Biochem Exam 2

preparative approaches

result in protein to "work with"

analytical approaches

reveal something about a protein

what physical properties do both preparative and analytical approaches make use of?

binding, charge, hydrophobicity, size, solubility, and density

What does PAGE stand for, what does it do, how are we able to see them, and what does it form?

Polyacrylamide Gel Electrophoresis (remember this)

monitor protein migration through a gel from the cathode to anode as current is applied

proteins seen via stain

forms fibers (like a net), so small things travel fast while large things get stuck

what charge do anodes have?

positive (+)

what charge do cathodes have?

negative (-)

Native PAGE

Protein(s) remain folded

Size, charge and shape

Can't estimate molecular weight (MW can’t be estimated)

fibular proteins are slow while globular proteins are fast

SDS PAGE

proteins are denatured (unfolded) and uniformly coded with negative charge, so:

oligomers are separated into monomers because they are denatured

proteins are separated mostly by size

NOT charge and shape

heat is applied

steps of SDS PAGE

1. denature sample with sodium dodecylsulfate

2. proteins placed on gel and electric field is applied

3. proteins are stained to visualize separated bands

a plot of protein mobility vs. log of MW can be used to determine what? (not the plot)

the apparent molecular weight of an unknown protein

A plot of protein mobility vs log of MW can create what plot?

Plot of log MW vs distance migrated

What happens if an oligomeric protein is separated on a SDS page?(e.g Homotetramer of 80kDa)

It will dissociate and run as a 20k monomer

is SDS-PAGE preparative or analytical?

both, but mainly preperative

about how big is transglutaminase?

Approximately 100 kDa

centrifugation is also known as what?

differential- not at equilibrium

what occurs in centrifugation?

- particles PELLET from solution at different speeds

- large and denser particles sediment faster (even if there was no centrifugation)

what is centrifugation used for?

- harvesting cells (1 spin)

- separating organelles (series of spins aka cell fractionation)

cell fractionation does what and what are the steps

shearing forces

1. tissue-sucrose homogenate (tube is moved up and down as pestle rotates to mince it + 0.25M sucrose buffer)

2. Strained homogenate (remove connective tissue and blood vessels)

3. Centrifuge homogenate (600g * 10min) (Nuclei and unbroken cells at the bottom of tube)

4. Centrifuge supernatant 1 (15,00g*5min) (Mitochondria, lysosomes, and microbodies at bottom of tube)

5. Centrifuge supernatant 2 (100,000g*60min) (Soluble fractions of cytoplasm in supernatant) (ribosomes and microsomes at bottom of tube)

How is protein solubility influenced?

By pH and ionic strength

Where are proteins least and most souble?

Least at their pI and most away from pI when they are charged?

salting in

when small amounts of salt is added to buffer charges

what do low salt concentrations do?

improve solubility of charged proteins due to reduced charge-charge interactions

salting out

initial purification

what do high salt concentrations do?

reduces solubility as salt competes for water which is required to solvate proteins

why is ammonium sulfate used?

it's soluble at 100%

- adding the right amount can precipitate the molecule of interest

S100 fraction and S100 proteins refer to

solubility in 100% ammonium sulfate at neutral pH

what does chromatography refer to?

separation techniques (via paper, gas, or liquid)

What does phases does Liquid Chromatography and what is the monitor absorbance?

• Colum has mobile (liquid) and stationary phase (resin)

• 280 nm

absorbance at 280 nm is

the UV absorbance of tryptophan (W)

stationary phase

- hydrated polymers are present

- mechanically stable due to high pressures

- chemically inert (don't interact with proteins)

- cheap

- physical support (can attach chemical groups for separation)

e.g. amylose, cellulose, agarose

what are the types of chromatography to remember?

- IEX (ion exchange- separates by charge)

- HIC (hydrophobic induced chromatography)

- affinity

- SEC (size exclusion chromatography)

in IEX, what occurs in the anion exchanger?

Has positive charge on resin so negative charge molecules will bind

in IEX, what occurs in the cation exchanger?

Has negative charged groups on resin so positive charged molecules will bind

the higher the charge on the molecule,

the stronger the binding

in IEX, interactions that are stronger require

more salt to compete

What do we have to consider for IEX?

pKa of exchangers

What exchanger works below pKA

Anion exchanger (DEAE and Q)

What exchanger works above pKA

Cation exchanger (CM and S)

chromatogram

graph of chromatography

in IEX, after salt is loaded in, what is used to elute and what does it elute? What is separated based on?

