biochem chapter 3 - working with proteins

a protein is defined by its sequence of _____ _____

each cell type has a distinct set of _____

the entire complement of proteins is called the ______

amino acids

proteins

proteome

~____ protein-coding genes (HGP)

~____ have been detected in at least one tissue

~____ are present in all tissues

~____ are essential

~20,000 protein-coding genes (HGP)

~18,000 have been detected in at least one tissue

~10,000 are present in all tissues

~2,000 are essential

~20,000 proteins are ____ 

~18,000 have ____

~10,000 proteins are ____

~2,000 are ____

~20,000 protein-coding genes (HGP) TOTAL #

~18,000 have been detected in at least one tissue

~10,000 are present in all tissues

~2,000 are essential

Proteome is actually much larger than genome because of:

• ____

• ____

• ____

• post translational modifications

• synthesis intermediates or precursors

• alternative splicing

protein functionality is enhanced through _____ _____

ligand binding

lipoproteins

nonprotein part: ____

examples: ____

lipids

blood lipoprotein complexes, HDL, LDL

nucleoproteins

nonprotein part: ____

examples: ____

RNA/DNA

Ribosomes, chromosomes

glycoproteins

nonprotein part: ____

examples: ____

carbohydrate groups

immunoglobulins, LDL receptor

metalloproteins and metal-activated proteins

nonprotein part: ____

examples: ____

Ca+,K+,Fe2+,Zn2+,Co2+, others

Metabolic enzymes, kinases, phosphates, others

hemoproteins

nonprotein part: ____

examples: ____

heme group (carries O₂)

hemoglobin, cytochromes

flavoproteins

nonprotein part: ____

examples: ____

FMN, FAD

electron transfer enzymes

peptides are short polymers of amino acids. individual amino acid units are called ___.

residues

2 residues - dipeptide

3 residues – tripeptide

12-20 residues – ____

>20 residues – ____

>50 residues – ____

oligopeptide

polypeptide

protein

protein sequences are unique and are conventionally read ____.

N → C (amino terminus→ carboxyl terminus)

preparative approaches result in protein to ____.

analytical approaches ____.

preparative approaches result in protein to “work with”.

analytical approaches reveal something about the protein.

what protein properties do both preparative and analytical proteins make use of?

• binding

• charge

• hydrophobicity

• size

• solubility

• density

the 3 steps for purifying proteins is 

first step = break open tissue or microbial cells

      –  ____ = releases proteins in solution

second step = ____ = separate proteins into fractions based on size or charge

     – “____” = lower solubility of proteins in salt to selectively precipitate proteins

third step = ____ = use semipermeable membrane to separate proteins from small solutes

1. crude extract

2. fractionation, salting out

3. dialysis

when purifying, what is the goal?

• highest purity with the least amount of steps

• more steps leads to less yield and more purity

centrifugation is what kind of process?

differential(non-equilibrium) - because its separated due to force

during centrifugation, particles ____ from solution at different speeds.

• ______ particles sediment faster.

PELLET

larger and denser

centrifugation is used for: (2)

1. harvesting cells(one spin)

2. separating organelles using a series of spins or cell fractionation

protein solubility is influenced by ___ and ___

pH, ionic strength

proteins are least soluble when?

when are they the most soluble?

least - at their pI (neutral)

most - away from pI, when charged

salting in vs salting out on solubility

salting in - low salt concentrations improve solubility 

salting out - high concentration decrease solubility

during ___ ____adding the right amount of salt(___ ___) can precipitate contaminants or molecules of interest.

salting out ; ammonium sulfate

S100 fraction and S100 proteins are _____.

solubility in 100% ammonium sulfate at neutral pH

during ____ molecules separate based on their interactions with the stationary phase

liquid chromatography

during liquid chromatography a column has a ____ phase(liquid) and a ____ phase (resin).

molar absorbance at  ____

during liquid chromatography a column has a mobile phase and a stationary phase (resin).

molar absorbance at 280 nm

stationary phase of liquid chromatography

are made up of ____

they are mechanically ____ (high pressures)

chemically ____

____

____ support for chemical groups

are made up of hydrated polymers (polysaccharides)

they are mechanically stable (high pressures)

chemically inert (don’t react with sample components)

cheap 

physical support for chemical groups

what are different stationary phases made of? (3 examples)

• agarose

• amylose

• cellulose

column chromatography has 2 steps:

• First step: The buffered solution (mobile phase) migrates through the porous solid material (stationary phase) to equilibrate the column.

• Second step: The buffered solution containing the protein mixture is added and migrates through the solid phase, allowing separation based on each protein’s interactions with the stationary phase.

protein properties affect _____ of column chromatography

migration rate

____ separates based on sign and magnitude of the net electric charge

ion-exchange chromatography (IEX)

during IEX, pH and concentration of free salt ions affect ____

protein affinity

IEX involves

• cation exchangers → ____ charged resin binds ____ charged proteins.

