biochemistry ch 3-exploring proteins

Biochemistry Chapter 3: Exploring Proteins and Proteomes-Kyle Minor 2009

1. The Purification of Proteins: Understanding Their Functiona. Pure proteins used to

i. determine their amino acids sequences1. Used to map evolutionary relationships between proteins in diverse

organisms ii. Understand its biochemical function

iii. Grow crystals to obtain x-ray data to provide picture of their tertiary structure which is the shape that determines its function

b. The Assayi. Used to Recognize the Protein that is desired by the biochemist purifying a

particular proteinii. A positive result indicates the protein

is presentiii. The more specific the assay the more

effective the purificationiv. Usually measure enzyme activity or the ability to promote a particular chemical

reactionc. Specific activity

i. Ratio of enzyme activity to the amount of protein in mixtureii. Overall goal of protein purification is to maximize the specific activity

iii. Pure enzyme specific activity will have constant valued. Releasing the Protein form the Cell to be Purified

i. Fractionate the cell into components and determine which component is enriched in the protein

1. Schemes developed on basis of expected properties of the protein or by trial and error

ii. Homogenate1. First step

a. Formed by disrupting the cell membrane and mixture is fractionated by centrifugation

i. Yields pellet of heavy material ii. Supernatant above pellet

iii. Differential Centrifugation1. Second Step

a. Supernatant is centrifuged at greater force to yield another pellet and supernatant if several fractions

b. Fractions are each separately assayed for desired activitye. Salting out

i. Proteins are usually less soluble at high salt concentrations1. Concentration of salt for protein to precipitate out differs from protein

to protein


2. Used to fractionate protein (e.g. 0.8M ammonium sulfate precipitates fibrinogen, 2.4M ammonium sulfate precipitates serum albumin)

ii. Useful for concentrating dilute solutions of proteinsiii. Dialysis used to remove salt

1. Separates Proteins from small molecules with certain dimensions through a semi-permeable membrane

2. Does not distinguish between proteins effectivelyf. Gel-Filtration Chromatography

i. More-discriminating technique ii. Separates proteins by size

iii. Sample applied to top of column consisting of porous beads made of insoluble, highly-hydrated polymers

1. Small molecules can enter beads

iv. Results in 1. small molecules

distributed in the aqueous solution both inside and between the beads

2. Large molecules only in the solution between the beadsv. Large molecules flow more rapidly through the column and emerge first due to

the smaller volume accessible to themg. Ion-Exchange Chromatography

i. Separating proteins on the basis of their net charge1. Example proteins with net

charge at pH of 7 will bind to column of beads containing carboxylate groups and negatively charged proteins will not

2. To release the protein from the bead by increasing the concentration of NaCl or other salt in eluting buffer due to the completion between the Proteins and Na+ to the carboxylate groups on the beads

h. Affinity Chromatographyi. Uses the advantage of high affinity of proteins for a

specific chemical group.


1. Crude extract of protein is placed in column with Beads that are covalently bound to a substrate that a protein has affinity for

2. Protein attaches to substrate on bead3. Enzyme is released by adding concentration of

substrateii. Powerful means of isolating transcription factors

i. High-Pressure Liquid Chromatography (HPLC)i. Enhanced verison of column techniques

ii. Column materials are much more finely divided making more interaction sites

iii. Pressure

must be applied due to finer material j. Gel Electrophoresis

i. Used to tell if a purification scheme is effective ii. Molecules with net charge move in a electric field

iii. Used to separate proteins DNA RNA1. Velocity of migration (v)2. Electric field strength (E)3. Net charge of a protein (z)4. Frictional coefficient (f)

a. V=Ez/fb. Ez is electrical force driving the charged moleculec. Fv d. Viscous drag

iv. Performed in a thin vertical slab of polyacrylamide1. Chemically inert and easily synthesized

v. Proteins are separated by mass under denaturing conditionsvi. Proteins are first dissolved in a solution of sodium dodecyl sulfate

