EXPERIMENT 5 – PROTEIN DEGRADATION

INTRODUCTION

A protein’s life cycle begins with its initial synthesis and correct folding. The second part of the
life cycle involves the control of the expression levels of a protein by regulating its degradation.
The lifespan of proteins varies greatly and are dependent on several factors. For example, the
half-lives of various enzymes in rat liver range from 12 minutes to 150 hours.

The “N-end” rule is the first factor to consider in the lifespan of a protein. Basically, the amino
acid residue on the N-terminus of a protein can sometimes be used to predict the protein’s
lifespan. On average, proteins with an N-terminal Met, Ser, Ala, Thr, Val, or Gly have half-lives
greater than 20 hours and with N-terminal Phe, Leu, Asp, Lys, or Arg have half-lives of 3 min or
less.

The “PEST” factor is for proteins that are enriched in Pro (P), Glu (E), Ser (S) and Thr (T) and
these are often degraded more rapidly than other proteins. The most common degradative
pathway for proteins is the ubiquitin-proteosome pathway. Proteins to be degraded are tagged
with chains of ubiquitin, a monomeric 76 amino acid proteins that becomes the signal that
directs the protein to the proteasome.

The proteasome is a multimeric protein that degrades proteins into short peptide sequences.
Ubiquitin is attached to the “condemned” protein in an ATP dependent manner, using a series
of 3 enzymes. Initially, the terminal carboxyl group of ubiquitin is joined in a thioester bond to a
cysteine residue on Ubiquitin-Activating Enzyme (E1). This is the ATP-dependent step. The
ubiquitin is then transferred to a sulfhydryl group on an Ubiquitin-Conjugating Enzyme (E2) and
finally an Ubiquitin-Protein Ligase (E3) then transfers the activated ubiquitin to the ε-amino
group of a lysine residue of a protein recognized by that E3, forming an isopeptide bond.
 Figure.1: The ubiquitin–proteasome system
(UPS)
Activated ubiquitin binds to E1 and is
transferred to the ubiquitin-conjugating enzyme
(E2). The E2 carries the activated ubiquitin to
the ubiquitin ligase (E3), which facilitates the
transfer of the ubiquitin from the E2 to a lysine
residue in the target protein (S). Poly-
ubiquitinated target proteins are degraded by
the 26S proteasome, consisting of a 19S
regulatory Particle (RP) and 20S core subunit
(CP).

The proteasome is a large multimeric protein found in the cytosol and nucleus of a cell. In
mammalian cells, the proteasome contains 2 copies of 14 different proteins that are arranged
into 4 ring-like structures. The proteins to be degraded pass through the center of the
proteasome where they are degraded. Specific cap proteins regulate protein entry to the
proteasome. This experiment uses protein electrophoresis to demonstrate the effects of
proteases on a protein.

In most cases, one has to determine total protein concentration in the solution. Protein
quantitation is an integral part of any laboratory workflow including protein extraction, analysis
of purification steps. Protein concentration determination allows to normalize multiple samples
for storage or side-by-side comparison. Moreover, proteins obtained from a purification
experiment are assayed to determine yield. It is also important for setting up the reactions
bearing proteins with appropriate stoichiometry. Depending on the accuracy required and the
amount and purity of the protein available, different methods are appropriate for determining
protein concentration.

The simplest and most direct assay method for proteins in solution is to measure the
absorbance at 280 nm. Amino acids containing aromatic side chains (i.e., tyrosine, tryptophan
and phenylalanine) exhibit strong UV-light absorption. Consequently, proteins and peptides
absorb UV-light in proportion to their aromatic amino acid content and total concentration.
There are many methods for measuring total protein concentration other than UV absorbance,
including Lowry assay, bicinchoninic acid assay (BCA Assay) and Bradford assay. Those listed
assays are also spectroscopic assays which are based on light absorption amounts of molecules
due to their amino acid content. With most protein assays, sample protein concentrations are
determined by comparing their assay responses by using serial dilutions of a Standard protein
whose concentration is known. Protein samples and standards are prepared in the same
manner by mixing them with assay reagent. Then the absorbance of sample with unknown
concentration and serial dilutions of known concentration are read by using a
spectrophotometer. The responses of the standards (serial dilutions of known sample) are used
to plot a standard curve. Absorbance values of unknown samples are then interpolated onto
the plot or formula for the standard curve to determine their concentrations.

