9B and 10 - Tertiary Structure and Protein Technology

Question 1

What are three contributors to the total negative free energy change in protein folding?

Answer 1

Conformational entropy (+)
Internal interactions with enthalpy (-)
Hydrophobic Effect on entropy (-)

Question 2

Why are proteins susceptible to loss of protein structure?

Answer 2

Because of small differences between the free energy of folded and unfolded proteins

Question 3

What is protein renaturation?

Answer 3

The reverse of denaturation

Question 4

List 8 denaturing conditions

Answer 4

1 Strong acid or base
2 Organic solvents
3 Detergents
4 Reducing agents
5 Salt concentration
6 Heavy metal ions
7 Temperature
8 Mechanical stress

Question 5

What order are Hsp70 and Hsp60 used in protein folding?

Answer 5

The nascent polypeptide is first folded with help of Hsp70 and then Hsp60 (chaperonin) has its hand at it. Both use ATP and are involved in refolding proteins. If it can’t be folded both promote protein degradation.

Question 6

Are fibrous proteins water soluble?

Answer 6

no

Question 7

How many families of collagen are there?

Answer 7

20

Question 8

What are the two most common amino acids in collagen?

Answer 8

Glycine and proline

Question 9

List 4 Classes of motor proteins

Answer 9

Classical motors (kinesis, myosin, dynein)

Timing devices (EF-Tu in translation)

Microprocessing Switching Devices (G proteins)

Assembly and Disassembly Factors (cytoskeleton assembly and disassembly)

Question 10

What are three rich sources of proteins

Answer 10

Certain cell types

Organellar compartmentalisation

Heterologous expression of cloned genes

Question 11

What is Isopycnic (sucrose density) centrifugation

Answer 11

A sucrose gradient is used to identify different densities. Draining different layers from the bottom of the type is called fractionation.

Question 12

List three extraction and solubilisation methods for proteins

Answer 12

Lysis of cells/organelles (osmotic, enzymatic, homogenisation or sonication)

Filtration or centrifugation to seperate soluble and insoluble fractions

Detergent solubilisation of insoluble (eg. membrane bound) proteins

Question 13

What three things can electrophoresis separate by?

Answer 13

Charge

Molecular weight

Shape

Question 14

What is the BLAST database and what is it used for?

Answer 14

Basic Local Alignment Search Tool - Using a polypeptides determined sequence, functions can be determined

Question 15

What are the steps one takes to study a protein?

Answer 15

1. Purification: isolation and purification of a protein, often by salting out of a protein fraction. Source of protein may be lysis of cells/organelles
2. Centrifugation: either differential (to isolate soluble proteins from other organelles) or isopycnic (sucrose density) centrifugation, which uses fractionation to separate different layers of proteins
3. Detergent solubilisation of insoluble proteins (eg. membrane bound)
4. Chromatography might be used as separater of protein size, shape, weight or binding affinity
5. Electrophoresis may be used to assay the protein by charge, molecular weight or shape (SDS-polyacrylamide gel electrophoresis commonly used to determine molecular weight)
6. Mass spectrometry may be used for determining mass of purified protein
7. BLAST may be used to infer function from similar proteins. At this point the protein must have been isolated, purified and sequenced
8. X-ray crystallography may be used to determine protein 3D structure.

Question 16

X-ray crystallography creates what type of map?

Answer 16

electron density map