Why is a protein’s conformation important for carrying out its function? What pr

Question

Why is a protein’s conformation important for carrying out its function?

What properties of the amino acids in a protein relate to protein folding?

Does the eluate containing your red fluorescent protein appear less bright
or brighter than it did in the cell lysate following centrifugation? If there is a noticeable difference in the intensity of the red color, what might account for that?

What characteristic of red fluorescent protein is used as the basis for separation by column chromatography?

How might the column chromatography procedure be adjusted or modified to increase the purity of the red fluorescent protein sample?

Explanation / Answer

Answers :-

1. The shape, conformation, affect its function by determining what the shape is some proteins are long and fibrous : those form hair and form blood clots ... Some are globular and can function as enzymes which transport oxygen. The shape of a protein affects the function.

2. As a result of all of these interactions, each type of protein has a particular three-dimensional structure, which is determined by the order of the amino acids in its chain. The final folded structure, or conformation, adopted by any polypeptide chain is generally the one in which the free energy is minimized.

Answer 3 ,4 & 5 refer this experiment:-

Purification of mFP from an Overnight Culture

When scientists at a therapeutics company,

like

Amgen, have successfully identified a promising therapeu

-

tic protein, two objectives would be to locate and isolate

the gene that encodes the protein. Once isolated, the gene

is inserted into a plasmid so that the gene can be cloned,

as additional copies of the gene will be needed for ongo

-

ing studies. The

rfp

gene was cloned in a plasmid called

pKAN-R. pKAN-R is a

cloning vector

, a plasmid that

has been engineered to replicate in high numbers within

the bacterial cell.

Later, cloned genes are inserted into plasmids that have

been engineered specifically for protein expression in

bacteria or other suitable organism. Such plasmids are

known as

expression vectors

. pARA-R is an

expression

vector

and carries the cloned

rfp

gene in a specific plas

-

mid location, which allows the bacterial cell to produce

mutant fluorescent protein.

Transformed cells are allowed to express the protein

in an overnight culture and then lysed (broken open) to

release the newly synthesized protein from the cell. The

protein is isolated from the other cytoplasmic proteins,

purified and tested for activity.

You have already completed

much of the work that parallels

this drug discovery scenario.

The bacterial cells that have

been growing in the LB/amp/

ara broth have been expressing

mFP and are now ready to

be lysed (day

one

of Lab 7)

and the mFP purified (day

two

of Lab 7) using column

ch romatog raphy.

Mutant fluorescent protein

is a molecule that is about

238 amino acids in size. The

native (as it exists in

Discosoma

) protein is shaped

like a cylinder with the fluorescent region, called the

fluorophore, located in the center of the cylinder.

In order to purify a molecule from other proteins

present in the cell, one needs to look at how groups

of molecules differ from one another and how these

differences can be used to effect separation.

One molecular attribute commonly used in purification

is protein

hydrophobicity.

The term

hydrophobicity

is related to the behavior of a molecule in water. If a

molecule is

hydrophobic

, it fears water while hydrophilic

molecules love water. For example, oils, waxes and fats

are hydrophobic; they do not dissolve in water. Table

sugar and table salt are hydrophilic, and they dissolve

quickly in water.

It is not uncommon for large molecules, such as

proteins, to have regions that are hydrophobic and other

regions that are hydrophilic. If these proteins are placed

in water, the hydrophobic regions tend to “bend away”

from water while their hydrophilic regions try to bend

toward the water. To a large extent,

it’s the bending of the protein’s

amino acid chain that is responsible

for its overall

conformation

or

molecular shape, with hydrophobic

regions “hiding” in the interior

of the molecule and water-loving

regions on the outside.

It’s important for you to know

that a bacterial cell contains many

different kinds of proteins. The

diagram below is greatly simplified

as it indicates only a few kinds. The

problem, however, is how do you

separate a single protein, like mFP,

from all of the others? A typical

bacterium may contain more than a

1000 different kinds of protein. The

use of the recombinant expression

vector, pARA-R, will make mFP isolation somewhat

easier:

The E. coli

cells you have cultured will have been

made to produce a disproportionately high concentration

of m F P.

Protein purification can use hydrophobicity to separate

and purify protein molecules. One common purification

procedure that uses differences in hydrophobicity to

separate proteins is called column

chromatography

.