(SDS) 1. Disrupts nearly all noncovalent interactions 2. Binds to main chains at a ½ amino acid residues3. Gives a large net negative charge proportional to mass of

protienvii. Mercaptoethanol or dithiothreito are added to reduce disulfide bonds

viii. Proteins are visualized by staining them with silver or Coomassie blue


k. SDS-PAGE (sodium dodecyl sulfate- polyacrylamide gel electrophoresis)i. Used for carbohydrate-rich and membrane proteins

ii. Rapid, sensitive and capable of high degree of resolutioniii. Used to gain a quick estimate of the molecular weight of a protein

l. Isoelectric Focusingi. Seperation of proteins based on

their relative contents of acidic and basic residues

ii. Isoelectric point (pI)- pH at which protein has net charge of zero

iii. Electrophoretic mobility is zero (z=0)iv. Proteins are moved thru a gel with a pH gradient

m. 2-D Electrophoresisi. Combination of SDS-PAGE and Isoelectric Focusing

ii. First Isoelectric Focusing seperates the proteins horizontally by isoelectric pointiii. Second sample is placed on SDS-polyacrylamide slab and undergo

electrophoresis in a vertical fashion based on there massn. Ultracentrifugation

i. Valuable for separating Biomolecules and Determining their masses1. S=(m(1-v*p)/f

a. S- sedimentation coefficienti. Expressed in Svedberg units (s)

ii. Smaller s slower molecule moves in a centrifugal fieldb. m- mass of particle

i. More massive particle sediments more rapidly c. v- partial specific volumed. p- density of the medium

i. Dense particles move more rapidly due to the opposing buoyant force (1-vp)

ii. Particles sink when vp<1 , float when vp>1 and does not more when vp=1

e. f- frictional coefficienti. Less for a compact particle then a extended particle of

same masso. Zonal, Band or Gradient centrifugation

i. Used to separate proteins with different sedimentation coefficientsii. First step- Form a density gradient in a centrifuge tube

1. Solution of low and high density solutions (5 and 20% sucrose) missed together to form a linear fradeint of sucrose concentration

2. Precents convective flow


iii. Second step- small volume of protein mixture placed at top of gradientiv. When spun down proteins move throught the gradient and separate according

to their sedimentation coefficients v. Separated bands of protein can be harvested but making a hole at the bottom of

the tube.p. Sedimentation-equilibrium technique

i. Used for determining massii. Very accurate and can be applied without denaturing the protein

2. Amino Acid Sequences a. Once protein is purified to homogeneity b. Determining the amino acid composition of the peptide

i. 1st peptide is hydrolyzed into its constituent amino acids by heating in 6 M HCL at 110C for 24 hours

ii. 2nd amino acids are separated by ion-exchange chromatography1. Identity of the amino acid is revealed by its elution volume (volume of

buffer used to remove the amino acid from the column)2. Quantity of amino acid is revealed by reaction with ninhydrin

a. Gives an intense blue color except proline which gives a yellow color do it its secondary amino group

3. Lower concentrations of amino acids can be seen with reaction with fluorescamine

c. Edman degradationi. Used to determine N-terminal amino acid

ii. Removes one amino acid at a time form the amino terminal endiii. Phenyl isothiocyanate reacts with the uncharged terminal amino acid group of

the peptide to from phenylthiocarbamoyl derivative.

iv. Next under mildly acidic conditions a cyclic derivative of the terminal amino acid is liberated

1. Phenylthiohydrantoin (PTH)-amino acid

v. This can be continued to the shortened peptide until every amino acid is determined thru chromatography

d. Proteins can be Specifically Cleaved into Small peptides to facilitate analysis

i. Using the Edman method peptide cannot be much longer than about 50 residues


ii. Efficiency of release of amino acid decreases exponentially after each round making very impure mixtures after multiple rounds

iii. Proteins are cleaved into smaller peptides that can be sequenced1. Cyanogen bromide (CNBr)- cleaves polypeptide chains only on carboxyl

side of methionine resides 2. Trypsin- enzymes that

cleaves polypeptide chains on carboxyl side of arginine and lysine

3. Chymotrypsin- enzymes that cleaves polypeptide carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine

a. Used to gain information about original primary structure after first cleavage reagent is used by forming overlap peptide

4. If the initial protein sample contains several polypeptide chains addition al steps are necessary

a. SDS-gel electrophoresis under reducing conditions displays the number of chains

b. Edman Method can be used to determine the number of distinct N-terminal amino acids

c. After the protein has been identified to be made up of more then one chain denaturing agents such as urea or guanidine hydrochloride are used to dissociate chains held together by noncovalent bonds

5. Dithiothreitol, Performic acid, Beta- mercaptoethanol

a. Used to break disulfide bonds between polypeptide chains if disulfide bonds are present

6. Cysteine residues are alkylated with iodoacetate to form stable S-carboxymethyl derivatives to prevent them from recombining

