biochem Week 2 lect 5

protein purification, property characterization, and sequence analysis

Heterologous expression

Clone and over express a gene from one species in a rapidly growing cell of another species (such as E. coli, insect cell lines, etc) to produce large amounts of protein

Salting out

Protein purification based on solubility differences, most commonly used salt is ammonium sulphate

solubility

Protein purification characteristic for salting out

Size

Protein purification characteristic for selective dialysis

Size

Protein purification characteristic for gel filtration chromatography

Selective dialysis

Protein purification method based on size, used for buffer exchange, ions pass through a membrane sometimes with small proteins

Ammonium sulphate

Commonly used salt in protein purification based on solubility differences aka salting out

Gel filtration chromatography

Protein purification method based on size, gel beads have cavities permeable to small molecules and not large ones, larger ones pass through and small get stuck

Larger

In Gel filtration chromatography, will larger or smaller proteins move faster and come off the column first?

Ion exchange chromatography

Protein purification method based on charge, molecules are passed through an ion exchange matrix

Positive

for ion exchange chromatography, what will the change be for a weak anion exchanger bound to the bead?

Isoelectric point

point at which a protein has no net charge at a certain pH

Negative

What is the overall charge of a protein when the pH is above its pI?

Positive

What is the overall charge of the protein when the pH is below it’s pI

Affinity chromatography

Protein purification method separates proteins based on binding some molecule of interest

Ni

common metal used to bind proteins of interest in affinity chromatography

affinity chromatography

which of the two protein purification methods (ion vs affinity chromatography) only requires one step to get a pure protein?

UV spectroscopy

method for determining protein concentration, based on UV spectroscopic absorption of aromatic sidechains

coomassie staining

method for determining protein concentration, sensitive measure based on a protein binding stain, can be more specific and use lower concentrations than UV spectroscopy

UV spectroscopy, coomassie staining

two methods for determining protein concentration, by checking for GENERAL protein concentration

enzyme linked immunosorbent assay (ELISA)

method for determining protein concentration, checks for presence and concentration of a SPECIFIC protein

Trp, Tyr, Phe

aromatic AA side chains that absorb UV light at 280nm and is often used for protein concentration determination by absorption spectroscopy

N and C backbone

nonspecific way/loci in which the dye coomassie brilliant blue binds to proteins changing the color from brown to blue in a Bradford assay

gel filtration chromatography, SDS-PAGE

protein characterization method(s) based on size

ion exchange chromatography

protein characterization method(s) based on charge

enzyme specificity, ELISA

protein characterization method(s) based on binding specificity

mass spectrometry

protein characterization method(s) based on amino acid sequence

sodium dodecyl sulfate (SDS)

used in SDS-PAGE denatures proteins and binds to denatured proteins with one SDS per two amino acids largely independent of AA sequence

SDS-PAGE

using control standard bands with known length, this characterization method can estimate the molecular mass within 10-20% accuracy

larger

during an SDS-PAGE, do larger or smaller proteins move slower?

mass spec, chemically

two ways in which AA sequence can be determined:

identities of adjacent AA

what identity of AAs determines where a protease will break a peptide bond?

false, only one or the other

true/false: a protease will break the bonds of the R groups of the N and C term residue based on the identities of the adjacent AA

trypsin

protease enzyme cleaves adjacent N term residue with a specificity for Arg/Lys

Arg/Lys, N term

protease enzyme trypsin specificity? which terminus?

Phe/Trp/Tyr/Leu, N term

protease enzyme chymotrypsin specificity? which terminus?

chymotrypsin

protease enzyme cleaves adjacent N term residue with a specificity for Phe/Trp/Tyr/Leu

Pro

which adjacent AA residue will make it so proteases will not cleave a bond?

electrospray (ESI), MALDO, fast atom bombardment

mass spectrometry techniques used to determine mass of purified proteins, requires only picomoles, time required is short, mass is accurate to ± 1 Dalton for proteins up to 300kD

electron spray mass spec (ESI)

dry N2 gas promotes evaporation of solvent from charged droplets containing protein of interest leaving gas phase ions, mass spectrometer determines mass to charge ratio of these ions, resulting mass spectrum consists of series of peaks corresponding to ions that differ by a single ionic charge and the mass of one proton

(m/z) charge ratios

how do resulting mass spec graph peaks from ESI differ?

tandem mass spectrometer (MS/MS)

used to determine protein sequence from broken up fragments, ESI generated gas phase peptide ions are sorted by mass charge ratios, peptides directed to collision He2 cell fragmenting it which is then directed to second spectrometer for determination of m/z values

protein fold identification

sequence information, if >25% of the protein sequence is conserved between proteins, it is highly likely the two proteins adopt the same overall fold

family sequence alignment

sequence information, invariant amino acids in a family are likely to be important for function or for structure

estimating evolutionary rates of proteins

since not all proteins are subject to the same evolutionary pressure, due to different functions, their rate of change in the course of time is not the same

phylogenetic tree

these can give profound insight into evolutionary relationships between species

indels

“-” inserted for protein sequence alignment in order to maximize identities

gene duplication and divergence of sequence and function

based on the sequence alignment of myoglobin and the human hemoglobin a-chain, it was found they were 27% identical and derived from a common ancestor, what is the likely even that caused their divergence?

AA at that location likely plays an important structural/functional role

looking at a family sequence alignment, the bottom will indicate the number of different amino acids per position, what does only a single AA identity at a position indicate?

number of inferred differences per 100 residues

computational clustering, what does the number beside the branch of a phylogenetic tree indicate?