Biochemistry Exam 2

Classify the two types of proteins based on structure.

fibrous proteins & globular proteins

Describe the structure of fibrous proteins.

• made up of multiple subunits

• not water soluble

Describe the function of fibrous proteins.

provide structural support to cells & tissues

List three examples of fibrous proteins.

• keratin

• collagen

• fibroin

Describe the structure of globular proteins.

• spherical

• made up of multiple subunit

• generally water soluble

List two examples of globular proteins.

• immunoglobin

• albumin

What is fibroin?

the main fibrous, structural protein found in silk from moths

What type of structure does fibroin contain?

secondary structure, antiparallel beta sheets

Describe the secondary structure of fibroin.

close packing of antiparallel beta sheets (which result in a stronger structure) allowed by small side chains (R groups)

What bonds help to stabilize the structure of fibroin, thus making it strong?

• hydrogen bonds between the beta sheets

• van der waals between atoms (many due to tighter packing of beta sheets)

What is collagen?

the most abundant fibrous, structural protein found within the human body (ie: skin, blood vessels, ligaments, bones)

Describe the structure of collagen.

triple helix structure formed by three left-handed helices intertwined togeter

What do the left-handed helices combine to form?

the overall right handed triple-helix of collagen

Describe the primary structure of collagen.

amino acid sequence contains a repeating pattern of three amino acids

Define the general formula for this repeating amino acid sequence.

(Gly-x-y)_n

Define the typical formula for this repeating amino acid sequence.

(Gly-Pro-4Hyp)_n

What does each piece represent?

gly - amino acid glycine

pro - amino acid proline

4hyp - 4 hydroxyproline (amino acid after hydroxylation or introduction of OH)

n - number of repeats

What does this amino acid sequence give rise to?

a left-handed helix

Describe the general shape of the individual helices within the triple-helix.

extended, meaning they are much more open than ordinary, highly-coiled alpha helices

To sum…list three defining characteristics of the helices that make up the triple-helix.

• left-handed

• repeating amino acid sequence

• extended structure

What bonds stabilize the triple-helix?

hydrogen bonds between the three polypeptide chains/helices

What allows the helices to stay separate from each other/maintain their own shape?

steric crowding of proline & 4hydroproline due to their ring structures

What allows the helices to be packed together?

their glycine r groups which are very small (H) allowing for tight packing

Draw the reaction from proline to 3-hydroproline.

What is the difference between proline & 4-hydroproline?

a hydroxyl group (OH)

Name the enzyme responsible for adding a hydroxyl group onto proline, resulting in 4-hydroproline.

proline hydroxylase

What does proline hydroxylase need in order to produce the reaction?

vitamin C as a co-factor

What increases the thermal stability of the triple-helix?

4-hydroproline being in the y position, or being the third amino acid in the repeating sequence

Explain what happens when there is a lack of vitamin C.

• proline hydroxylase cannot convert proline to 4-hydroproline

• without 4-hydroproline, the structure of collagen becomes less stable

• this can lead to scurvy, a disease associated with the breaking down of blood vessels & connective tissues as a result of defective collagen production

Describe the structure of keratin.

coiled-coil

What bonds stabilize the structure of keratin (form between the coils)?

disulfide bonds

Describe the location of disulfide bonds.

between the sulfur atoms on two cysteine amino acids

Explain the directionality of the helices that make up the coiled-coil structure.

two right-handed helices twist together to form the left-handed coiled-coil

What does the extent or number of disulfide bonds in keratin depend on?

the function of keratin

Explain how the extent of disulfide bonds affects the structure of keratin.

• more disulfide bonds → more rigid structure

• less disulfide bonds → more flexible structure

List two examples of where keratin is found.

• hair

• nails

Explain the process of perming in regard to keratin.

• a reducing agent breaks disulfide bonds in keratin, converting them into thiol groups (SH) which causes more flexibility and allows for reshaping

• the hair is then placed in curlers to force a new shape

• an oxidizing agent reverses reaction so thiol groups then form disulfide bonds again, causing rigidity to stabilize the new shape

Draw the corresponding reactions.

What is immunoglobin?

an antibody

Describe the general structure of immunoglobin.

composed of four polypeptide chains

• 2 heavy chains (longer)

• 2 light chains (shorter)

What kind of bond holds together immunoglobin?

disulfide bonds

List the two locations of disulfide bonds in immunoglobin.

• between the two heavy chains

• between the constant domains of the light chain & the heavy chain

What are the abbreviations for the heavy chains & light chains of immunoglobin?

H₂ & L₂

Draw the immunoglobin structure with all parts labeled.

What is a domain in regard to proteins?

a region within a protein that folds into a structural unit

What domains do the heavy & light chains consist of?

constant domains & variable domains

Describe the constant regions of the heavy chain.

specific to antibody class

What makes up the antigen-binding site?

the variable domains of one heavy chain and one light chain

How many antigen-binding sites are there per immunoglobin?

2

What is a protease?

an enzyme that breaks peptide bonds in proteins

What fragments does protease treatment of immunoglobin result in?