• anion exchangers → ____ charged resin binds ____ charged proteins.

• cation exchangers → negatively charged resin binds positively charged proteins.

• anion exchangers → positively charged resin binds negatively charged proteins.

during IEX, proteins are loaded in ___, which means there is low salt, and use gradient of ___ salt to elute(release from column)

salt ex: NaCl, KCl

low

increasing

what does a chromatogram represent in IEX and how is it interpreted?

• A chromatogram is a graph showing absorbance at 280 nm (y-axis) versus elution volume or time (x-axis).

• Peaks correspond to proteins or other molecules eluting from the column.

• Early peaks = weakly bound or unbound proteins (elute at low salt).

• Later peaks = strongly bound proteins (require higher salt to elute).

• The salt gradient (NaCl or KCl) is often shown on a secondary axis, rising as elution progresses.

what are other names for size-exclusion chromatography(SEC)?

also called gel filtration chromatography

what’s the order of protein emergence during SEC?

large proteins emerge from the column before small proteins

• small proteins have to go through the beads but the big ones just pass freely around beads

which molecules are voided during SEC?

the larger macromolecule is is voided because it never actually travelled through the resin

affinity chromatography separates based on ___ affinity

binding

affinity chromatography properties

• resin carries ligands that bind the target protein

• high specificity! >95% purity in a single step

• to isolate the target protein: elute with a solution that disrupts the protein-ligand interaction, typically an excess of competing ligand

examples of affinity chromatography:

1. GST tag binds GSH

2. His tag binds Ni-NTA

how does Ni²⁺ affinity chromatography purify His-tagged proteins?

• The resin contains nitrilotriacetic acid (NTA) groups that coordinate(bonds) Ni²⁺ ions.

• The target protein is engineered with 6–8 histidine residues (His-tag).

• The imidazole rings of histidines bind to the Ni²⁺ on the resin.

• After washing away unbound proteins, the His-tagged protein is eluted by adding imidazole, which competes with histidines for Ni²⁺ binding sites.

how does hydrophobic interaction chromatography (HIC) work?

• Hydrophobic groups covalently-linked to polar resin (HIC)

• Reverse of salting out

• Load in high [salt] and use gradient of decreasing [salt] to elute

hydrophobic proteins don’t like water, so high salt → high solubility

where in the cell are most hydrophobic proteins?

the inner cell membrane, so lots of transmembrane proteins are purified by HIC

what are the principles of dialysis

• diffusible solutes in the dialysis bag equilibrate across the membrane

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

what are the principles of ultrafiltration

• Upper and lower chambers 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

is dialysis or ultrafiltration faster?

ultrafiltration is faster than dialysis

• because dialysis is passive diffusion while ultrafiltration is force driven by pressure of a vacuum actively pushing water and small solutes

sequential purification steps increase/decrease sample size

decrease

how to calculate purification factor?

purification factor = (final specific activity) / (starting specific activity)

• final specific activity: after a purification step

• starting specific activity: in the crude extract (or previous step if cumulative)

• meaning: How many times purer the protein is compared to the crude sample

how to calculate percent yield

%final activity / %starting activity activity = percent yield

___ visualizes and characterizes purified proteins

electrophoresis

what can electrophoresis be used to estimate?

• number of different proteins in a mixture

• degree of purity

• isoelectric point (pI)

• approximate molecular weight

what is beer lambert’s law and what is it used to determine?

• used to determine protein concentration

• A = εcl

  • A=absorbance at 280nm

  • ε=extinction coefficient at 280nm (will be given)

  • c=concentration

  • l=path length

What are the 2 types of Electrophoresis?

1. Native PAGE

2. SDS-PAGE

What are the characteristics of Native PAGE?

• Proteins remain folded and functional

• Migration determined by size, charge, and shape

• Cannot reliably estimate molecular weight, because shape and charge effect mobility, not just weight

What are the characteristics of PAGE?

• General method to separate proteins using an electric field through a polyacrylamide gel.

• Proteins migrate from cathode (-) to anode (+). 

• ‘See’ proteins by using a stain

how does electrophoresis separate and visualize proteins?

• Uses cross-linked polyacrylamide gels (PAGE).

• Proteins migrate based on their charge-to-mass ratio under an electric field.

• Migration is from cathode (-) to anode (+).

• Proteins are visualized using Coomassie blue dye, which binds to proteins.

what is SDS and why is it important in SDS-PAGE?

sodium dodecyl sulfate (SDS) = a detergent

• binds and partially unfolds proteins

• gives all proteins a similar charge-to-mass ratio

• electrophoresis in the presence of SDS separates proteins by molecular weight

  • because same MW but diff charge travel differently, once all charges are same(negative), move only based off of MW

• smaller proteins migrate more rapidly

how to calculate electrophoretic mobility(μ)?