• a gradient of increasing salt

• (NaCl, and KCl)

• this shakes the salt off so it doesn't "compete" with the resin

• Separation based on charge so stronger interactions = more salt

what occurs in HIC?

- hydrophobic interactions

- hydrophobic groups are covalently linked to polar resin

- reverse of |EX- load in high salt and use gradient of DECREASING salt to elute

Affinity Chromatography

• Resin carries ligand that binds target protein

• High specificity so >95% purity in a single step

• elute with a solution that disrupts the protein-ligand interaction typically an excess of competing ligant

• Eg: GST tag and GSH, Hig tag and Ni-NTA

as water increases

salt decreases causing proteins to fold back

what metal does affinity use?

nickel (Ni2+)

what does resin use to coordinate nickel?

nitrilotriacetic acid (NTA)- this interacts with histidine

how many histidine residues do proteins usually have?

6-8 consecutively

how does histidine elute proteins?

by adding imidazole

how do molecules separate in SEC?

on their own

void

molecules that never entered the column

elution

molecules separated based on size

Dialysis- where are the solutes and what are the sizes of membrane pores?

• Diffusible solutes in the dialysis bag equilibrate across the membrane

• Membrane pores should be smaller than the size of the protein

Ultrafiltration- how are chamber separated and what does centrifugation do?

• Upper and lower chambers are separated by a semipermeable membrane

• Centrifugation or vacuum is used to force water and small solutes through the membrane, leaving concentrated target protein in the upper chamber

Multi-step Purification

• Typical protein purification uses a series of separation methods

• (note Dramatic increase in the activity of the enzyme per mg of protein though a series of five different purification procedures)

as more protein purification occurs,

less percentage is recovered

Fred Sanger

- first to sequence a protein

-insulin-1953

- figured out how to sequence DNA

- showed how all molecules of a protein have a fixed amino acid composition

- only person to ever receive 2 Nobel prizes in chemistry

steps of protein sequencing

1. separate non-covalently linked chains

2. break and block disulfide bonds

3. cleave proteins with either proteases or chemicals (using i sequence specie proteases and/or ii chemicals)

4. isolate and separate peptides

5. sequence peptides form N (Edman degradation) and C (Carboxypeptidases) termini

6. repeat steps 3-5 with different cleavage (until good overlap between fragments)

7. assemble full sequence

how are non-covalently linked chains separated?

- high salt

- chaotropes like 8 M urea or 4-6 M guanidine-HCl

- reduce the hydrophobic effect by disrupting water, allowing buried hydrophobic residues to be soluble (exposed)

how are disulfide bonds broken?

reducing agents and modifiers

e.g. TCEP, β-mercaptoethanol, or dithiothreitol (DTT)

What do modifiers do to disulfides?

Prevent them from reforming

how are proteins cleaved and what are the 5 example proteases?

Using (i) sequence specific proteases and/or (ii) chemicals

Trypsin- C-term side of basic AAs (Arg or Lys)

V8 proteases- after acid AAs (Glu and Asp in some phosphate)

chymotrypsin- after aromatic AAs (Phe or Tyr)

pepsin

thermolysin- before hydrophobic AAs (Leu, Ile, Val)

Chemical Cleavage- what does it do

• Acts on Substrate in methionine residues to cleave C-term peptide bond

• Useful as proteins have few methoinine residues

• One product as C-term homoserine lactone where the met once was

where does hydroxylamine act and what does it do?

between asparagine and glycine

chemical cleavage

where does cyanogen bromide act and what does it do?

- on sulfur in methionine residues so they can cleave the C-terminal peptide bond

- proteins have a few methionine residues

Chemical cleavage

what's typically used to isolate and separate peptides?

reverse phase liquid chromatography

what occurs in reverse phase liquid chromatography?

- resin contains hydrocarbon chains of differing lengths

- longer chain = more hydrophobic

- polar solution (water) loaded and eluted with gradient of non-polar solution (Organic solvent like acetonitrile)

- separated based on hydrophobicity

- non polar solutions denature proteins, so more widely applied to peptides

Edman degradation

the Edman reagent, phenylisothiocyanate, reacts with the α-amino group to produce PTH derivative with a UV absorbance of 254 nm

- this is less efficient with larger proteins (too slow)

• immobilize peptide on matrix via C-term

• Cycle 1 identifies N-term residues and so forth

How are amino acids separated

Gradient separation of common PTH-amino acids by reverse phase chromatography

Areas under peaks are proportional to moles of each amino acid

how are the full sequences assembled?

they will check for an overlap in two sets of fragments

what was protein sequencing replaced with?

mass spectrometry

- peptide mass fingerprinting

- peptide sequencing using tandem MS

what occurs in mass spectrometry?

