Chapter 3(4) Exploring Proteins and Proteomes

  • proteome is functional representation of the genome

  • proteome is not fixed

3.1 The Purification of Proteins is an Essential First Step in Understanding their Function

assay

  • test for some unique identifying property of the protein

  • The more specific, the more effective the purification

  • for enzymes, measure activity indirectly

  • to analyze, need amount of protein present

  • enzyme activity + protein present → calculate specific activity

  • ideally, s.a. will rise as purification proceeds

Proteins must be released from the cells to be purified

  • homogenate: formed by disrupting cell membrane

  • differential centrifugation

Proteins can be purified according to solubility, size, charge, and binding affinity

  • salting out

    • most proteins less soluble at high salt concentrations

  • dialysis

    • protein mix in dialysis bag, put in buffer solution, small molecules and ions diffuse out

    • does not distinguish between proteins effectively

  • gel-filtration chromatography

    • molecular exclusion chromatography

    • more discriminating

    • put sample on column with porous beads, only small molecules can enter

    • bigger molecules get to the bottom first

  • ion-exchange chromatography

    • used for high purity

    • one chromatography step is usually not enough

    • separates based on net charge

    • cation/anion exchange

  • affinity chromatography

    • highly selective

    • uses high affinity of proteins for specific chemical groups

    • ex. plant protein concanavalin A (carb. binding protein), has affinity for glucose

    • used to isolate protein by covalently attaching group to a column or added mix of proteins to column and washing or eluting by adding high conc. of soluble

    • ex. His tag binds tightly to immobilized metal ions

  • high-performance liquid chromatography

    • enhanced column technique

    • column materials more finely divided, so possess more interaction sites

    • needs pressure

    • high resolution and rapid separation

Proteins can be separated by gel electrophoresis and displayed

  • used to show whether or not purification was effective

  • porous gel (polyacrylamide)

  • larger proteins move slower

  • dissolve in SDS to disrupt non-covalent interactions

  • add beta-mercaptoethanol or dithiothreitol to reduce disulfide bonds

  • different from gel filtration because of electric field so all move through same matrix

  • after electrophoresis, can stain with silver nitrate or coomassie blue

  • as purification goes on, there will be less bands and one band should be more prominent

Isoelectric focusing

  • isoelectric point

    • pH at which the protein’s net charge is 0

    • has zero electrophoretic mobility

    • different for proteins

  • mix of proteins → electrophoresis in pH gradient (no SDS)

  • separates proteins based on isoelectric point

2D electrophoresis

  • isoelectric focusing combined with SDS-PAGE

  • isoelectric first

A protein purification scheme can be quantitatively evaluated

  • monitor each step of purification

  • total protein

    • protein concentration of fraction x total volume

  • total activity

    • enzyme activity in fraction x total volume

  • specific activity

    • total activity/total protein

  • yield

    • activity retained after each purification step (percent of activity in crude extract)

  • purification level

    • measure of increase in purity

    • specific activity after each step/initial specific activity

  • good purification scheme looks at both purification and yield

ultracentrifugation is valuable for separating biomolecules and determining their masses

  • centrifugation also useful for analysis of physical properties of biomolecules

    • mass and density, shape, interactions

  • need math description of how particle behaves with centrifugal force

    • sedimentation coefficient

      • mass, partial specific volume, density, frictional coefficient

      • expressed in Svedberg units

      • smaller the S, more slowly it moves in centrifugal field

    • S velocity depends partially on mass

      • bigger sediments faster

    • S velocity impacted by shape

      • elongated particles sediment slower than spherical ones

    • S velocity impacted by density

      • more dense particle moves faster

      • density of solution also affects it

  • zonal/band/gradient centrifugation separates proteins with different sedimentation coefficients

    • first form density gradient in centrifuge tube

      • mix high and low density solution to make linear gradient of sucrose concentration (denser at bottom)

    • put proteins on top of density gradient

    • proteins move through gradient and separate according to sedimentation coefficients

      • time and speed of centrifuge determined empirically

    • bands can be harvested by making hole in bottom of tube and collecting drops

  • protein mass can be determined by sedimentation equilibrium

  • sedimentation equilibrium

    • sample centrifuged at low speed

    • concentration gradient formed

    • counterbalanced by diffusion of sample from high to low conc.

