09/02/08Biochemistry: Proteins Proteins and Protein Methods Andy Howard Introductory Biochemistry,...

09/02/08Biochemistry: Proteins

Proteinsand Protein Methods

Andy HowardIntroductory Biochemistry, Fall 2008

2 September 2008

09/02/08Biochemistry: Proteins 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


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


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%


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-


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!


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


Acid-base reactivity in histidine It’s easy to protonate and deprotonate

the imidazole group


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


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:


Resonance-stabilized ion


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)


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


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


Growth of oligo- or polypeptideCN+HHHHCOO-R1CN+HHHCOO-+H2OCN+HHHHCOR1CNCOO-HR2HR2H


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


Double-bond character of peptideCN+HHHHCOR1NCHR2HCOCN+HHHHCO-R1N+CHR2HCO


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


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 ,


Ramachandran angles

G.N. Ramachandran


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


Ramachandran plot

Cf. fig. 4.9 in Horton

Exceptions are rare except with glycine


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


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


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


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.


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


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!


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


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


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


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.


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


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.


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


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


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


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


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

09/02/08Biochemistry: Proteins Proteins and Protein Methods Andy Howard Introductory Biochemistry,...

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Transcript of 09/02/08Biochemistry: Proteins Proteins and Protein Methods Andy Howard Introductory Biochemistry,...