μ = V/E = Z/f

μ = electrophoretic mobility

V = velocity

E = electrical potential

Z = net charge

f = frictional coefficient

how to estimate the molecular weight of a protein?

plot of log M_r of marker proteins vs relative migration during electrophoresis = linear

what happens if an oligomeric proteins is separated on a SDS page (e.g. homotetramer of 80kDa)?

it will dissociate and run as a 20kDa monomer

using ____ to determine the pI of a protein 

isoelectric focusing

**not used anymore

two-dimensional electrophoresis characteristics

• permits resolution of complex protein mixtures of proteins

• more sensitive than individual methods

hemoglobin is a ____ - it has two alpha chains and two beta chains

heterotetramer

what is protein databank and pymol?

• Protein Data Bank (PDB):

  • A public database of experimentally determined 3D structures of proteins, nucleic acids, and complexes.

• PyMOL:

  • A molecular visualization software used to view and manipulate 3D protein structures..

what are the 2 general shape classifications for proteins

1. fibrous

2. globular

fibrous vs globular protein shapes

• fibrous proteins have an extended structure and are structural proteins like collagen, elastin, and keratin

• globular proteins are sphere-like in structure. most cytosolic enzymes have this shape. 

  • because cytosol is packed and globular proteins take up less space than fibrous proteins

protein classification by solubility

• soluble proteins:

  • hydrophilic residues on the surface, hydrophobic residues buried inside

  • easily dissolved in aqueous buffers

• insoluble proteins (e.g., membrane proteins):

  • hydrophobic residues exposed on the surface

  • soluble in detergents

what are the four levels of structure in proteins?

1. primary structure - amino acids linked by covalent bonds in a polypeptide chain

2. secondary structure - recurring structural patterns like alpha or beta helices

3. tertiary structure - 3D folding of polypeptide

4. quaternary structure - 2+ polypeptide subunits

what are the two principal secondary structure found in proteins?

α-helix

β-pleated strand

sequencing of the first protein

• fred sanger was the first to sequence a protein

• insulin - 1953

• he went on to figure out how to sequence DNA

• showed that all molecules of a protein have a fixed amino acid composition

what method is used to sequence proteins today?

mass spectrometry

what are the steps of protein sequencing?

1. separate non-covalently linked chains

2. break and block disulfide bonds

3. cleave proteins (fragmentation) into peptides using sequence specific proteases or chemicals

4. isolate and separate peptides

5. sequence peptides from N-terminus and C-terminus

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

7. assemble full sequence

edman degradation and carboxypeptidases in protein sequencing?

Edman Degradation: sequencing from N-terminal → C-terminal

Carboxypeptidases: sequencing from C-terminal → N-terminal

how do we separate non-covalently linked chains? (1st step of protein sequencing)

Overcome weak forces using:

• high salt (ammonium sulfate)

• chaotropes like 8 M Urea or 4-6 M Guanidine-HCl

• interfere with H-bonds and reduce “the hydrophobic effect” a.k.a they disrupt water and allow otherwise buried hydrophobic residues to be soluble and thus exposed

how do we break and block disulfide bonds linked chains? (2nd step of protein sequencing)

to break:

• use reducing agents and then modifiers

• in the lab prevent with reducing agents such as TCEP, β-

  mercaptoethanol and dithiothreitol (DTT)

to block from reforming:

• because reaction between the newly reduced –SH groups to reestablish disulfide bonds is a likelihood, S-S reduction must be followed by –SH modification

  modifiers prevent disulfides re-forming!!!

how do we cleave proteins into peptides w/proteases and chemicals? (3rd step of protein sequencing)

Protease	Cleavage Specificity	Notes
Trypsin	After Arg (R) or Lys (K)	Cleaves on the C-terminal side of basic residues
V8 Protease (Glu-C)	After Glu (E) in ammonium buffer; sometimes after Asp (D) in phosphate buffer	C-terminal cleavage
Chymotrypsin	After Phe (F) or Tyr (Y) (aromatic residues)	C-terminal cleavage
Pepsin	Preferentially cleaves aromatic and acidic residues in acidic pH	Works under low pH
Thermolysin	Cleaves before Leu, Ile, Val, and other hydrophobic residues	N-terminal cleavage; prefers hydrophobic residues

proteases = catalyze ____ of peptide bonds

proteases = catalyze hydrolytic cleavage of peptide bonds

___ immobilizes peptide on matrix via C-terminus, leaving the n-terminal free to react.

edman degradation

edman degradation

cycle 1 - identifies ____

cycle 2 - identifies ____

less efficient with ___

• can do 50 cycles for a 100-200 aa protein

• can do 10-20 cycles for a 2000 aa protein

cycle 1 - identifies N-terminal residues

cycle 2 - identifies N+1 residue

less efficient with large proteins

• can do 50 cycles for a 100-200 aa protein

• can do 10-20 cycles for a 2000 aa protein

reverse phase chromatography

• resin contains hydrocarbon chains of ____ ____(C4, C8, C12)

