Transcript Protein Structure - Illinois Institute of Technology
Proteins and Protein Methods
Andy Howard Introductory Biochemistry, Fall 2008 2 September 2008 Biochemistry: Proteins 09/02/08
Plans for Today
pKa’s for main chain atoms Side-chain Reactivity Acid-base reactivity Other reactions Peptides The peptide bond Main-chain torsion angles , , Proteins Protein Purification Salting Out Chromatographic Techniques Biochemistry: Proteins 09/02/08 Page 2 of 39
Why does p
K
a
the side chain?
depend on
Opportunities for hydrogen bonding or other ionic interactions stabilize some charges more than others More variability in the amino terminus, i.e. the p
K
a of the carboxylate group doesn’t depend as much on R as the p group
K
a of the amine
Biochemistry: Proteins 09/02/08 Page 3 of 39
How do we relate p
K
a
percentage ionization?
Derivable from Henderson-
to
Hasselbalch equation
If pH = p
K
a , half-ionized One unit below:
90% at more positive charge state,
10% at less + charge state
One unit above: 10% / 90%
Biochemistry: Proteins 09/02/08 Page 4 of 39
Don’t fall into the trap!
Ionization of leucine:
pH %+ve % neutral %-ve Main species 1.3
90 2.3
50 10 50 0 NH 3 + CHR COOH 0 3.3
10 90 8.7
0 90 0 10 NH 3 + C HR COO NH 3 + CHR COO 9.7
0 50 50 10.7
0 10 90 NH 2 CHR COO -
Page 5 of 39 Biochemistry: Proteins 09/02/08
Side-chain reactivity
Not all the chemical reactivity of amino acids involves the main-chain amino and carboxyl groups Side chains can participate in reactions:
Acid-base reactions Other reactions In proteins and peptides, the side-chain reactivity is more important because the main chain is locked up!
Biochemistry: Proteins 09/02/08 Page 6 of 39
Acid-base reactivity
on side chains
Asp, glu: side-chain COO :
Asp sidechain p
K
a = 3.9
Glu sidechain p
K
a = 4.1
Lys, arg: side-chain nitrogen:
Lys sidechain –NH 3 + p
K
a = 10.5
Arg sidechain =NH 2 + p
K
a = 12.5
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Acid-base reactivity in
histidine
It’s easy to protonate and deprotonate the imidazole group Biochemistry: Proteins 09/02/08 Page 8 of 39
Cysteine: a special case
The sulfur is surprisingly ionizable Within proteins it often remains unionized even at higher pH H H+ S S H H C C H H+ p
K
a = 8.4
O H H C C H O C H H N+ Biochemistry: Proteins H O H H N+ 09/02/08 C H O Page 9 of 39
Ionizing hydroxyls
X –O–H X –O + H + Tyrosine is easy, ser and thr hard: Tyr p
K
a = 10.5
Ser, Thr p
K
a = ~13 Difference due to resonance stabilization of phenolate ion: Biochemistry: Proteins 09/02/08 Page 10 of 39
Resonance-stabilized ion
Biochemistry: Proteins 09/02/08 Page 11 of 39
Other side-chain reactions
Little activity in hydrophobic amino acids other than van der Waals Sulfurs (especially in cysteines) can be oxidized to sulfates, sulfites, … Nitrogens in his can covalently bond to various ligands Hydroxyls can form ethers, esters Salt bridges (e.g. lys - asp) Biochemistry: Proteins 09/02/08 Page 12 of 39
Phosphorylation
ATP donates terminal phosphate to side chain hydroxyl of ser, thr, tyr: ATP + Ser-OH ADP + Ser-O-(P) Similar activity adds P to his N Often involved in activating or inactivating enzymes Under careful control of enzymes called
kinases
and
phosphatases
Biochemistry: Proteins 09/02/08 Page 13 of 39
Peptides and proteins
Peptides are oligomers of amino acids Proteins are polymers Dividing line is a little vague: ~ 50-80 aa.
All are created, both formally and in practice, by stepwise polymerization Water eliminated at each step Biochemistry: Proteins 09/02/08 Page 14 of 39
Growth of oligo- or polypeptide
H H H N+ R 1 H C H C O O + H H N+ H C R 2 C O O H H H N+ Biochemistry: Proteins R 1 C H O H H 2 O C H N C O C R 2 O 09/02/08 Page 15 of 39
The peptide bond
The amide bond between two successive amino acids is known as a
peptide
bond The C-N bond between the first amino acid’s carbonyl carbon and the second amino acid’s amine nitrogen has some double bond character Biochemistry: Proteins 09/02/08 Page 16 of 39
Double-bond character of peptide
R 1 H O H H N C C C H N+ C H R 2 H O O R 1 H C H N+ H Biochemistry: Proteins H C H N+ H C O C R 2 09/02/08 Page 17 of 39
The result: planarity!