7. To determine the positions of the orginial disulfide bondsa. Diagonal electrophoresis


b. 1st protein is cleaved into peptide under conditions in which the disulfides remain intact

c. 2nd mixture of peptides is applied to a corner of a sheet of paper and subject to electrophoresis in a single lane along one side

d. 3rd resulting sheet is exposed to vapors of performic acid which cleaves disulfides and converts them into cysteic acid residues

i. Peptides originally linked by disulfides are now independent, more acidic

e. 4th mixture is subject to electrophoresis in a perpendicular direction under same conditions as step 1

i. Peptides devoid of disulfides will be located in the single diagonal line because their mobility has not changed

ii. Newly formed peptides containing cysteic acid migrate differently and lie off the diagonal

e. Insight from Amino acid sequencesi. Sequence of a protein of interest can be compared with all other known

sequences to ascertain whether significant similarities exist; If the protein belongs to an established family

1. Gives information about its structure and functionii. Comparison of sequences of the same protein in different species yields a

wealth of information about evolutionary pathwaysiii. Amino acid sequences can be searched for the presence of internal repeats

1. Reveal history of an individual proteiniv. Many proteins contain amino acid sequences that serve as signals designating

their destinations of controlling their processingv. Sequence data provide a basis for preparing antibodies specific for a protein of

intrest.vi. Amino acid sequences are valuable for making DNA probes that are specific for

the genes encoding the corresponding proteins1. Permits the use of reverse genetics

f. Recombinant DNA technologyi. More effective then edmans degradation for proteins with 1000 plus residues

3. Immunology


a. Antibodies to specific Proteins can be generatedi. Antibodies recognize a specific group or cluster of amino acids on a target

molecule called antigenic determinant or epitopeb. Depend on the ability to generate antibodies to a specific antigen

4. Monoclonal Antibodiesa. Isolates a clone of cells that produce a single identical antibodyb. Immortal cell lines are derived from multiple myeloma cells.

i. Divide uncontrollably generating very large number of cells of a single kindii. Secrete large amounts of normal immunoglobulin of a single kind generation

after generationc. Cesar Milsteing and Georges Kohler discovered that fusing short-lived antibody-

producing cell with an immortal myeloma cell can produce large amounts of homogeneous antibody of nearly any desired specificity

i. Antigen is injected into a mouse , its spleen is removed several weeks later. ii. A mixture of the plasma cells from the spleen are fused with myeloma cells

resulting in hybridoma cellsiii. Hybridoma cells are screened using specific antigen-

antibody assays to determine which ones produce antibodies having the desired specificity

1. Collection of cells shown to produce the desired antibody are subdivide and reassayed and repeated until a pure cell line producing a single antibody is isolated

5. Enzyme-Linked Immunosorbent Assay (ELISA)a. Used to quantify the amount of a protein or other antigen

i. Uses an enzyme that reacts with a colorless substrate to produce a colored product.

ii. Enzyme is linked covalently to a specific antibody that recognizes a target antigen

1. If antigen is present the reaction will produce a color

iii. Is rapid and convenient and can detect less than a nanogram of protein

iv. Performed with either polyclonal or monoclonal antibodies

1. Monoclonal antibodies produce more reliable results

b. Indirect ELISAi. Used to detect the presence of a antibody

ii. Used to test for HIV infection


1. Detects the presence of antibodies that recognize viral core proteins (antigen)

2. Antibodies from person being tested are added to a coated well3. Enzyme linked antibodies to human antibodies (goat antibodies that

recognize human antibodies) allowed to react will the well4. Unbound antibodies are removed by washing5. Substrate is then applied6. Enzyme reaction suggests that the enzyme-linked antibodies were

bound to human antibodies which implies that the patient has antibodies to the viral antigen

c. Sandwich ELISAi. Used to detect antigen rather than antibody

1. Antibody to particular antigen is first absorbed to the bottom of the well2. Blood or urine containing the antigen is added to the well and binds to

the antibody3. A second different antibody to the antigen is added to the well

a. Antibody is enzyme linked and processed like indirect ELISA4. Extent of reaction is directly proportional to the amount of antigen

present and permits the measurement of small quantities of antigen6. Western Blotting

a. Used to detect small quantities of protein of interest in a cell or in a body fuildb. Small sample is subjected to electrophoresis on as SDS-polyacrylamide gelc. Resolved proteins on the gel are transferred to surface of a polymer sheet by blotting to

make them more accessible for reactiond. Antibody specific for the protein of interest is added to the sheet and reacts with the

antigen e. The antibody-antigen complex on the sheet can be detected by rinsing the sheet with a

second antibody specific for the first (goat antibody that recognizes mouse antibody)i. Radioactive

label on the second antibody produces a dark band on x-ray film

(autoradiogram)ii. Enzyme on second antibody generates a colored product (like ELISA)


f. Makes it possible to find a protein in a complex mixture g. Used to test for Hepatitis C detects core protein of the virus and monitoring protein

