BIOC 503 - Amino Acids

proteins biological funcitons

• catalysis (enolase, DNA polymerase etc.)

• transport (hemoglobin, lactose permease etc.)

• structure (collagen, keratin etc.)

• motion (myosin, actin)

proteins

linear heteropolymers of alpha amino acids

common features of amino acids

• alpha amino group

• alpha carboxy group

• alpha hydrogen

• tetrahedral alpha carbon

• a side chain/R substituent of some kind

L

proteins only contain ___ amino acids

nonpolar aliphatic amino acids

• glycine

• alanine

• proline

• valine

• leucine

• isoleucine

• methionine

polar uncharged amino acids

• serine

• threonine

• cysteine

• asparagine

• glutamine

aromatic amino acids

• phenylalanine

• tyrosine

• tryptophan

hydrogen

polar uncharged amino acid side chains can form ___ bonds

cysteine

___ can form disulfide bonds

positively charged amino acids

• lysine

• arginine

• histidine

negative amino acods

• aspartate

• glutamate

2.34

pKa of alpha carboxy group

9.6

pKa of alpha amino group

6.0-6.5

average pI of uncharged amino acids

more

alpha carboxy is ___ acidic than normal carboxy group

slightly less

alpha amino group is ___ basic than normal amino group

zwitterion

a single molecule having both positive and negative charges

form of amino acids found at the pI aka isoelectric point

isoelectric point

Point at which the net charge is 0, the amino acid is the least soluble in water and does not migrate through electric field.

aka pI or average of pKas

pI = (pKa1 + pKa2)/2

buffering region

plateau on titration curve

peptides

small condensation products of amino acids

small compared to proteins aka <10kDa

N-terminal

naming and numbering of proteins and peptides start at the ___

functions of peptides

• hormones and pheromones (insulin, oxytocin, sex-peptide in fruit flies)

• neuropeptides (substance P)

• antibiotics (polymyxin B, bacitracin)

• protection/toxins (amanitin in mushrooms, conotoxin in cone snails, chlorotoxin in scorpions)

Components of proteins

• cofactors (metal ions)

• coenzymes (organic factor)

• prosthetic groups (heme in myoglobin)

• posttranslational modification (phosphorylation)

fish them out

Some polypeptides bind specific targets, which can be used to ____ of a complex mixture

Separation relies on a lot of chemical and physical properties

• charge

• size

• affinity for a ligand

• solubility

• hydrophobicity

• thermal stability

column chromatography

allows separation of a mixture of proteins over a solid phase (porous matrix) using a liquid phase to mobilize the proteins

Proteins with a LOWER affinity for the solid phase will wash off first, the ones with the HIGHER affinity will retain on the column longer and wash off later

Ion exchange

inside polymer resin often made of silica is charged and retain OPPOSITELY CHARGED molecules. So, the molecules with the same type of charge (negative vs positive) as the resin will be repelled and elude FIRST.

size exclusion

cross-linked polymer will retain SMALLER molecules because they fit in all the small passages and thus take longer to elude. So, BIGGER molecules elude FIRST.

Binding affinity

polymer resin with the ligand binding to protein of interest, retains protein of interest, washed off in later faction with a ligand solution `

electrophoresis

• electric field pulls proteins according to their charge

• gel matric hinders mobility of proteins according to size and shape

• gel is commonly polyacrylamide = polyacrylamide gel ____ = PAGE

SDS

sodium dodecyl sulfate

detergent

micelles bind to proteins and facilitate unfolding in electrophoresis`

rate of movement will only depend on size aka small proteins move faster

UV

aromatic amino acids absorb ___ light around 275-280 nm absorbance maxima

Lambert-Beer law

A = E*c*l = extinction coeff * concentration * path length

activity

function of the protein

specific activity

ratio of activity to total protein concentration

DNA

actual protein sequence usually determined from the ___ sequence

Edman degradation

classical method

• successive rounds of N-terminal modification, cleavage, and identification

• used to identify protein with known sequence

Mass spectrometry

modern method

identify the mass of a peptide, and thus the amino acid sequence

used to determined posttranslational modifications