I. Protein Isolation -- Power Point

A. Selection of a Protein Source-often can obtain the same or similar protein from several different sources chose a source with

1. which can be obtained in large amounts and has..

2. high concentration of the protein

3. molecular cloning techniques allow production and purification of proteins from E. coli, yeast or other cells.

B. Method of solubilization

1. serum proteins or secreted proteins are already soluble

2. otherwise cells must be broken up

  • osmotic lysis; perhaps aided by enzymes or chemicals to weaken cell membranes (detergents, solvents, or lysozyme for bacteria)
  • mechanical disruption by grinding, blending, homogenizing or ultrasonic disruption

3. filter or centrifuge crude lysate to remove cell debris (membranes, cell walls etc.)

  • protein may be in souble fraction
  • protein may be in particulate fraction which may be purified by centrifugation techniques and then solubilized by treatment with detergents or other chemicals

C. Stabilization of proteins--proteins are delicate

1. may be denatured by high temperature
  • keep solution at appropriate temperature, usually fairly cold.
  • can use denaturation of some proteins to help purify a protein which is stable at high temperature

2. proteases are enzymes which break peptide bonds

  • maintain conditions which inhibit proteases ==> low temperature or change in pH or addition of chemical inhibitors
  • may use proteases to digest labile proteins if the protein of interest is stable to proteases

3. many proteins are unstable at air-water interfaces or at low concentration

  • keep protein solutions concentrated
  • keep solution from "frothing" ==> don't mix vigorously

D. Assay of Proteins-need some way of measuring the concentration of a specific protein so we know when we're doing something right

1. enzymes can be measured by the reactions they catalyze--either measure products produced or reactants used up.

2. other proteins may be measured by their biological effects: ability to bind specific molecules or the effect of a hormone on cells, tissue or organism.

3. Immunochemical techniques-can produce antibodies which bind specifically to particular proteins--see Fig. 5-1 for enzyme linked immunosorbent assay.

E. General Strategy of Protein Purification-fractionate based on different characteristics

Proteins are purified by fractionation procedures in a series of steps. The goal is not necessarily to preserve all of the protein of interest, although it is important to preserve as much as possible. One must, however, eliminate all of the other proteins in the mixture, and this usually means that much of the protein of interest will be lost. One needs a criterion of purity; with enzymes this is the specific activity = amount of catalytic activity per unit mass (mg). Similar criteria can be established for other proteins based upon their biological activities.

1. Charge
  • ion exchange chromatography
  • electrophoresis
  • isoelectric focusing

2. Polarity

  • adsorption chromatography
  • paper chromatography
  • reverse-phase chromatography
  • hydrophobic interaction chromatography

3. Size

  • dialysis and ultrafiltration
  • gel electrophoresis
  • gel filtration chromatography
  • ultracentrifugation

4. Specificity of binding-affinity chromatography

II. Solubilities of Proteins

A. Effects of Salt Concentration

1. salting in - proteins are less soluble at very low salt concentrations (ionic strength); salt ions helps shield protein's multiple charged groups --> Fig. 5-3

2. salting out - proteins are also less soluble at salt concentrations (high ionic strength) because the salt ions bind most of the water molecules --> Fig 5-2

3. These two effects are different for different proteins ==> fractionate by raising the [salt] just below point where protein to be purified becomes insoluble, removed other precipitated proteins by centrifugation, then raise the [salt] to precipitate protein and collect it by centrifugation.

B. Effects of organic solvent

can selective precipitate some proteins by increasing the concentration of water miscible organic solvents

C. Effects of pH

All proteins have an isoelectric point, pI, a pH at which they have no net charge, and they are least soluble at their pI because there are no net electrostatic repulsions between protein molecules: Fig. 5-4. Different proteins have different pI's, so one can manipulate the relative solubilities of a mixture of proteins by changing the pH.

D. Crystallization

Once a protein is reasonably pure, one may try to crystallize it which is the ultimate criterion of purity.

