Lecture 5-8 Flashcards
(27 cards)
Describe the Sanger Method.
It is the first method developed to sequence proteins. The N-terminus of the proteins becomes a nucleophile at high pH and the HF of the fluorodinitrobenzene acts as a leaving group to a C atom of the fdnb can act as an electrophile. The fdnb has a bight yellow colour which identifies the N terminus so the tagged protein can be hydolysed
What is the limitations of the Sanger Method?
it hydrolyses the entire peptide so only the tagged AA can be identified and the rest of the chain is destroyed
Describe Edman degradation.
- Basic phase
- the N-terminal acts as a nucleophile which bonds to the electrophilic C of the phenylisothiocyanate
- the C atom breaks the C to S double bond on the phenylisothiocyanate to a single bond and the electron goes to the N-terminal of AA #2 - Acidic phase
- the N-terminal of AA #2 acts as the leaving group taking the rest of the peptide with it
- the amino acid attached to the phenylisothiocyanate cyclizes to form its final product
Where does trypsin cut?
Arg and Lys
except: in the presence of proline
Where does chymotrypsin cut?
Phe, Tyr, and Trp
except: in the presence of proline
What is cyanogen bromide?
a selective chemical hydrolysis that converts methione to homoserine
What is significant about the behaviour of the peptide bond?
it behaves more a double bond
What are some properties of the alpha-helix secondary structure of proteins?
The CdbO group of amino acid #1 will be exactly inline with the N-H group of amino acid #5 so they can hydrogen bond, and every AA is 1.5A apart
What AA prefer alpha helix secondary structure?
FMLKAREHQ
What AA prefer beta sheet secondary structure?
WYFVITC
What AA form secondary structure breakers?
GPSND
What force causes a protein to fold back on itself to form a tertiary structure?
the hydrophobic effect
How is the “jigsaw” interlocking formed in tertiary structures?
Through Van Der Waal forces forming between atoms in perfect contact. Atoms too close together will repel, atoms too far apart will not have any force between them
What determines the whether a secondary structure folds into an alpha helix bundle or a beta antiparallel sheet?
an alpha helix bundle will form if the sequence contains mostly groups of alpha AA
a beta antiparallel sheet will form if the sequence contains mostly groups of beta AA
What is the significance of non-polar and polar AA alternating in a beta sheet?
it allows for polar AA and non-polar AA to be on separate sides of the sheet so it can wrap in an oval shape with the NP AA on the inside and the P AA on the outside
What tertiary structure forms when beta and alpha helix segments alternate in a sequence?
An alpha-beta barrel is formed if the helices lie on one side of the beta sheets and an alpha-beta sandwich is formed if the helices lie on both sides of the sheet
What is the interaction of oxygen with disulfide bonds?
oxygen helps forms disulfide bonds by removing the hydrogen from the SH
Describe the experiment that determined that folding information is present in the primary protein structure.
Starting with ribonuclease, the enzyme is treated with urea which weakens the hydrophobic interactions causing the proteins to denature
Ribonuclease contains 4 disulfide bonds so mercaptoethanol is added to reduce the bonds to SH groups
Experiment showed that the unfolded ribonuclease could refold if urea was first removed and then the enzyme was exposed to air to allow the disulfides to reform
If the unfolded ribonuclease is exposed to air and disulfide bonds form before the urea is removed, the enzyme cannot rearrange itself to its original form
What functions allow proteins to recognize and bind to other molecules?
- non-polar patch for the hydrophobic effect
- complementary shapes to max contact
- charged or H-bonding groups line up with partners
Why does chymotrypsin cleave only phe, trp, and tyr?
because the hydrophobic pocket of chymotrypsin holds AA with aromatic rings
What does trypsin cleave only arg and lys?
it has a narrow binding pocket with Asp at the end so only negative AAs and long side chains
Using the rate of reaction equation, how are reactions sped up?
by increasing Z or p and decreasing Ea
What are the proximity and orientation effect?
enzyme binds substrates by holding them in the active site to the groups are aligned in the correct orientation
What is an electrophilic catalysis?
the use of a prosthetic group that binds to an enzyme and acts as a electrophile