Column chromatography uses a plastic or glass cylinder

into which a separating medium, referred to as “resin,”

is placed. The specific type of resin used will vary

depending on what type of protein is being purified. In

this lab, we will be using a resin bed consisting of small

hydrophobic beads. Mutant fluorescent protein is highly

hydrophobic and when mFP is placed into a solution of

high salt concentration

, the mFP molecule is distorted

in a way that will cause the hydrophobic regions of

the molecule to adhere to the hydrophobic resin in the

chromatography column. The hydrophilic proteins made

by the cell continue down the column, through the resin

without sticking to the resin bed and are flushed away.

Once the mFP is trapped in the resin bed, the column

can be washed with a solution of lower salt concentration

to elute (wash out) moderately hydrophobic molecules

from the column. This column

wash buffer

will have a

slightly

lower salt

concentration than the solution used

to bind mFP to the resin but not so low as to wash the

mFP from the resin. Finally, we can use a solution of

very low salt

concentration to elute or release the mFP

from the resin beads. Under low salt concentration, the

hydrophobic regions of the mFP molecule point toward

the interior of the molecule, thus releasing the mFP from

the hydrophobic resin in the column.

Industrial protein purification is much more complex

than this mFP purification protocol, but the principles

employed by industry are similar. The mFP sample that

you obtain from this purification does contain other

proteins. The procedure, however, has removed many of

the other proteins present in the bacterium’s cytoplasm

Materials

Reagents

2 mL LB/amp/ara culture of

E. coli

(Lab 6)

Lysis buffer (TE, NaCl, SDS)

Binding buffer, 4 M (NH

4

)

2

SO

4

Column equilibration buffer, 2 M

(NH

4

)

2

SO

4

Column wash buffer, 1.3 M (NH

4

)

2

SO

4

Elution buffer, 10 mM TE

10% Bleach or other disinfectant

TE (same as elution buffer)

e

quipment & supplies

Centrifuge

P-200 pipette and tips

P-1000 pipette and tips

Chromatography column

Microfuge tube rack

1.5 mL microfuge tubes

Permanent marker

6 mL waste collection tube

Cell-contaminated waste bag

Methods

Preparation of cell lysate from the overnight liquid culture

Obtain 1 mL LB/amp/ara culture from your teacher.

Examine this culture. What color is the culture?

Place this tube into the centrifuge.

Important:

You or your teacher will need to make certain

the tubes have been placed in the rotor in a balanced

configuration before the centrifuge is turned on. Centrifuge

the microfuge tubes for

5 minutes.

After the rotor has stopped,

carefully

remove your tube to

avoid disturbing the cell pellet.

Determine the location

of the mFP. Is it in the bacterial cell

pellet, or in the supernatant (the liquid

above

the cell pellet)?

Once you’ve determined the location of the mFP, carefully

decant (pour off) the supernatant into the beaker containing

disinfectant. Do this without disturbing the cell pellet.

Obtain a second 1 mL aliquot of the overnight culture and

repeat steps 3–6.

Pick up a tube of “Elution buffer” and “Lysis buffer” from your

teacher.

Invert the microfuge tube containing the cell pellet and,

using a small piece of paper towel, try to wick away as much

of the liquid as you can from your microfuge tube

without

touching

the cell pellet. Discard the used towel in the “cell-

contaminated waste” bag.

Using the P-200 pipette (set at “1-5-0”) and a clean tip,

transfer 150L of elution buffer to the cell pellet. Close

the cap tightly.

Resuspend the cells by dragging the tightly capped

microfuge tube briskly across the surface of the microfuge

tube rack. You may need to do this several times to

resuspend the cells. Examine the tube carefully to make

certain there are no visible clumps of cells.

Using the P-200 pipette (set at “1-5-0”), transfer 150L

of lysis buffer to the resuspended cells. Lysis buffer will

dissolve E. coli’s plasma membrane which helps to break

open the cells. After adding the lysis buffer to the cells,

cap the tube tightly and drag the tube vigorously across

the plastic tube rack several times to mix.

Check to see if you have labeled this tube with your group

number and class period. Give the tube to your teacher.

The cells will be left to incubate at room temperature overnight.

