Biochem Lab Quiz #6

Front 1

Why proteins fold?

Back 1

"Hydrophobic affect" in primary sequence of AA's
Hydrophobic side chains form the core of the protein.
Charged/polar side chains are then exposed to solvent

Front 2

How are folded proteins stabilized?

Back 2

1)Hydrogen bonds formed in the secondary structure.
2)Covalent (disulfide) bond and salt bridges stabilize final structure.

Front 3

Levinthal's Paradox

Back 3

Since there are numberous ways a protein can fold, it could potentially take longer than the age of the universe to sample thru these to finally reach its conformation.

Front 4

Levinthal's Paradox solved...

Back 4

protein folding is assisted by HSPs (Heat Shock Proteins) called chaperones. They protect the protein from heat.

Front 5

Why study protein folding?

Back 5

Which bonds are important
How folding affects activity (kinetics)
Biotechnology:
-Design proteins with better stability
-Better in vivo folding and production
Medicine:
-destabilization/misfolding leads to disease

Front 6

Misfolding of proteins leads to:

Back 6

misfolded prions which causes other prions to misfold; alzheimers; cystic fibrosis; inherited emphysema; cancer

Front 7

Prion Diseases

Back 7

BSE
Kuru
Chronic Wasting Disease
CJD (consumption of cattle with BSE)

Turns alpha helix to B pleated sheets (PrP^C to PrP^Sc)

Front 8

Alzheimers

Back 8

Neuron disease
Makes beta amyloid plaques and neuroaxilary triangles

Front 9

Two ways to study protein stability

Back 9

1) Thermodynamic
2) biochemical: What conditions can a protein survive?

Front 10

5 factors involved in Protein Unfolding

Back 10

1) temperature
2) pH
3) Solvents (acetonitrile, alcohol)
4) Reducing Agents
5) cosolutes (urea, GdnCl, iodide, detergent)

Front 11

pH

Back 11

Denaturation by acid or base (salt bridges break)

Front 12

Solvents

Back 12

Hydrolyze H-Bonds

Front 13

Reducing agents

Back 13

Break disulfide bonds

Front 14

Ways to stabilize Protein

Back 14

Crowding - immobilization
Cosolutes
Engineering

Front 15

3 ways of Crowding (immobilization)

Back 15

1)adsorption onto/covalent attachment to a polymer support
2)encapsulation within a membrane sphere
3) entrapment within a porous matrix

Front 16

Measuring Protein Stablity

Back 16

You can use enzyme activity or thermodynamics

Front 17

Types of probes for measurin protien stability

Back 17

1. Fluorescence (FRET)
2. Circular dichrism
-far UV
-near UV
3. NMR
4. SEC
5. visible light spectroscopy

Front 18

NMR for measuring protein stability

Back 18

requires pure protein

Front 19

Cyt c has three state transitions in a ____ salt buffer at pH's between __ and __

Back 19

High, 2 and 3

Front 20

Characteristics of molten globule

Back 20

1) native like secondary structures
(assesed by far UV circular dichroism)
2) decreased tertiary structure
(flurescent labeling)
3) lack of tight side chain packing
(NMR or soret band absorbance)
4) radius is 10-30% greater
(SEC)

Front 21

Why does immobilization of an enzyme, for example in an acrylamide matrix,
stabilize the protein to thermal denaturation?

Back 21

contrains the area available to the protein so the protein cannot expand when it unfolds.

Front 22

Are there any other advantages to using
immobilized enzymes as biocatalysts in biotechnology applications?

Back 22

1) limits diffusion of catalyst
2) which allows removal of catalyst from product
3) recovery for re-use

Front 23

How can you demonstrate that immobilization enhances an enzyme’s stability
toward thermal denaturation? What experimental set-up would you employ? What controls or
normalization would you need? Show a plot that clearly describes the results expected from
your experimental set-up.

Back 23

COMPARE the activities of the immobilized enzyme and the free enzyme at low
temperature (e.g. 25 °C) and high temperature where unfolding is expected, e.g. 70 °C. The
activity of the free enzyme may be greater than the immobilized enzyme at low
temperature. HOWEVER the relative or percent activity at high temperature should be
greater for the immobilized enzyme, i.e. (Activity immobilized high T)/( Activity
immobilized low T) > (Activity free high T)/( Activity free low T). The more active enzyme
present, the greater the activity and product accumulation.

Front 24

Why would organic solvents, such as 50% ethanol, denature a protein?

Back 24

As a hydrophobic co-solvent, ethanol will solubilize the hydrophobic side chains, reducing
the favorability of burying these groups away from aqueous solvent (i.e. minimizing the
hydrophobic effect).

Front 25

What is the NAME of the absorbance band you monitored for WHAT functional
group on cytochrome c to assess the relative concentrations of unfolded and native protein as a
function of GdmCl or pH?

Back 25

The Soret band absorbance between 350 and 450 nm of the covalently bound heme.

Front 26

What is the relationship between ΔG and Keq, the equilibrium constant for a given
reaction?

Back 26

ΔG = -RT ln(Keq)

Front 27

Define the Keq for the unfolding reaction of cytochrome c

Back 27

Keq = [U]/[N]

Front 28

What is the definition of Fapp? Define in terms of the concentration of the
appropriate species, such as unfolded, native or total protein concentration.

Back 28

What is the definition of Fapp? Define in terms of the concentration of the
appropriate species, such as unfolded, native or total protein concentration.

Front 29

Further define Fapp in terms of the spectral signals of the relevant species.
Define any variables that used in your Fapp definition.

Back 29

Fapp =
(Yi " YN )
(YU " YN )
Yi = signal observed at a given condition;
YN = signal expected for N, from the folded baseline;
YU = signal expected for U, from the unfolded baseline.