III. Chromatographic Separations

One of the most power class of separation procedures is chromatography; this technique can take various forms based upon the physical apparatus--column chromatography, paper chromatography, or thin layer chromatography for example--and upon the physical principle--ion exchange chromatography, partition between phases of different polarity, size exclusion chromatography etc. All depend upon having a mobile phase, usually a liquid, and a stationary phase, usually a solic or a solid coated with a liquid. The sample is applied in the mobile phase which passes down the column (or paper or thin layer plate) and the different solutes move at different speeds depending upon their relative affinities for the mobile and stationary phases; we say that they partition between the mobile and stationary phases.

A. Ion Exchange Chromatography--adsorption chromatography

1. Stationary Phase -- chemically bound charged groups with counter ions bound; these may be positively charged groups which bind anions (anion exchanger) or negatively charged groups which bind cations (cation exchanger) Table 5-2

2. Mobile Phase -- an aqueous buffer solution characterized by: pH and ionic strength

3. Eluting Ion Exchange Columns -- molecules usually adsorb tightly in the buffer in which they're applied ==> must weaken this interaction. Fig. 5-6

  • Increase ionic strength (most common method): F = q1 q2 / D r2
    q1and q2 are the charges on 2 groups, r is the distance between the groups, and D is the dielectric constant of the solvent which is increased with higher ionic strength thus weakening the force between the solute and the ion exchanger. Another way to look at this is that other ions in the buffer compete for the ion exchanger binding site.
  • change pH -- changes the charges on the molecules being separated; also can change the charge of a weak ion exchanger
  • These changes can be made stepwise by changing the buffer reservoir (step gradient) or as gradient -- by mixing two buffers

B. Gel Filtration Chromatography Fig. 5-6

1. Column Packing -- spherical porous beads of defined size
crosslinked dextrans -- Sephadex (Pharmacia)

crosslinked polyacrylamide -- Bio-Gel P (Bio-Rad)

crosslinked agarose -- Sepharose (Pharmacia) or Biogel A (Bio-Rad). Agarose beads have very large pores and are, therefore, good for separating very large molecules

other materials developed by other companies

2. Gel beads are designed to have a distribution of pore sizes around a mean pore size. The mean pore size and the distribution determines the size range of molecules which can be separated.

3. Dialysis is a form of molecular Filtration Fig. 2-12

C. Affinity Chromatography

based upon specific binding of the target protein to a particular ligand which is bound to an inert matrix. Fig. 5-7,8

D. Other Chromatographic Techniques

1. Reverse Phase Chromatography
stationary phase is more hydrophobic liquid adsorbed to inert matrix

mobile phase is more hydrophilic liquid

2. Hydrophobic Interaction Chromatography--similar to reverse phase chromtography but with less densely packed hdrophobic groups ==> less denaturing.

3. HPLC = High Performance Chromatography--a form of column chromatogrpahy with very small particles in the stationary phase to increase resolution; speed increased by using very high pressures. Commonly used in reverse phase mode but also with ion exchange, gel filtration, or hydrophobic interaction.

IV. Electrophoresis

A. Macromolecule is accelerated by a force (like sedimentation)

1. F = q E ==> [q is the net charge on the molecule; E is the electric field strength experienced by the molecule]

2. This causes an acceleration until the velocity, v, causes a frictional force equal to but opposite in direction to the applied force ==> F = q E = f v

3. Zonal: most common mode used

  • sample is applied in a zone (small region: a spot on moistened paper or a band on a gel) and an applied field causes the molecules to separate into zones based upon different U's.
  • Like Zonal sedimentation, need some way of stabilizing the zones to prevent mechanical mixing (from vibrations) or convection mixing (from temperature differences -- a particularly severe problem with resistance heating caused by the electric field).

4. Gel Electrophoresis Media -- Three types

  • Starch Gel -- swollen potato starch granules (little used now except for prep isoelectric focusing)
  • Agarose Gel -- purified large MW polysaccharide (from agar) ==> very open (large pore) gel used frequently for large DNA molecules
  • Polyacrylamide Gels -- most commonly used gel because they are very stable and can be made at a wide variety of concentrations or even with a gradient of concentrations ==> large variety of pore sizes
  • Acrylamide Concentrations -- typically 5-20% by weight (5%, 7.5%, 10%, 12.5%, 15%, 20% are commonly used values) ==> gel is mostly water. Acrylamide polymerizes in head-to-tail fashion to form long polymers which form a complex network held together by bis-acrylamide crosslinks. The cris-crossing polymers create pores in the gel; the size of pores is determined by the acrylamide concentraion.