purification and the cloning of genes

7. Fluorescent Markers a. Powerful means of examining proteins in their biological contextb. Cells are stained with fluorescence-labeled antibodies and examined by fluorescence

microscopy to reveal the location of a protein of interesti. Example

1. Arrays of parallel bundles are evident in cells stained with antibody specific for actin

a. Protein that that polymerizes into filamentsb. Actin filaments are constituents of the cytoskeleton that

controls their shape and movement2. Tracking protein location with fluorescent markers procide clues to

protein functiona. Glucocorticoid receptor protein binds to steroid hormone

cortisoneb. Receptor linked to green fluorescent protein (GFP) (naturally

fluorescent protein isolated from jellyfish)c. Fluorescence microscopy revealed that in the absence of the

hormone the receptor is located in the cytoplasmd. Addition of the hormone the receptor is translocated to the

nucleus where it binds to DNAi. Results suggested that glucocorticoid receptor protein is

a transcription factor that controls gene expression3. Highest resolution of fluorescence microscopy is about 200nm

(wavelength of visible light)a. Finer spatial resolution can be achieved by electron microscopy

if antibodies are tagged with electron-dense markersi. Antibodies conjugated to clusters of gold or to ferrite

(has electron-dense core rich in iron) are highly visible under electron microscope

8. Importance’s of Synthesized Peptidesa. Peptides of a defined sequence can be synthesized to assist in biochemical analysis


i. Can serve as antigens to stimulate the formation of specific antibodies1. IF you want to isolate a protein expressed by a specific gene. Peptides

can be synthesized that match the translation of a part of the genes nucleic acid sequence and antibodies can be generated that target these peptides .

a. Antibodies can be used to isolate the intact protein or localize it within the cell

ii. Can be used to isolate receptors for many hormones and other signal molecules1. White blood cells are attracted to bacteria by formylmethionyl (fMet)

peptides released in the breakdown of bacterial proteins2. Synthetic formylmethionyl peptides have been useful in identifying the

cell-surface receptor for this class of peptide3. Synthetic peptides can be attached to agarose beads to prepare affinity

chromatography columns for purification of receptor proteins that specifically recognize the peptides

iii. Can serve as drugs1. Vasiopressin is a peptide hormone that stimulates the reabsorption of

water in the distaltubules of the kidney a. Leads to the formation of more-concentrated urineb. Patients with diabetes insipidus are deficient in vasopressin

(antidiuretic hormone) i. Treated by administering 1-desamino-8-D-arginie

vasopressin iv. Studying them can help define the rules governing the three-dimensional

structure of proteins1. Ask whether a particular sequence by itself tends to fold into an alpha

helix or beta strand a hairpin turn or behaves as a random coilb. Synthesizing Peptides

i. To prevent unwanted reactions between amino acids the alpha-amino group of first amino acid of the desired peptide is blocked with a protecting group ( tert-butyloxycarbonyl (t-Boc)

1. Carboxyl group of same amino acid is activated by reaction with dicyclohexylcarbodiimide (DCC)

ii. Free amino of next amino acid to be linked attacks the activated carboxyl group leading to the formation of a peptide bond and the release of dicuclohexylurea

iii. Repeated until desired peptide is synthesizediv. Exposing the peptide to dilute acid removes the t-Boc protecting group but

leaves the peptide bonds intactc. Soild-phase method

i. Peptides containg more than 50 amino acids can be synthesized


1. Linking the growing peptide chain to an insoluble matrix (polystyrene beads) greatly increases efficiency

2. Desired product at each stage is bound to the beads that can be rapidly filtered and washed so there is no need for purification of intermediates

3. Carboxyl-terminal amino acid of desired peptide is first anchored to the polystyrene beads

4. The t-Boc protecting group of this amino acid is removied5. The next amino acid (in the t-Boc form) and DCC are added together6. After peptide bond is formed excess reagents and dicyclohexylurea are

washed away leaving the desired dipeptide product attached to the beads

7. Additonal Amino acids are attached by repeating the process8. At the end of the synthesis the peptide is released from the beads by

adding hydrofluoric acid (HF) a. Cleaves the carboxyl ester anchor without

disrupting the peptide bondsb. Protecting groups of potentially reactive

side chains (lysine) are also removed at this time

9. Cycle of reactions can be automated10. Used to synthesize

a. interferons (155 residues)b. ribonuclease (124 residues)