BRADFORD ASSAY

Bradford reagent contains Coomassie Blue G-250 dye which has equilibrium between three
forms: red, green and blue. This dye forms strong, noncovalent complexes with proteins via
electrostatic interactions with amine and carboxyl groups via van der Waals forces. Under
strongly acidic conditions the dye is stable in the red form and after binding of a protein dye
becomes stable in the blue form. This Coomassie Blue G-protein complex causes a shift in the
absorption of dye from 465 (red) nm to 595 nm (blue). Figure 2. below shows the reaction
between proteins and Coomassie dye.

Development of color in Bradford assay depends on the certain basic amino acid content
including arginine, lysine and histidine residues. Binding of Coomassie dye to the protein is
proportional to the number of those positively charged residues. Free amino acids, peptides
and smaller proteins than 3000 daltons do not produce color with Coomassie dye.
 Figure.2: Reaction schematic for the Braford assay

MATERIALS

 100 μl Bovine Protein

 100 μl Protease K
 120 μl Stop Solution
 330 μl PAGE: Sample Loading Buffer
 dH2O
 Centrifuge Tubes (1.5ml)
 Protein assay mixture (Bradford dye)
 Microplate reader
 BSA standards (0,5 mg/mL, 1 mg/mL, 2 mg/mL, 4 mg/mL, 6 mg/mL, 8 mg/mL, 10 mg/mL,
12 mg/mL)
 96 well transparent plates

PROCEDURE

1. Label 10 tubes (1-10) and aliquot 10 μL Bovine Protein to each tube.

2. Add 20 μL Stop Solution to tube #10 and mix content by pipetting up and down 3-4 times.

3. Add 10 μL Protease K to only the tube marked #1. Mix content by pipetting up and down 3-4
times. After mixing, start the timer.
4. Add 10 μL Protease K to tubes #2-9 when the timer reaches the time points indicated in the
table below.

Tube # 1 2 3 4 5 6 7 8 9 10
Bovine Protein 10 l 10 l 10 l 10 l 10 l 10 l 10 l 10 l 10 l 10 l
Timer (min) 0 15 30 40 50 55 58 59 60 --
Incubation time
60 45 30 20 10 5 2 1 0 --
(min)

5. After adding Protease K to the tube 9, immediately add 10 μL Stop Solution to tubes 1-9. Mix
content by pipetting up and down 3-4 times.

6.1. Label new set of 10 tubes (1-10) and add 5 μL protein samples according to sample
number.

6.2. Add 20 uL dH2O (distilled water) on to each tube to diute your samples in to 1:5.

6.3. Add 30 μL PAGE: Sample Loading Buffer to remaining protein samples and keep the
samples at -20 C for the SDS-PAGE experiment next week.

7. Add 150 µL of the Bradford Reagent into 96 well transparent plate in duplicates (2
wells/sample).

8. Mix 10 uL, 1:5 diluted protein samples with Bradford Reagent, mix well by pipetting and
incubate 5 mins on bench. (Do NOT bubble the mixture)

9. For Standard Samples; prepare 150 uL Bradford Reagent and 10 uL BSA standards (0, 0,5, 1,
2, 4, 8, 10,12 mg/mL) into 96 well transparent plate in duplicates.

10. Read 96 well plate in the microplate reader at 595 nm.

11. Make a graph using your measurements for the standard samples in which x axis shows
concentration and y axis shows absorbance 595nm value. You can use Microsoft Office Excel
program. Make a linear regression analysis to obtain the equation for calculation. The R
squared value of your standard curve should be higher than 0.9 for accurate measurements.

12. Calculate the average concentration of your unknown samples by using the equation that
you obtained from standard curve. Compare and discuss your results