5. Acrylamide can be polymerized into any desired shape -- two shapes used for electrophoresis

  • Tube Gels -- polymerize in glass tubing ==> cylindrical shape
  • Slab Gels -- polymerize between glass plates

B. SDS PolyAcrylamide Gel Electrophoresis -- SDS PAGE

1. Sodium Dodecyl Sulfate = Sodium Lauryl Sulfate: CH3(CH2)11SO3-Na+
This is a detergent because it contains a hydrophobic region, the CH
3(CH2)11 tail, attached to a hydrophilic group, SO3-Na+, making it amphipathic. It is a very strong detergent which denatures proteins by binding to the polypeptide backbone.

2. Measurements show that most proteins bind 1.4 gm SDS/gm protein with very little variation (except for some membrane proteins; membrane proteins are very hydrophobic and may bind more SDS) ==> The charge on an SDS-protein complex is determined almost entirely by SDS --> -1 charge for each SDS. Since the amount of SDS bound is determined by the size of the protein and all protein/SDS micelles are anionic ==> z is directly proportional to M and in the absence of a gel all proteins will move at the same rate

3. Hydrodynamic studies show that the shape of an SDS/protein complex is a rod or prolate ellipsoid--called a micelle--of ~18Å diameter and a length proportional to M

4. Electrophoresis of SDS/protein micelles through a polyacrylamide gel, however, will separate them according to M since small proteins will move more easily through the gel
==> Rf = b - a log M

Fig. 5-10,11

C. Immunoblotting

D. IsoElectric Focusing: All protein charges vary from a net positive charge at low pH (-COOH and -NH3+ forms of acidic and basic functional groups) through 0 at some intermediate pH to a net negative charge (-COO- and -NH2 forms) at high pH.

1. pI - IsoElectric Point: pH at which a protein has a net 0 charge (positive and negative charges balance). Depends mostly on the amino acid composition and a little on the tertiary structure

2. Create a pH gradient in a gel: Can be done on a slab (vertical or horizontal) or a tube.

the final positions of each band depend only on an intrinsic property of the proteins, their pI's, and not on where they started in the gel

F. Two-Dimensional Electrophoresis: There are many variations; all basically combine 2 types of electrophoresis

Most common combines Isoelectric Focusing in tube gels and SDS-PAGE in slab gels

Final gel has a complex mixture or proteins separated by pI along the horizontal axis and by log M along the vertical axis. Can resolve thousands of spots (proteins) by this technique. Analysis is now automated by computer so that one can do 2D gels on whole cell extracts and monitor how each protein changes during: a) Development; b) Transformation; c) Excitation -- e.g. by a hormone etc.

Chapter 6: Covalent Structures of Proteins

  1. Primary structure (1° structure) is the amino acid sequence of its polypeptide chain(s).
  2. Secondary (2°) structure is the local spatial arrangement of a polypeptide's backbone atoms without regard to the conformations of its side chains.
  3. Tertiary (3°) structure refers to the three-dimensional structure of an entire polypeptide. The line between 2° and 3° structure is somewhat vague.
  4. Quaternary (4°) structure is present in only some proteins, those which contain more than one polypeptide chain, referred to as subunits, held together by noncovalent bonds or, in some cases, disulfide bonds.

I. Primary Structure Determination

A. Endgroup Analysis ==> how many different types of subunits?

1. N-terminus Identification
  • dansyl chloride Fig 5-13
  • Edman degradation: Phenylisothiocyanate Fig. 5-15; can be used to sequence peptides by repeating sequence

2. C-Terminus Identification: exopeptidases, e.g. carboxy-peptidase

  • release amino acids sequentially from C-terminus
  • rate varies for different amino acids

B. Cleavage of Disulfide Bonds- permits separation of polypeptides connected by disulfide bonds

1. Performic Acid: oxidizes sulfhydryl groups to cysteic acid, -SO3-
  • stable in acid or base
  • also oxidizes methionine ==> can't use cyanogen bromide cleavage
  • partially destroys Trp sidechains

2. Reduce disulfide to sulfhydryl groups Fig. 5-17

  • mercaptoethanol, HS-CH2CH2OH, or dithiothreitol
  • stabilize sulfhydryl groups by reacting with iodoacetate

C. Separation, Purification, and Characterization of Polypeptide Chains

1. Denature polypeptides by changing pH, [salt], or adding chemicals such as urea or guanidinium which disrupt structure.