11. Synthetic peptides can be linked to create even longer molecules uses specially developed peptide ligation methods9. Mass Spectrometry

a. Can determine protein masses with accuracy of one mass unit or less

b. Mass of a peptide or protein can be used as a name tag for picking out specific molecule in a vast database of amino acid sequences

c. Used to analyze ionized forms of molecules in the gas phasei. Mass measurements are obtained by determining how readily an ion is

accelerated in an applied electric fieldii. More massive ions have a lower acceleration compared to a ion less massive

with the same chargeiii. Biggest challenge is to generate a sufficiently high concentration of ionized but

intact protein molecules in the gas phase because they are not volatiled. Matrix-assisted Laser Desorption-ionization (MALDI)

i. Protein or Peptide under study is coprecipitated with an organic compound that absorbs laser light of an appropriate wavelength


ii. Flash of laser on preparation expels molecules from the surface1. Molecules capture electrons as they exit the matric and leave as

negatively charged ionse. Electrospray Ionization (ESI)

i. Solution of protein or peptide flow thru a fine metallic tip held at nonzero electrical potential.

ii. Fine electrically charged droplets are released containing both protein and the solvent

iii. Solvent evaporates from the droplet concentrating the chargeiv. Mutual repulsion of the like-charged molecules increases and individual

molecular ions fly free1. Ions are usually multiply charged

v. After gas-phase ions have been generated different approaches may be used to determine their mass

1. Time of Flight (TOF) a. Ions are accelerated in a electric field toward a detector

i. Lighter ions arrive at the detector first f. MALDI-TOF Mass Spectrometry

i. After cleaving molecules in individual spots from gel electrophoresis the mixture of fragments can be analyzed using MALDI-TOF

1. Peptide masses are matched against proteins in a database that have been “electronically cleaved” by a computer simulating the same fragmentation technique used for the experimental sample

a. Proteome within a given cell type or other sample can be analyzed in considerable detail

b. Consider the analysis of the nuclear-pore complex from yeast (facilitates transport of large molecules into and out of the nucleus)

c. Nuclear-pore complex is fractionated by HPLC followed by gel electrophoresis

d. Individual bands from the gel were isolated cleaved with trypsin and analyzed by MALDI-TOF

e. Fragments produced were compared with amino acid sequences deduced from DNA sequence of the yeast genome

f. 174 proteins were identified in this manneri. 40 were confirmed to be components of the nuclear-

pore complex by other methodsii. Mass spectrometric methods are sensitive enough to

detect essentially all components of the pore if they are present in the sample

g. X-ray Crystallography


i. First method developed to determine protein structure in atomic detail and still the clearest visualization of the protein structure

ii. Reveals precise 3-D postions of most atoms in a protein moleculeiii. Provides best resolution because the wave length of x-rays is about the same

length of covalent bonds.iv. Three components

1. Protein Crystal a. Crystals are obtained by slowly adding ammonium sulfate or

another salt to a concentrated solution of protein to reduce its solubility favors the formation of highly ordered crystals (salting out)

b. Myoglobin crystallizes in 3 M ammonium sulfatec. Hundreds of conditions must be tested to obtain crystals fully

suitable for crystallographic studiesd. Proteins usually crystallize in the biological active configuratione. Enzyme crystals may display catalytic activity if the crystals are

suffused with substrate2. Source of X-ray

a. Wavelength of 1.54 A is produced by accelerating electrons i. against a copper target

ii. In circular orbits at speeds close to the speed of light (synchrotron radiation)

b. Narrow beam of x-ray strikes the protein crystali. Some goes thru the crystal while some scattered in

various directions1. Scattered light is detected by x-ray film and

blackening of the emulsion being proportional to the intensity of the scattered x-ray beam or by a solid state electronic detector

ii. Electron scatter x-rays1. Amplitude of the wave scattered by an atom is

proportional to its number of electrons (carbon scatters 6x stronger then hydrogen atom)

iii. Scattered waves recombine1. Each atom contributes to each scattered beam.

Scattered waves reinforce one another at the film or detector if they are in phase (in step) there. And they cancel one another if they are out of phase

iv. The way in which the scattered waves recombine depends only on the atomic arrangement


3. Detectorv. Protein crystal is mounted and positioned in a precise oritentation with respect

to the x-ray beam and the film. 1. Crystal is rotated so the beam can strike from many directions2. Rotational motion results in an x-ray photograph consisting of a regular

array of spots called reflectionsvi. The intensity of the spots are measured

1. Intensity and position of the spots are the experimental datavii. A reconstruction image of the protein is observed from the intensities by a

Fourier transformh. Light Microscopy or electron microscopy the diffracted beams are focused by lenses to

directly form an imagei. Lenses for focusing x-rays do not exist

10. NMRa. Reveals the Structures of Proteins in Solution at high concentrationsb. Used for proteins that do not readily crystallize

biochemistry ch 3-exploring proteins

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