bis 102 protein stuff

after mt1

separation of protein chains with:

• extreme pH

• 8M urea

• 6M HCl

• high salt concentration (like ammonium sulfate)

mer-capto-ethanol (MCE)

HS-CH2-CH2OH

• protonates one cysteine and the other stays attached to MCE

• sulfhydryl reducing agent

di-thio-threital (DTT)

C4H10O2S2

• sulfhydryl reducing agent

phenyl-iso-thio-cyanate (PITC)

N-terminal analysis

• Edman’s reagent

• produces phenyl-thio-hyantoins (PTH-aa)

carboxypeptidase A

C-terminal analysis (exo-peptidase)

• cleaves any residue EXCEPT Pro, Arg, Lys

carboxypeptidase B

C-terminal analysis (exo-peptidase)

• ONLY works with Arg, Lys

trypsin

cleaves Arg or Lys (proteolytic enzyme)

chymotrypsin

cleaves Phe, Trp, or Tyr, Leu (proteolytic enzyme)

clostripain

cleaves Arg (proteolytic enzyme)

staphylococcal protease

cleaves Asp or Glu (proteolytic enzyme)

endopeptidase Lys-C

cleaves Lys (proteolytic enzyme)

cyanogen bromide

cleaves Met (proteolytic enzyme)

phosphorylation

modifies: S, T, Y

• hormone receptors

• regulatory enzymes

acetylation

modifies: K

• histone

• metabolic enzymes

methylation

modifies: K, R

• histones

acylation

modifies: C

• G-protein coupled receptors

prenylation

modifies: C

• Ras p21

ADP-ribosylation

modifies: H, R

• G proteins

• eukaryotic elongation factors

adenylylation

modifies: Y

• glutamine synthetase

peptide planar bond

six atoms of the peptide group in a plane; C-N partial double bond and can’t rotate; two degrees of freedom

rotation parameters

phi: 180 degrees

psi: 180 degrees

ramachandran plot

phi vs. psi

• proline: most tightly constrained and can’t react (no free N)

• glycine: least sterically hindered

alpha-helix

• hydrogen bonds

• phi: -60 degrees

• psi: -45 to -50 degrees

• residues: 1.5 amps (0.15 nm)

• 3.6 residues per long-axis turn

• 6 amps in diameter

• rise per turn (pitch): 5.4 amps

beta-pleated sheet

• rise per residue:

  • 3.47 amps ANTIPARALLEL

  • 3.25 amps PARALLEL

• 2 residues per long-axis turn

• hydrophobic interactions on one side of sheet

radial strands

strong/rigid and have high percentage of beta-sheets

circumferential strands

flexible and have high percent of alpha-helices

hydrogen bonding between…

carbonyl oxygen and amide in i+3 position

beta-turn

a tight loop from an H-bond between carbonyl O and amide H three positions down

• makes this stable

• allows protein to reverse direction of the peptide chain

• types 1 (more common) and 2

• involves four residues without specific phi and psi