BCH4024 Lecture 3

Which terminal is written first

N terminal

peptide bond reaction

O=-OH-NH2-R > O=-N-R;

loses 1 H2O

condensation reaction catalyzed by the ribosome

who analyzed peptide bond geometry?

Pauling and Corey

what is unique about the carbonyl-N peptide bond

its shorter because of double bond character, it cannot rotate

angles of polypeptide backbone

! phi = CN

psi = carbonyl-C

omega = carbonyl-N

peptide bond prefers (cis/trans) config, with the exception of ___

trans, bc they are planar and reduce steric;

proline, bc it is cyclic

in aq sol, peptide bond is (favored/not), hydrolysis is (favored/not)

not favored, favored

how to name peptides

list every aa in order N-terminus - C-terminus;

every name replaces -ine with -yl except C-terminus

oligopeptides

short; few residues

proteins

long chains of of fully folded aas or polypeptides; >10,000 daltons

polypeptide

chain of amino acids, not folded

ionizable side groups

ser, tyr, lys, arg, his, glu, asp

calculating isoelectric point

!!!!

when charge is neutral, use pka before and after

avg weight of aa

138, 128 in a protein, 110 residue in protein

number of sequence possibilities

number of aa^(peptide length)

ex: 20 aa, tetrapeptide = 20^4 = 160,000

how many sequences do organisms rely on

30-35,000

conjugated proteins

contain chemical components called prosthetic groups

lipoproteins contain lipids

glycoproteins contain sugars

metalloproteins contain metals

steps to separate proteins

1. break open tissue/cells

   crude extract = release proteins in sol

2. fractionation = separate based on size and sol.

   salting out = lower sol of salt to precip

3. dialysis = semipermeable membrane

ion exchange chromatography

based on pH and free salt conc. impact on protein affinity.

uses bound cat/anions to elute proteins

size-exclusion chromatography

gel filtration, large go before small;

small get trapped in porous beads while large go around

affinity chromatography

binding affinity, eluted by high conc. of salt or ligand

as purification goes on, fraction volume ____, total proteins _____, activity ____, and specific activity _____.

decreases, decreases, decreases, increases

electrophoresis can be used to estimate:

number of proteins in a mix, purity, isoelectric pt, weight, conc., oligomerization

electrophoresis uses

cross-linked polymer polyacrylamide gels and coomassie blue dye

proteases

catalyze hydrolytic cleavage of peptide bonds

trypsin cleave at Lys Arg

chymotrypsin cleave at Phe Trp Tyr

cyanogen bromide cleave at Met

mass spec

can sequence short aas of 20-30 res.

can document entire cellular proteome.

uses mass:charge ratio

tandem MS

two mass filters in tandem

first sorts peptides by cleavage

seconds measures mass/charge m/z

consensus sequence

most common aa at each position

homologs

members of protein families

para = parallel = same species

ortho = different species