Couple of lab things
1. In calculating rate (mmol/min/mg) from
absorbance/time slope, take the absolute
value (rates always (usually?) positive)
2. For write-up: in your table of rates,
please include the raw data (Dabs/time)
as well as the calculated rate
+1
+2
0
+
-
+
+
+1.5
+
-1
0
+
+0.5
-
0
0
-0.5
0
pI between pKR
and pK2
Free amino acid vs. polymers
Pentapeptide (five a carbons!)
What’s the charge at pH=7?
Different polypeptides have different
charge characteristics
1. Amino terminus (if ‘free’)
2. Carboxyl terminus
3. Charged side chains
Calculated pI ~7.0
http://www.embl-heidelberg.de/cgi/pi-wrapper.pl
• Free amino acids vs. polymerized
– Side chains may have different pKas
• pKa affected by charges on amino/carboxyl groups
• pKa may be affected by interactions with other side
chains in the larger molecule
Nucleophilic B: strengthens the acidic character
of the adjacent serine
Look at protein structure at a
low resolution
• Primary (1°) structure
– Sequence of amino acids
• Total number: anywhere from two to
tens of thousands
– Few (2 to tens) amino acids: oligopeptides
» Typically hormones, etc.
– Hundreds or more
» “Proteins”
» Enzymes, structural proteins, etc.
Look at protein structure at a
low resolution
• Non-amino acid chemical groups can
enhance protein function
– “Prosthetic groups” “Enzyme co-factor”
– Associated or covalently-bound
– eg. Metals
• Iron, Calcium, Zinc, Magnesium, etc.
• Structural components
• Good nucleophiles: enzymatic ‘activation’ of water,
for example
• Redox chemistry: accept/donate electrons
Look at protein structure at a
low resolution
• Prosthetic groups
– Lipids (lipoproteins) or sugars (glycoproteins)
• Enhance protein stability
• Alter interactions with other biological molecules
http://www.liv.ac.uk/physiology/ncs/conform.html
Look at protein structure at a
low resolution
• Primary (1°) structure
• Secondary (2°) structure
– Arrangement of local stretches of amino acids
– Stabilized predominantly by hydrogen bonds
between backbone N-H and O=C
Common elements of 2° structure
a helix
b strand
Look at protein structure at a
low resolution
• Primary (1°) structure
• Secondary (2°) structure
• Tertiary (3°) structure
– Three-dimensional fold of a polypeptide
– How do 2° structure elements interact?
• Non-covalent interactions
– Hydrophobic interactions
– H-bonds
– van der Waals
• Covalent bonds: disulfide
a-helices interact
to give the overall
3D fold of a
polypeptide
Look at protein structure at a
low resolution
•
•
•
•
Primary (1°) structure
Secondary (2°) structure
Tertiary (3°) structure
Quaternary (4°) structure
– Interaction between subunits of a multisubunit protein
• Non-covalent interactions
• Disulfide (covalent) bonds
SUBUNITS (INDIVIDUAL POLYPEPTIDES)
PROTEIN
Different proteins have different
chemical characteristics
• Caused by 1°, 2°, 3°, 4° structures
• Define their biological roles
• Can be exploited to separate proteins from
each other
– Purify a single protein of interest
Different proteins have different
chemical characteristics
1. Charge
•
Isoelectric point
•
•
At pH > pI, net charge ? 0
At pH < pI, net charge ? 0
2. Size
•
•
Sum of masses of all amino acids (minus
18)
Effective size can be influenced by 3°, 4°
structure
Effective sizes can be influenced
by protein shape
Globular
Filamentous
Different proteins have different
chemical characteristics
3. Ligand-binding/Affinity for other
molecules
4. Exposed hydrophobic patches
Exploitation of chemical characteristics
• Purification
– Chromatography
• Separation/analysis
– Electrophoresis