Biol 3461 Exam 1

what is a protein and what do cell types have to do with them

sequence of amino acids, each cell type has a distinct set of proteins

what is a proteome

entire complement set of proteins

why is the proteome actually larger?

- post-translational modifications

- synthesis intermediates or precursors

- alternative splicing

what determines a protein's structure?

sequence of amino acids

how is the human proteome classified?

by function

how is protein functionality enhanced?

ligand binding

- protein-protein interactions

- protein-DNA interactions

- protein co-factors (metals and coenzymes)

peptides are what and what is each unit called

short polymers of amino acids. Each unit is called a residue

12-20 residues

oligopeptide

over 20 residues

polypeptide

over 50 residues

protein

what is a sequence and where does it read from

- unique characteristic of every protein

- reads from N terminus to C terminus

hemoglobin is a what and as what sort of chains

heterotetramer- two alpha chains and two beta chains

protein shapes NOT STRUCTURE

fibrous and globular

fibrous shape

extended structure

e.g. collagen, elastin, keratin

globular shape

sphere-like structure

e.g. most cytosolic enzymes (3D)

soluble protein

most hydrophobic amino acids are on the inside

insoluble protein made up of

hydrophobic amino acids are on the outside, soluble in detergents

what are the protein structures

primary, secondary, tertiary, quaternary

primary structure

sequence of amino acids

secondary structure

local regular structures (folding of primary sequences) helices and sheets

tertiary structure

3D shape as secondary structures pack together

quaternary structure

3D structures interact via side chains (oligomers)

when proteins move into a solution, they undergo

conformational change

- doesn't require covalent bonds to be broken

when proteins undergo conformational change, do their bonds typically break?

no

α-helix β-pleated strand what type of structure and where are they found

two principal secondary structures found in proteins

what determines the frequency of amino acids?

the number of steps

what is cysteine good for?

binding metals and signaling

what size amino acids are more common?

smaller (Glycine, Alanine, Serine)

how do amino acids join head to tail? (bonds)

formation of covalent peptide bonds

what does peptide bond formation result in? (what kind of reaction)

release of water (dehydration)

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 is PAGE?

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

- 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

- proteins are separated mostly by size

- 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 molecular weight can be used to determine what?

the apparent molecular weight of an unknown protein

if an oligomeric protein is separated on an SDS page with a homotetramer of 80 kDa, what will happen?

it will dissociate and run as a 20 kDa 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 applies

shearing forces

(refer to diagram on lecture 10)

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)

FPLC

Fast Protein Liquid Chromatography

what are the phases in FPLC?

the mobile phase (solution) and the stationary phase (resin)

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?

it's trying to catch positive charges causing negative charges to bind

in IEX, what occurs in the cation exchanger?

it's trying to catch negative charges causing positive charges to bind

the higher the charge on the molecule,

the stronger the binding

in IEX, interactions that are stronger require

more salt to compete

chromatogram

graph of chromatography

in IEX, after salt is loaded in, what is used to elute?

a gradient of increasing salt

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

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

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

all amino acids have a chiral orientation of S except

cysteine

priorities of functional groups

SH > OH > NH2 > COOH > CHO > CH2OH > CH3

(SON C/KAM)

what is the main contributor in deciding if an amino acid is essential or nonessential?

the number of steps required to make it. the cutoff is 6, but cysteine is the exception

leucine isn't made in the human body, but why is it considered essential?

it's present in proteins which helps arrive hydrophobic reactions

which amino acid is only partially positive?

histidine

histidine's side chain has a pH of

6

what's used to get a molecule to have a net charge of 0?

titration

what influences pKa values?

the environment

e.g. nearby groups, nearby residues, solvent accessibility

what's more important when deciding pKa values?

charge > aromaticity

what does linking affect?

pKa

post-translational modification

changes made in polypeptides following the making of proteins

Phosphorylation

- adds negative charge by adding PO3

- phosphate groups come from ATP

- Kinases attach phosphate group

- Phosphatases remove phosphate group

e.g. hormone receptors and regulatory enzymes

acetylation

adds acetyl group to neutralize the positive charge

e.g. histones, metabolic enzymes

methylation

- only occurs in histones by adding methyl group

- uses methyl donors like SAM

- does NOT neutralize the positive charge

what does basic mean?

positively charged

GFP

- naturally fluorescent protein

- oxidative product of FSYGVQ

- the GFP chromophore lies in the center of the β-barrel protein structure

- SYG found in other proteins does NOT form chromophore

- No enzymes, but oxygen required

colocalization

- 2 things can be in the same place

amino acid substitutions

GFP can tune to color of emitted light

which amino acids absorb UV light?

Phenylalanine, tyrosine, and tryptophan

(Tryptophan has the highest absorption of over 250 nm which makes it the easiest to measure)

Beer-Lambert law

A=εcl

l is always 1

what happens when Edman reagent reacts with the α-amino group?

a PTH derivative is produced at uv absorbance of 254 nm

HPLC

High Performance/Pressure Liquid Chromatography

gradient separation for common PTH-amino acids

what does ninhydrin degrade amino acids into?

aldehydes, ammonia, and carbon dioxide

what does OPA produce?

fluorescent moiety

cysteine residue forms what?

disulfides

this is known as a oxidation-reduction reaction

reactions of amino acids are

more common in extracellular proteins than cytosolic proteins