2. Purify proteins using techniques described above: chromatography, electrophoresis, etc.

3. Mass spectrometry

D. Amino Acid Composition

1. Hydrolyze polypeptides
6N HCl, 120°C, 10-100 hrs; destroys Trp and hydrolyzes Gln to Glu and Asn to Asp

2-4 N NaOH 100°C 4-8 hrs destroys Cys, Ser, Thr, and Arg, but leaves Trp intact

2. Separate amino acids and measure concentrations

ion exchange chromatography or HPLC

quantitate amino acids with dansyl chloride or o-pthalaldehyde (OPA)

E. Specific Peptide Cleavage Reactions&endash;can sequence polypeptides 40-80 amino acid residues long ==> longer polypeptides and proteins must be cut into smaller pieces at specific locations

1. Endopeptidases: see Table 5-5 for specificities of endopeptidases
  • Trpsin cleaves peptide bonds whose carboxyl group is provided by positively charged residues (e.g. Lys or Arg)
  • Chymotrypsin cleaves after bulky hydrophobic residues (Phe, Trp, Tyr)
  • Endopeptidase V8 from Staph aureus cleaves after Glu

2. Cyanogen Bromide (CNBr) cleaves after Met residues

F. Separate and purify fragments : generally by reverse phase HPLC

G. Sequence determination--Edman degradation; generally automated

H. Ordering the Peptide Fragments: fragment in two different ways which produce two sets of fragments which overlap&endash;e.g. trypsin cleavage at Lys & Arg residues vs. CNBr cleavage at Met residues. Compare fragment sequences. Fig. 5-16

Chapter 6 -- Three Dimensional Structures of Proteins - PowerPoint

Levels of Protein Structure - Fig. 6-1

I. Secondary Structure

A. The Peptide Group - Fig. 6-2,3,4

1. Linus Pauling and Robert Corey studied the structures of peptides and deduced that the C-N bond has approx. 40% double bond character
  • no rotation about C-N bond ==> usually in the trans conformation which is approx 8 kJ/mole more stable than the cis conformation in which atoms tend to bump into one another
  • All atoms involved in peptide bond plus the alpha C's are coplanar ==> peptide conformation can be defined by the torsion angles, f between N and the alpha C and y between the alpha C and carboxyl C. Fig. 6-4

2. Ramachandran Diagram ( Fig. 6-6) shows values of torsion angles which are allowed by steric interactions (i.e. which keep atoms apart ; Fig. 6-5)

B. Helical Structures

1. a-Helix : discovered by Linus Pauling in 1951 by bulding models to fit data from X-ray diffraction of fibrous proteins
  • helix make 1 turn every 3.6 amino acid residues
  • residues are spaced 1.5Å along the helix ==> 1.5 Å x 3.6 residues/turn = 5.4 Å/turn ==> the pitch of the helix
  • for L-amino acids the helix is righthanded with torsion angles are f = -57° and y = -47°

2. Other Polypeptide Helices : Fig. 6-17

  • 310 helix : 3 residues / turn with a pitch of 6 Å; mildely forbidden region of Ramachandran diagram
  • p Helix : 4.4 residues / turn; also in mildly fobidden region
  • collagen helix = polyproline helix : 3.0 residues/turn and pitch = 9 Å (also adopted by polyglycine).

C. Beta Structures - b-pleated sheets also discovered by Pauling and Corey in 1951. Conformations are slightly different from extended polypeptide giving them a rippled or pleated appearance.

1. Antiparallel b-pleated sheet-neighboring hydrogen-bonded polypeptides run in opposite directions Fig. 6-9a

2. Parallel b-pleated sheet-neighboring hydrogen-bonded polypeptides run in the same direction Fig. 6-9b 

3. Supersecondary Structures - grouping of 2° structure elements forming motifs

bab motif
b-hairpin motif
aa motif