and protein A. GST and protein
A have the same MW and pI. How could you
separate them?
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Peptide Sequencing
http//www.ncbi.nlm.nih.gov/BLAST/
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Peptide Sequencing -Chemistry
Edman Degradation
Phenylisothiocyanate (PTC)
PTC
PTC derivative
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Protein Structure
Primary (1) amino acid sequence, disulfide
bridges
Secondary (2) regular structure
- alpha helix (a)
-beta sheet (b)
Tertiary (3) fold of structure
Quaternary (4) interaction of sub-units
-higher order structures
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Primary (1) Structure
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Primary (1) Structure
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Elements of Secondary Structure -helices -sheets
-turns -loops
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a-helix
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a-helix - properties
-4-40 residues -right-handed helix -3.6
residues/turn -i, i 4 interaction
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Helical Packing
heptad repeat
amphipathic helices
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GCN4
MKELEKLVERLLNENYHLESKVAELKDK
VGQLKSKVAELEDKNYHLLNEVERLEKL
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GCN4
MKELEKLVERLLNENYHLESKVAELKDK
VGQLKDKVAELESKNYHLLNEVERLEKL
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K E L E
L E K K
G A Y E
E R H E
V V N V
L L L L
K N S D
DSNK
L L L L
MVN V
K E Y A
Q E H R
E L E K
K K E L
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K E L E
L E K K
G A Y E
E R H E
V V N V
L L L L
K N S D
DSNK
L L L L
MVN V
K E Y A
Q E H R
E L E K
K K E L
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K E L E
L E K K
G A Y E
E R H E
V V N V
L L L L
K N S D
DSNK
L L L L
MVN V
K E Y A
Q E H R
E L E K
K K E L
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Leucine Zipper Coiled Coil
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Recall...
d-
d
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Helix Dipole
-

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b - Sheet 5-10 amino acids
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(No Transcript)
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Parallel
Anti-parallel
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Turns