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09/02/08Biochemistry: Proteins
Proteinsand Protein Methods
Andy HowardIntroductory Biochemistry, Fall 2008
2 September 2008
09/02/08Biochemistry: Proteins Page 2 of 39
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
09/02/08Biochemistry: Proteins Page 3 of 39
Why does pKa depend on the side chain? Opportunities for hydrogen bonding
or other ionic interactions stabilize some charges more than others
More variability in the amino terminus, i.e. the pKa of the carboxylate group doesn’t depend as much on R as the pKa of the amine group
09/02/08Biochemistry: Proteins Page 4 of 39
How do we relate pKa to percentage ionization? Derivable from Henderson-
Hasselbalch equation If pH = pKa, half-ionized One unit below:
90% at more positive charge state, 10% at less + charge state
One unit above: 10% / 90%
09/02/08Biochemistry: Proteins Page 5 of 39
Don’t fall into the trap! Ionization of leucine:
pH 1.3 2.3 3.3 8.7 9.7 10.7
%+ve 90 50 10 0 0 0
% neutral 10 50 90 90 50 10
%-ve 0 0 0 10 50 90
Main species
NH3+-
CHR-COOH
NH3+C
HR-COO-
NH3+
CHR-COO-
NH2-
CHR-COO-
09/02/08Biochemistry: Proteins Page 6 of 39
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!
09/02/08Biochemistry: Proteins Page 7 of 39
Acid-base reactivity on side chains
Asp, glu: side-chain COO-: Asp sidechain pKa = 3.9
Glu sidechain pKa = 4.1
Lys, arg: side-chain nitrogen: Lys sidechain –NH3
+ pKa = 10.5
Arg sidechain =NH2+ pKa = 12.5
09/02/08Biochemistry: Proteins Page 8 of 39
Acid-base reactivity in histidine It’s easy to protonate and deprotonate
the imidazole group
09/02/08Biochemistry: Proteins Page 9 of 39
Cysteine: a special case The sulfur is surprisingly ionizable Within proteins it often remains unionized
even at higher pHCN+HHHHCOO-CHHSHCN+HHHHCOO-CHHS-H+H+pKa = 8.4
09/02/08Biochemistry: Proteins Page 10 of 39
Ionizing hydroxyls X–O–H X–O- + H+ Tyrosine is easy, ser and thr hard:
Tyr pKa = 10.5
Ser, Thr pKa = ~13 Difference due to resonance stabilization
of phenolate ion:
09/02/08Biochemistry: Proteins Page 11 of 39
Resonance-stabilized ion
09/02/08Biochemistry: Proteins Page 12 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)
09/02/08Biochemistry: Proteins Page 13 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
09/02/08Biochemistry: Proteins Page 14 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
09/02/08Biochemistry: Proteins Page 15 of 39
Growth of oligo- or polypeptideCN+HHHHCOO-R1CN+HHHCOO-+H2OCN+HHHHCOR1CNCOO-HR2HR2H
09/02/08Biochemistry: Proteins Page 16 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
09/02/08Biochemistry: Proteins Page 17 of 39
Double-bond character of peptideCN+HHHHCOR1NCHR2HCOCN+HHHHCO-R1N+CHR2HCO
09/02/08Biochemistry: Proteins Page 18 of 39
The result: planarity!
This partial double bond character means the nitrogen is sp2 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
09/02/08Biochemistry: Proteins Page 19 of 39
Rotations and flexibility
Planarity implies = 180, where is the rotation angle about N-C bond
Free rotations are possible about N-C and C-C bonds Define = rotation about N-C Define = rotation about C-C
We can characterize main-chain conformations according to ,
09/02/08Biochemistry: Proteins Page 20 of 39
Ramachandran angles
G.N. Ramachandran
09/02/08Biochemistry: Proteins Page 21 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
09/02/08Biochemistry: Proteins Page 22 of 39
Ramachandran plot
Cf. fig. 4.9 in Horton
Exceptions are rare except with glycine
09/02/08Biochemistry: Proteins Page 23 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
09/02/08Biochemistry: Proteins Page 24 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
09/02/08Biochemistry: Proteins Page 25 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 + Pi + (n+1)-length peptide
09/02/08Biochemistry: Proteins Page 26 of 39
What happens at the ends?
Usually there’s a free amino end and a free carboxyl end:
H3N+-CHR-CO-(peptide)n-NH-CHR-COO-
Cyclic peptides do occur Cyclization doesn’t happen at the
ribosome: it involves a separate, enzymatic step.
09/02/08Biochemistry: Proteins Page 27 of 39
Reactivity in peptides & proteins Main-chain acid-base reactivity
unavailable except on the ends Side-chain reactivity available but
with slightly modified pKas. Terminal main-chain pKavalues
modified too Environment of protein side chain is
often hydrophobic, unlike free amino acid side chain
09/02/08Biochemistry: Proteins Page 28 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!
09/02/08Biochemistry: Proteins Page 29 of 39
Disulfides
In oxidizing environments, two neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains
CHHSHCHHSH+(1/2)O2SSHCHHCHH2O
09/02/08Biochemistry: Proteins Page 30 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
09/02/08Biochemistry: Proteins Page 31 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
09/02/08Biochemistry: Proteins Page 32 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.
09/02/08Biochemistry: Proteins Page 33 of 39
How to do it Dissolve protein mixture in highly soluble
salt like Li2SO4, (NH4)2SO4, 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
09/02/08Biochemistry: Proteins Page 34 of 39
Dialysis Some plastics allow
molecules to pass through if and only ifMW < Cutoff
Protein will stayinside bag, smaller proteins will leave
Non-protein impurities may leave too.
09/02/08Biochemistry: Proteins Page 35 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
09/02/08Biochemistry: Proteins Page 36 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
09/02/08Biochemistry: Proteins Page 37 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
09/02/08Biochemistry: Proteins Page 38 of 39
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
09/02/08Biochemistry: Proteins Page 39 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