• longer chain is more ____

• load in ____ solution (water) and elute with gradient of ____ solution (organic solvent such as acetonitrile)

• separation based on ____

• non-polar solutions often ____ proteins so more widely applied to ____

reverse phase chromatography

• resin contains hydrocarbon chains of differing lengths (C4, C8, C12)

• longer chain is more hydrophobic

• load in polar solution (water) and elute with gradient of non-polar solution (organic solvent such as acetonitrile)

• separation based on hydrophobicity

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

the seven step protein sequencing steps have been replaced with _____: (2 types of it)

• _____

• _____

mass spectrometry approaches:

• peptide mass fingerprinting (with databases)

• peptide sequencing using tandem MS

_____ = measure molecular mass with high accuracy

• can sequence short amino acid sequences (20 to 30 amino acid residues)

• can document the entire cellular proteome

mass spectrometry

short

proteome

mass spectrometry

• separates molecules (in this case peptides) based on their ___

• detects ___ or ___ molecules in the ___ phase

• mostly ___

• cannot detect uncharged molecules

• ___ or ___ ___ maintains covalent bonds

• separates molecules (in this case peptides) based on their mass-to-charge ratio (m/z)

• detects charged or ionized molecules in the gas phase

• mostly protonation

• cannot detect uncharged molecules

• weak or soft ionization maintains covalent bonds

what are the 3 components of MS?

source: evaporate and ionize molecules in a vacuum to create gas phase ions e.g. ESI or MALDI

mass analyzer: Separate ions in space or time based on their m/z ratios

detector: measure the amount of ions with specific m/z ratios

source: ESI - ___

• solution sample is sprayed as fine droplets from a glass capillary under a ___ ___ ___

• often inline with ___ ___ ___

Electrospray Ionization

strong electric field

liquid chromatography (LC-MS)

what does an ESI spectrum look like?

x-axis: m/z units

y-axis: relative intensity

each peak are different charge states

• tallest peak is the base peak

what is the base peak on an ESI spectrum?

• The base peak is the tallest peak in a mass spectrum.

• Represents the most abundant ion detected in the sample.

• Its intensity is set to 100%, and all other peaks are measured relative to it.

• Does not necessarily correspond to the molecular ion (the intact molecule); it could be a fragment or adduct that is most easily detected.

what is the second type of source for MS?

MALDI (matrix-assisted laser desorption ionization)

characteristics of MALDI

• the matrix is a light absorbing substance that is excitable by a laser

• sample is mixed with a matrix and dried on a surface

• lasers ionize the matrix and facilitate the transfer of a proton

• not inline with chromatography (off-line)

• most molecules pick up only a single H+

• limited mass range

what is the analyzer for MS?

TOF (time of flight) analyzer

TOF -

• ion's mass-to-charge ratio is determined via a____. Ions are accelerated by an _____ of known strength.

• acceleration results in an ion having the same ___ as any other ion that has the same charge.

• the velocity depends on the _____ (heavier ions of the same charge reach lower speeds, although ions with higher charge will also increase in velocity).

• ion's mass-to-charge ratio is determined via a time of flight measurement. Ions are accelerated by an electric field of known strength.

• acceleration results in an ion having the same kinetic energy as any other ion that has the same charge.

• the velocity depends on the mass-to-charge ratio (heavier ions of the same charge reach lower speeds, although ions with higher charge will also increase in velocity).

Peptide Mass Fingerprinting

• identification of a protein based off the mass of its ____

• can predict tryptic peptides of all proteins in a ____

• ___ and ___ databases

• identification of a protein based off the mass of its tryptic peptides

• can predict tryptic peptides of all proteins in a database

• genomic and proteomic databases

peptide mass fingerprinting

1) Peptides are generated by ___ ___ using a specific protease

2) Peptide masses are determined using ___ or ___

3) ___ ___ ___ are calculated for each entry in the protein database using the same protease

4) A ___ is calculated to measure the fit between experimentally derived and calculated peptide masses

1) Peptides are generated by protein digestion using a specific protease

2) Peptide masses are determined using MALDI-MS or ESI-MS

3) Theoretical peptide masses are calculated for each entry in the protein database using the same protease

4) A ranking is calculated to measure the fit between experimentally derived and calculated peptide masses

T/F all peptides can be detected by MS

False

Why can’t all peptides be detected by MS?

• Convoluted by peptides with similar masses but different sequences

• Convoluted by post-translational modifications