    • when equilibrium reached, gradient solely based on mass

    • very accurate

    • doesn’t denature

    • native quaternary structure preserved

Protein purification can be made easier with use of recombinant DNA technology

  • used to only be able to use native proteins

    • took 10lbs of beef pancreas to get 1g of deoxyribonuclease

  • advantages with technology

    • large quantitates

      • purification can start with a homogenate enriched with desired molecule

      • protein can be easily obtained

    • affinity tags can be fused to proteins

    • can generate variants of native protein sequence

3.2 Immunology Provides Important Techniques with which to Investigate Protein

  • purification removes protein from context

  • antibodies help tag protein so it can be isolated, quantified, or visualized

antibodies to specific proteins can be generated

  • antibody (Ig)

    • protein synthesized against antigen

    • high affinity for antigens

    • recognizes epitope

  • polyclonal antibody

    • give rabbit protein, wait for it to make antibodies, take its blood

    • take all antibodies

    • derived from multiple antibody-producing cell populations

    • antibodies for multiple antigens on the protein

monoclonal antibodies with virtually any desired specificity can be readily prepared

  • immortal cell lines

    • myeloma

  • fuse antibody producing cell with immortal cell line

    • hybridoma

  • screen for specific antibody

  • can be used as precise analytical and preparative reagents

    • tags

    • affinity columns

proteins can be detected and quantified by using an enzyme-linked immunosorbent assay (ELISA)

  • uses enzyme that reacts to produce colored product

  • enzyme linked to antibody that recognizes target antigen

  • antigen present: binds, creates color

  • can use either polyclonal or monoclonal antibodies, but monoclonal is more accurate

  • indirect ELISA

    • detects presence of antibody

    • ex. test for HIV

    • quantitative

  • sandwich ELISA

    • detects antigen

    • antibody put on bottom

    • antigen added, binds to antibody

    • secondary antibody (enzyme-linked) added

western blotting permits the detection of proteins separated by gel electrophoresis

  • immunoassay technique

  • first SDS-PAGE

  • polymer sheet pressed against gel, transfering resolved proteins on gel to sheet

  • antibody for protein of interest (primary antibody) added

  • secondary antibody added

    • usually has enzyme that produces color

  • makes it possible to find protein in a complex mixture

  • basis for hep C testing

  • useful in monitoring protein purification and gene cloning

Co-immunoprecipitation enables the identification of binding partners of a protein

  • sample of interest incubated with specific antibody

  • agarose beads with antibody-binding protein added

  • protein recognizes antibody

  • antibody now bound to beads

  • centrifugation → antibody on beads aggregates at bottom of tube

  • analysis by SDS-PAGE enables identification of binding partners

Fluorescent markers make the visualization of proteins in the cell possible

  • shows proteins in bio context

  • fluorescence labeled antibodies

  • fluorescence microscopy

  • shows location of protein

  • location helps determine function

  • better resolution with electron microscopy

3.3 Mess Spectrometry is a Powerful Technique for the Identification of Peptides and Proteins

  • gives measurement of molecule without prior knowledge of its identity

  • highly accurate and sensitive detection of mass

  • used to determine identity and chemical state

  • convert to gaseous, charged forms

  • ratio of mass to charge measured

  • ion source

    • conversion into gas-phase ions

    • MALDI and ESI make it so proteins can be ionized

    • MALDI

      • analyte evaporated in presence of volatile, aromatic compound that can absorb light at specific wavelength

      • laser excites and vaporizes matrix

      • gaseous collisions enable intermolecular transfer of charge

    • ESI

      • analyte passed through electrically charged nozzle

      • charged droplets emerge into low pressure chamber, evaporating

  • mass analyzers

    • distinguishes based on mass-to-charge ratios

    • Time of flight mass analyzes (TOF)

      • ions are accelerated through elongated chamber under fized electrostic potential

      • mass determined by time required for each ion to pass through chamber

peptides can be sequenced by mass spectrometry

  • old methods

    • Edman degradation

      • N-terminal labeled with phenyl isothiocyanate

      • cleavage yields derivative, which can be identified by spec methods

      • surpassed by mass spec

  • fragments (product ions) can be passed through second mass analyzer fro further mass characterization

  • tandem mass spectrometry

    • 2 mass analyzers

  • product ion framgents formed in ways that give clues to aa sequence of precursor ion

proteins can be specifically cleaved into small peptides to facilitate analysis

  • Edman limited to 50 aa peptides

  • sequencing long peptides by mass spec is hard to interpret

  • cleave protein into smaller peptides to sequence easier

  • cleavage can be done by chemical reagents or proteolytic enzymes

    • chemical reagents

      • cyanogen bromine (carboxyl side of methionine residues)

      • O-Iodosobenzoate (carboxyl side of tryptophan residues)

      • hydroxylamine (asparagine-glycine bonds)

      • 2-nitro-5-thiocyanobenzoate (amino side of cysteine residues)

    • proteolytic enzymes

      • trypsin (carboxyl side of lysine and arginine residues)