(Incubate cells overnight at room temperature.

Cells can then be frozen until the next lab.)

Purification of mutant fluorescent protein from the cell lysate

Getting the materials

Organize your group for multi-tasking.

Person A

checks to see if the following reagents are at

your workstation. These reagents will be shared with

another group.

Binding buffer

Equilibration buffer

Wash buffer

Person B

collects the lysed cells from your teacher; these

cells were frozen overnight. This person should take the

cells to the centrifuge to pellet the cell debris.

Person C

collects the following supplies:

2•

1.5

mL

microfuge

tubes.

Label

one

tube

“mFP

”

and

the other “

super

”

1•

6

mL

waste

collection

tube

(This

may

be

already in the plastic tube rack.)

Preparing the column

Set up your chromatography column as directed by

your teacher, being careful not to dislodge the stopcock

attached to the lower portion of this tube.

Set the waste collection tube or container under the

stopcock. Carefully open the column by turning the

stopcock valve and allow the equilibration buffer to begin

draining from the column.

Leave about 1mm of this liquid

above the resin bed to avoid drying out the resin in the

column.

Your chromatography column is now ready for the mFP

sample. While you are waiting for the mFP sample, be

certain that the fluid is not draining from the column. If the

waste collection tube is filled with liquid, this is a good time

to dump the liquid down the sink.

Preparing the mFP sample

Centrifuge the cell lysate for

five minutes

to pellet the cell

debris. You or your teacher will need to check the rotor to be

certain it is balanced before closing the lid and spinning.

Balancing these tubes before centrifugation is very

important.

After centrifugation, pick up your microfuge tube. Examine

the microfuge tube. Where is the mFP: supernatant or cell

pellet?

Without disturbing the cell debris pellet

,

carefully remove

200

L of supernatant using the P-200 pipette (set at

“2-0-0”) and a clean tip. Do this without transferring any

cell debris. If you dislodge the debris pellet, you will have

to centrifuge the tube again. Dispense the 200

L of clean,

debris-free supernatant into a 1.5 mL microfuge tube

labeled “

super

”(one of your group members should have

labeled this tube).

Replace the pipette tip on the P-200 and add 200

L of

binding buffer

to the supernatant you dispensed in the tube

labeled “super.” Mix the binding buffer with the supernatant

by gently pumping the solutions in and out using this pipette.

Using the p-1000 pipette (set at “0-4-0”) and a clean tip, add

400

L of this solution, mFP supernatant/binding buffer, to

the prepared column using the same pipette you used to mix

the solutions. Do this without disturbing the surface of the

resin bed by dispensing the solution down the side of the

column.

Without allowing the column to run dry, open the stopcock

and allow the solution in the column to drain into the waste

collection tube.

Leave about 1or 2 mm of buffer above the

resin bed.

Examine the column and locate the mFP. Is the mFP spread

throughout the resin bed or does it appear to be restricted

to a single band?

Using the P-1000 pipette (set at “1-0-0”), add 1000

L

(=1ml) of

wash buffer

gently down the side of the column.

Without allowing the column to run dry, allow this buffer to

drain from the column,

leaving 1 or 2 mm of buffer above

the resin bed.

Examine the column and locate the mFP. Has the location

of the mFP changed in the resin bed? The wash buffer will

elute some of the less hydrophobic proteins off the column.

The wash buffer’s salt concentration is less than the binding

buffer but not so low as to cause the mFP to release from

the resin.

Using the P-1000 pipette (set at “1-0-0”) and a clean tip, add

2 x 1000

L (=2ml total) of

elution

buffer

gently down the

side of the column. As the mFP begins to drip from the tip of

the stopcock, collect the protein in the tube labeled “

mFP

.”

Collect only the red eluate into this tube. Cap the tube when

you have collected all the mFP.

After all the mFP has been collected, add 2000

L (=2ml) of

equilibration buffer

to the column using the P-1000 pipette

and a clean tip. This will help prepare the column for the next

class.

Cap the column tightly.

The solution in the waste collection tube can be discarded

down the sink.

All microfuge tubes, except the one containing your mFP,

should be discarded in the cell-contaminated waste bag.

Compare your tube with mFP tubes from other groups.

Is there a difference in intensity of color from sample to sample?

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