More on proteins...

Question 1

• Temperature (heat), pH and solvent conditions can

Answer 1

be adjusted to unfold a
protein back into a more extended form.

Question 2

When the unfolding conditions are reverted, many proteins

Answer 2

have enough
information stored in their sequence of amino acids to refold back to exactly
the same tertiary structure. Other proteins get stuck along the way (e.g. curdled
milk stays curdled after heat/cool treatment

Question 3

The essence of correct protein
folding is the…

Answer 3

retention of partially
correct intermediates

Question 4

Molten globules containing…

Answer 4

native secondary structure but
not tertiary structure are formed early in folding

-Molten refers to fluctuating nature of interactions between
secondary structures

-Globule refers to a condensed state

Question 5

HYDROPHOBIC COLLAPSE
is the

Answer 5

driving force for
formation of molten globules

Question 6

An unfolded polypeptide chain is often …

Answer 6

unstable in water
(in absence of denaturing agents) because many
nonpolar residues may come into contact with water.

-The hydrophobic groups tend to come together to avoid
water (hydrophobic collapse)

Question 7

Nonpolar amino acids form a

Answer 7

hydrophobic core hidden from water

Question 8

Amino acid residues have different

Answer 8

tendencies for forming (a)-helices,
(b)sheets or (b)turns

Question 9

promote helix formation + other formations:

Answer 9

Some amino acid side chains (like glutamate,
alanine and leucine)

Proline and glycine do not favour helix
formation but have a tendency to form turns

Some amino acids favour b-strand formation
(like leucine, isoleucine, and valine)

Question 10

Secondary structure prediction is

Answer 10

only about
60% accurate (but getting better.

Question 11

Supersecondary structure:

In general more than 60% of

Answer 11

the
polypeptide backbone comprises
a-helices and b-sheets

Question 12

The overall free energy change on
folding is

Answer 12

negative–so favorable.
Free energy is a balance of several
thermodynamic factors:

-Conformational entropy

-Enthalpy contribution

-Entropy contribution from hydrophobic
effect

Question 13

Conformational entropy:

Answer 13

Unfavorable
energy favors random chains
conformation due to burying of
hydrophobic residues interacting with
water.

Question 14

Enthalpy contribution:

Answer 14

Favorable
energy from intramolecular side
groups interaction.

Question 15

Entropy contribution from hydrophobic
effect:

Answer 15

Favorable energy due to the
burying of hydrophobic R group

Question 16

Local folding through…

Answer 16

nucleation of small clusters of residues due
to the hydrophobic effect

Question 17

A General Order of Folding:

Answer 17

1. Random polypeptide: hydrophobic residues stabilized by water forming a
   cagelike structure, DS low.
2. Secondary structure starts to form:

-hydrophobic effect (hydrophobic residues buried inside the protein)

->favorable energy DS.

-release of water due to hydrophobic effect

->randomness conformational entropy increase (unfavorable energy DS).

3. Secondary structure are formed, domains, protein folded: Intramolecular
   side chain interactions create a negative enthalpy (favorable energy)

Question 18

Motif:

Answer 18

-simple combinations of a few secondary structure
elements with specific geometric arrangement

• b-a-b and calcium calcium binding “hand” are examples

-A variety of structures are seen in globular protein
domains

-Looking at possible similarities can provide insight
into how structure and function are related

Question 19

Domain:

Answer 19

polypeptide chain or part of polypeptide chain
that can fold independently into stable 3o structure

Question 20

Many proteins have several compact…

Answer 20

compact globular regions. Each globular unit is called a domain.

• domains tend to have ~50 up to 200-300 amino acids
• less than 50 is difficult to fold stably
• more than 300 is difficult to fold correctly

Question 21

A single domain is typically…

Answer 21

made of a single stretch of
primary sequence – there are many exceptions though

Question 22

Tertiary folding is stabilized by

Answer 22

the same non-covalent interactions as found
in the secondary structures but involves amino-acid side chain interactions only not main chain atoms interactions.

• H-bonding
• ionic interactions
• van der Waals forces
• ‘hydrophobic’ interactions

Question 23

disulphide linkages (Bonds)

Answer 23

covalent bonds that can stabilize protein tertiary structure

Question 24

Salting Out:

Answer 24

The solubility of protein in an aqueous solution depends
on many factors, including:

• Size of protein
• Surface charge on protein
• Polarity of protein
• pH and ionic strength of the solution.

Question 25

Protein Purification:

Answer 25

This “purification”
process involves
separating proteins
based on their ionic
properties, their sizes,
their hydrophobicity, and
their affinities for certain
molecules (ligands).

*Each successive step
is referred to as
fractionation.

Question 26

Ion-Exchange Chromatography

Answer 26

*The bead is
negatively charged, and
therefore the rate of
mobility of proteins
loaded onto the resin is
proportional to the
degree of negative
charge that they bear.

Question 27

Size Exclusion Chromatography:

Answer 27

In size-exclusion chromatography
(gel-filtration chromatography),
proteins migrate as a function of their
molecular mass.
* Notice in the figure at let left that the
large molecules elute first.

Question 28

Affinity Chromatography:

Answer 28

In affinity chromatography, proteins
are separated according to their ability
to bind to a specific ligand that is
connected to the beads of the resin.

• After the proteins that do not bind the
  ligand are washed through the column,
  the bound protein of interest is eluted
  by a solution containing free ligand.

Question 29

Electrophoresis
SDS-polyacrylamide gel electrophoresis

Answer 29

Electrophoresis is based on the migration of
proteins in a charged field.
The trick is to carry out the electrophoresis in
the presence of sodium docecyl sulfate, which
is a detergent.
SDS binds to every protein in roughly the same
proportion, which is about one molecule for
every two amino acid residues.
SDS carries with it a negative charge, and the
cumulative negative charge renders the intrinsic
net charge of the protein insignificant.
Therefore, every protein will have the same
charge to mass ratio, which will cause all
proteins to migrate towards the cathode with a
rate that is dependent on their sizes.
In contrast to gel-filtration, smaller molecules
migrate faster than larger molecules

Question 30

Isoelectric Focusing:

Answer 30

Isoelectric focusing is a procedure that allows the pI of a
particular protein to be determined, and that separates
proteins based on their respective pI values.

Question 31

2D-Electrophoresis:

Answer 31

2D-electrophoresis allows separation of proteins
by both size and isoelectric point (pI). Each spot
represents a different protein. The horizontal
represents the isoelectric focusing direction,
while the vertical represents the SDS-PAGE
direction.

Question 32

n order to purify a protein …

Answer 32

or any other substance, there
must be a means of quantitatively detecting its presence