      • clostripain (carboxyl side of arginine residues)

      • staphylococcal protease (carboxyl side of aspartate and glutamate residues, glutamate only under certain conditions)

      • thrombin (carboxyl side of arginine)

      • chymotrypsin (carboxyl side of tyrosine, tryptophan, phenylalanine, leucine, and methionine)

      • carboxypeptidase A (amino side of C-terminal amino acid, not arginine, lysine, or proline)

    • sequence specific methods

      • disrupt protein backbone at particular aa residue

  • peptides obtained separated by chromatography

  • aa sequence known, segment order unknown

  • overlap peptides

    • second enzyme used to split polypeptide at different linkages

    • find order of peptides

  • more steps needed if initial protein has several polypeptide chains

    • denature to break chains apart

Genomic and proteomic methods are complementary

  • for proteins with more than 1000 aa’s, DNA tech is often more efficient

  • still need to work with proteins

    • post translational modifications

The amino acid sequence of a protein provides valuable information

  • sequence can be compared with other sequences

  • can give info on evolutionary pathways

  • can show internal repeats

  • can see signal sequences

  • basis for preparing antibodies

  • DNA probes

Individual proteins can be identified by mass spec

  • mass spec with chromatographic and peptide cleavage techniques → highly sensitive protein identification

  • peptide mass fingerprinting

    • protein cleavage followed by chromatographic separation and mass spec

    • rapid identification and quantitation

  • identification of nuclear pore complex from yeast

3.4 Peptides can be Synthesized by Automated Solid-Phase Methods

  • synthetic peptides can serve as antigens to make antibodies

  • can be used to isolate receptors

  • used as drugs

    • diabetics don’t have enough vasopressin

  • help define rules of 3D proteins

  • how → solid phase method

    • amino group of linked to carboxyl group of another

    • product only favorable if a single a group and c group available

    • block some groups and activate others

    • carboxyl terminal aa attached to insoluble resin

    • amino group blocked with protecting group (ex. t-Boc)

    • t-Boc removed

    • next amino acid (with t-Boc) and DCC added together

    • only c group of new aa and a group of first aa free to form bond

    • DCC reacts with c group of incoming aa

    • after bond formed, excess reagents washed away

    • repeat for more aa’s

    • at the end, use HF to release from beads

    • remove protecting groups from side chains

  • solid phase method

    • desired product at each stage is bound to beads

    • no need to purify intermediates

3.5 3D Protein Structure can be Determined by X-ray Crystallography, NMR Spectroscopy, and Cryo-Electron Microscopy

  • knowing 3D structure is important

    • predict mechanism of action

    • predict effects of mutations

    • predicts desired features of drugs

X-ray crystallography reveals 3D structure in atomic detail

  • developed to determine protein structure in atomic detail

  • provides clearest visualization

  • x-rays best resolution because wavelength corresponds to length of covalent bond

  • protein crystal

    • protein needs to be in crystal form

    • fixed, repeated arrangement with respect to one another

    • slowly add ammonium sulfate (or another salt) to protein to reduce solubility

      • salting out

    • challenging

    • highly pure material required

  • source of x-rays

    • beam of x-rays 1.54 A produced by accelerating electrons against copper target

    • synchrotron radiation

      • acceleration of electrons in circular orbits at speeds close to speed of light

      • more intense

      • high quality data from small crystals over shorter exposure time

  • detector

    • x-ray fild or solid-state electronic detector

  • electrons scatter x-rays

  • scattered waves recombine

    • in or out of phase

  • how they recombine depends on atomic arrangement

  • intensities and positions of reflections is data

  • reconstruct image from reflections

  • Fourier transform

    • math relation used to form image from x-rays

  • electron-density map

    • obtained image from Fourier transform

Nuclear magnetic resonance spectroscopy can reveal the structures of proteins in solution

  • not all proteins crystallize easily

  • NMR reveals atomic structure in solution

  • need highly concentrated solution

  • depends on how certain atomic nuclei are intrinsically magnetic

  • limited number of isotopes have spin

    • ex. H does

    • see table

  • examine chemical surroundings of H nucleus

  • chemical shifts

    • different frequencies

  • NOESY graphically displays pairs of protons in close proximity

  • detect location of atoms relative to one another

  • can get family of related structures

    • not enough constraints may be accessible

    • NOESY only approximate

    • structures may be slightly different at any given moment

Cryo-electron microscopy is an emerging method of protein structure determination

  • thin layer of protein put on fine grid and then frozen, trapping molecules

  • sample put in transmission electron microscope under vacuum conditions and exposed to incident electron beam

  • gives 2D image

  • computers generate 3D image