This partial double bond character means the nitrogen is sp 2 hybridized Six atoms must lie in a single plane: First amino acid’s alpha carbon Carbonyl carbon Carbonyl oxygen Second amino acid’s amide nitrogen Amide hydrogen Second amino acid’s alpha carbon Biochemistry: Proteins 09/02/08 Page 18 of 39
Rotations and flexibility
Planarity implies = 180, where the rotation angle about N-C bond is Free rotations are possible about N C and C -C bonds Define Define = rotation about N-C = rotation about C -C We can characterize main-chain conformations according to , Biochemistry: Proteins 09/02/08 Page 19 of 39
Ramachandran angles
Biochemistry: Proteins G.N. Ramachandran 09/02/08 Page 20 of 39
Preferred Values of
and
Steric hindrance makes some values unlikely Specific values are characteristic of particular types of secondary structure Most structures with forbidden values of and turn out to be errors generally between 180º and -60º generally between 30º and 200º or -30 to -80 Biochemistry: Proteins 09/02/08 Page 21 of 39
Ramachandran plot
Cf. fig. 4.9 in Horton Exceptions are rare except with glycine Biochemistry: Proteins 09/02/08 Page 22 of 39
How to remember
and
Proteins are synthesized N to C on the ribosome Therefore the natural way to draw an amino acid is (NH-CHR-CO) is the first of those angles is the second is earlier in the Greek alphabet, and phi comes before psi in Roman spelling Biochemistry: Proteins 09/02/08 Page 23 of 39
Why bother with mnemonics?
Very few textbooks provide memory aids like these You’re grown-ups; you can read the actual answers in your textbook This is intended as a study aid, which is what an instructor should be providing We’ll do several during the semester Biochemistry: Proteins 09/02/08 Page 24 of 39
How are oligo- and
polypeptides synthesized?
Formation of the peptide linkages occurs in the ribosome under careful enzymatic control (the enzyme is an RNA molecule) Polymerization is endergonic and requires energy in the form of GTP (like ATP, only with guanosine): GTP +
n
-length-peptide + amino acid GDP + P i + (
n
+1)-length peptide Biochemistry: Proteins 09/02/08 Page 25 of 39
What happens at the ends?
Usually there’s a free amino end and a free carboxyl end: H 3 N + -CHR-CO-(peptide)
n
-NH-CHR-COO Cyclic peptides do occur Cyclization doesn’t happen at the ribosome: it involves a separate, enzymatic step.
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Reactivity in peptides & proteins
Main-chain acid-base reactivity unavailable except on the ends Side-chain reactivity available but with slightly modified p
K
a s.
Terminal main-chain p
K
a values modified too Environment of protein side chain is often hydrophobic, unlike free amino acid side chain Biochemistry: Proteins 09/02/08 Page 27 of 39
iClicker: What’s the net charge in ELVIS at pH 7?
(a) 0 (b) +1 (c) -1 (d) +2 (e) -2 You have 60 seconds here so you can look up the 1-letter codes again!
Page 28 of 39 Biochemistry: Proteins 09/02/08
Disulfides
In oxidizing environments, two neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains H C H S C H H H S H + (1/2)O 2 H C S H S H 2 O H C H Biochemistry: Proteins 09/02/08 Page 29 of 39
What could this do?
Can bring portions of a protein that are distant in amino acid sequence into close proximity with one another This can influence protein stability Biochemistry: Proteins 09/02/08 Page 30 of 39
Protein Purification
Why do we purify proteins?
To get a basic idea of function we need to see a protein in isolation from its environment That necessitates purification An instance of
reductionist
science Full characterization requires a knowledge of the protein’s action in context Biochemistry: Proteins 09/02/08 Page 31 of 39
Salting Out
Most proteins are less soluble in high salt than in low salt In high salt, water molecules are too busy interacting with the primary solute (salt) to pay much attention to the secondary solute (protein) Various proteins differ in the degree to which their solubility disappears as [salt] goes up We can separate proteins by their differential solubility in high salt. Biochemistry: Proteins 09/02/08 Page 32 of 39
How to do it
Dissolve protein mixture in highly soluble salt like Li 2 SO 4 , (NH 4 ) 2 SO 4 , NaCl Increase [salt] until some proteins precipitate and others don’t You may be able to recover both: The supernatant (get rid of salt; move on) The pellet (redissolve, desalt, move on) Typical salt concentrations > 1M Biochemistry: Proteins 09/02/08 Page 33 of 39
Dialysis
Some plastics allow molecules to pass through if and only if MW < Cutoff Protein will stay inside bag, smaller proteins will leave Non-protein impurities may leave too.
Biochemistry: Proteins 09/02/08 Page 34 of 39
Gel-filtration chromatography
Pass a protein solution through a bead containing medium at low pressure Beads retard small molecules Beads don’t retard bigger molecules Can be used to separate proteins of significantly different sizes Suitable for preparative work Biochemistry: Proteins 09/02/08 Page 35 of 39
Ion-exchange chromatography
Charged species affixed to column Phosphonates (-) retard (+)charged proteins: Cation exchange Quaternary ammonium salts (+) retard (-)charged proteins: Anion exchange Separations facilitated by adjusting pH Biochemistry: Proteins 09/02/08 Page 36 of 39
Affinity chromatography
Stationary phase contains a species that has specific favorable interaction with the protein we want DNA-binding protein specific to AGCATGCT: bind AGCATGCT to a column, and the protein we want will stick; every other protein falls through Often used to purify antibodies by binding the antigen to the column Page 37 of 39 Biochemistry: Proteins 09/02/08
Metal-ion affinity chromatography
Immobilize a metal ion, e.g. Ni, to the column material Proteins with affinity to that metal will stick Wash them off afterward with a ligand with an even higher affinity We can engineer proteins to contain the affinity tag: poly-histidine at N- or C-terminus Biochemistry: Proteins 09/02/08 Page 38 of 39
High-performance liquid
chromatography
Many LC separations can happen faster and more effectively under high pressure Works for small molecules Protein application is routine too, both for analysis and purification FPLC is a trademark, but it’s used generically Biochemistry: Proteins 09/02/08 Page 39 of 39