- separates molecules based on mass to charge ratios

- detects charge or ionized molecules in the gas phase

- mostly protonation

- weak or soft ionization maintains covalent bonds

- uncharged molecules NOT detected

source of mass spectrometry

evaporate and ionize molecules in a vacuum to create gas phase ions (ESI or MALDI)

mass analyzer of mass spectrometry does what

separate ions based on mass to charge ratios

detector of mass spectrometry

measure the amount of ions with specific mass to charge ratios

Electrospray ionization (ESI)

- solution sprayed with fine droplets from glass capilarry under a strong electric field

- liquid chromatography (LC-MS)

Matrix-Assisted Laser Desorption Ionization (MALDI)

- matrix is a light-absorbing substance that is excitable by a laser

- sample mixed with matrix and dried on a surface

- lasers ionize the matrix, facilitating the transfer of a proton

- not inline with chromatography (Off-line)

-Most molecules pick up only single H+( Limited mass range)

How is an Ion’s mass to charge ratio determined

Time of flight

Ions accelerated by electric field of known strength

Time of flight (TOF) analyzere

- ions accelerated by an electric field leading to all ions having the same kinetic energy

- velocity depends on the mass to charge ratio (heavier ions of same charge reach lower speeds, but ions with high charge also increase in velocity)

Peptide Mass fingerprinting (PMF)

identifying proteins based on the mass of tryptic peptides (except proline)

can predict tryptic peptides of all proteins in database

genomic and proteomic databases

steps of peptide mass fingerprinting

1. peptides generated by protein digestion (specific protease)

2. Peptide mases are determined by MALDI-MS or ESI-MS

3. masses calculated

4. ranking is calculated to measure fit between experimental and calculated masses

What are disadvantages to peptide mass fingerprinting?

Not all peptides detected

Convoluted by peptides with similar masses but different sequences

Convoluted by post-translation modifications

(We should also sequence peptides using Tandem Mass Spec)

Tandem Mass Spectrometry (MS/MS)

- two MS joined together

- first MS is a filter to choose which peptides or proteins need further analysis- no covalent bonds are broken

- second MS fragments the ion by collision with neutral gas (helium or argon)- covalent bonds broken in predictable way also called collision induced dissociation

-Gives full MS2 spectra for each MS1

what occurs in collision induced dissociation

- fragmentation occurs at the peptide bond

- produces y-ions (C-terminal charge) and b-ions (N-terminal charge)

- other fragments are likely neutral

- mass difference between adjacent ions will determine the AA

- proline is still difficult to cleave

- difficult to cleave over 15 AAs

- convoluted with post-translational modifications

- combined with peptide mass fingerprinting

Nature of Protein Sequences

• # of possible sequences is large

• Probability that 2 proteins will by chance have similar sequences is negligible

what does protein sequence similarity imply?

evolutionary relatedness

homologous

proteins with similar sequence

orthologous

proteins from different species (ancestral gene)

paralogous

proteins from the same species (gene duplication)

what are mutations?

changes at one or more sites in the amino acid sequence of a protein

what does it mean when the protein is conserved in sequence alignments?

they are the same in both sequences

What computer programs align sequences

BLAST

What are sequence alignment scored based on

Matching or similar amino acids

What do gaps in alignments do

Bring penalties

What are the two types of alignments

Global and local

what is the number of amino acid differences between two cytochrome c sequences proportional to?

phylogenetic difference between the species from which they are derived

- this observation can be used to build phylogenetic trees of proteins (molecular evolution)

What are numbers of cytochrome c sequences

Amino acid changes

protein function depends on _______ which depends on _________

structure, sequence

similar protein structure indicates common function all the time (true or false)

false

Proteins with different structures can carry out similar functions (true false)

True

what is a peptide bond?

2 amino acids bonded(linked) via dehydration

Partial double bond character is and has? What does NH and Odo?

• Coplaner O,C, and N

• Limited rotation

• Nh acts as a doner and O as acceptor