More on proteins... Flashcards

1
Q
  • Temperature (heat), pH and solvent conditions can
A

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

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2
Q

When the unfolding conditions are reverted, many proteins

A

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

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3
Q

The essence of correct protein
folding is the…

A

retention of partially
correct intermediates

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4
Q

Molten globules containing…

A

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

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5
Q

HYDROPHOBIC COLLAPSE
is the

A

driving force for
formation of molten globules

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6
Q

An unfolded polypeptide chain is often …

A

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)

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7
Q

Nonpolar amino acids form a

A

hydrophobic core hidden from water

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8
Q

Amino acid residues have different

A

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

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9
Q

promote helix formation + other formations:

A

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)

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10
Q

Secondary structure prediction is

A

only about
60% accurate (but getting better.

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11
Q

Supersecondary structure:

In general more than 60% of

A

the
polypeptide backbone comprises
a-helices and b-sheets

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12
Q

The overall free energy change on
folding is

A

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

-Conformational entropy

-Enthalpy contribution

-Entropy contribution from hydrophobic
effect

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13
Q

Conformational entropy:

A

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

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14
Q

Enthalpy contribution:

A

Favorable
energy from intramolecular side
groups interaction.

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15
Q

Entropy contribution from hydrophobic
effect:

A

Favorable energy due to the
burying of hydrophobic R group

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16
Q

Local folding through…

A

nucleation of small clusters of residues due
to the hydrophobic effect

17
Q

A General Order of Folding:

A
  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).

  1. Secondary structure are formed, domains, protein folded: Intramolecular
    side chain interactions create a negative enthalpy (favorable energy)
18
Q

Motif:

A

-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

19
Q

Domain:

A

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

20
Q

Many proteins have several compact…

A

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
21
Q

A single domain is typically…

A

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

22
Q

Tertiary folding is stabilized by

A

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
23
Q

disulphide linkages (Bonds)

A

covalent bonds that can stabilize protein tertiary structure

24
Q

Salting Out:

A

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.
25
Protein Purification:
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.
26
Ion-Exchange Chromatography
*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.
27
Size Exclusion Chromatography:
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.
28
Affinity Chromatography:
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.
29
Electrophoresis SDS-polyacrylamide gel electrophoresis
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
30
Isoelectric Focusing:
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.
31
2D-Electrophoresis:
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.
32
n order to purify a protein ...
or any other substance, there must be a means of quantitatively detecting its presence