• 2 fab fragments

• 1 F_c fragment

What does fab stand for?

fragment antigen binding

What does F_c stand for?

fragment crystallizable

What does each fragment contain?

• fab fragment: antigen binding site

• F_c framgent: constant domains of heavy chains

Draw each fragment.

What is the main force behind protein folding?

the hydrophobic effect

What is the hydrophobic effect?

when hydrophobic amino acids cluster together within the inside of a protein to minimize their interaction w/ water

Describe the stability of folded proteins v.s. unfolded proteins.

folded proteins are more energetically favored, thus more stable, than unfolded proteins

What dictates the protein folding pathway?

the primary structure or amino acid sequence

Define the relationship between the free energy of folded proteins & the free energy of unfolded proteins.

free energy of folded proteins < free energy of unfolded proteins

Do systems favor states with lower or higher free energy?

lower (so folded proteins)

Define the equation for ∆G in regard to folded/unfolded proteins.

∆G = G_{folded proteins} - G_{unfolded proteins}

Is ∆G negative or positive in regard to folded protein?

negative

What are the two protein folding pathways suggested?

• secondary & tertiary structures formed simultaneously

• secondary structures formed before tertiary structures

Why is there no confirmation of the correct pathway?

laboratory conditions do not reflect cellular conditions, so in-cell folding can be very different from folding under laboratory conditions

What are chaperone proteins?

proteins that facilitate the formation of 3D stable structures of other proteins

What are the 2 ways in which chaperone proteins help to form stable, 3D proteins?

• by helping them to fold correctly

• by rescuing misfolded proteins

What are the 2 types of protein folding diseases?

• loss of function

• gain of function

Describe each.

• loss of function reduces level of functional proteins

• gain of function result from proteins forming aggregate that interferes w/ cellular processes

What is aggregate?

a collection of proteins that abnormally associate

List examples of both types of diseases.

• loss of function: cystic fibrosis

• gain of function: alzheimer’s, huntington’s

List the 3 steps involved in protein purification.

• isolate protein from lysed cells/tissues

• use differences in biochemical properties of protein to separate from others

• use biochemical assays to check protein activity

Now list the 5 more specific steps in order to purify a protein.

• cell/tissue lysis

• preparative centrifugation

• salting out

• dialysis

• chromatography/purification

How is cell/tissue lysis usually achieved?

mechanically

What is preparative centrifugation?

a form of centrifugation which focuses on collecting, isolating, and purifying bulk components from an, often large, sample

What is the principle of preparative centrifugation?

high density molecules collect a the bottom of centrifuge as opposed to low density molecules

What is the principle of salting out?

exploit solubility of target protein w/ respect to other proteins in the mixture

Draw the salt-in/salt-out graph.

Describe what is happening to the protein during salting-in.

ions from the salt in the water interact w/ charged groups on the protein to dissolve it, increasing solubility

Describe what is happening to the protein during salting-out.

the numerous ions from the salt added are now competing w/ proteins for interaction w/ water

What happens when the salt concentration exceeds the protein’s solubility?

the protein precipitates

What is dialysis?

a diffusion-based technique used to separate small molecules from large ones using a semi-permeable membrane bag

Let’s say the MW cutoff for dialysis was 10k, what would happen to molecules smaller & larger than that weight?

• smaller: diffuses out of semi-permeable membrane bag & into buffer

• larger: remains in semi-permeable membrane bag

What else does dialysis allow for? When can this be applicable?

removal of impurities such as salt or imidazole, which makes dialysis a useful step after salting-in/salting-out or affinity chromatography

What is chromatography?

a technique used to separate proteins within a mixture based on how they interact w/ the separation material

Describe the overall set-up of chromatography.

protein mixture is added to to the top of the column, which contains a material called the stationary phase (often a resin), a liquid buffer called the mobile phase flows through the columns allowing different proteins to move through the columns at different rates which are collected in separate portions of liquids called fractions

What is it called when individual proteins exit the column at different times?

elution

List 3 different types of chromatography.

• gel filtration/size exclusion

• ion exchange

• affinity/ligand chromatography

What is gel filtration chromatography or size exclusion chromatography (SEC) based on?

size of protein

Describe the material used in SEC.

resin w/ many porous beads that only small molecules can enter

Differentiate between small & large proteins in SEC.

• small: enter porous beads, so have a longer path to get through gel and thus elute later

• large: cannot enter beads, so have a shorter path and elute first

What is ion exchange chromatography (IEC) based on?

charge of protein

Differentiate between positively charged & negatively charged matrix resin for IEC.

• positively charged: holds onto negatively charged proteins, so elutes positively charged proteins

• negatively charged: holds onto positively charged proteins, so elutes negatively charged proteins

Differentiate between anion & cation exchange chromatography.

anion: positively charged matrix

cation: negatively charged matrix

List an example of a matrix for each type of IEC.

• anion exchange chromatography: diethyl amino ethyl cellulose (DEAE)

• cation exchange chromatography: carboxymethyl cellulose (CMC)

What is the relationship between pI and pH?

if the pH of a solution is above the pI of a protein, that protein is negatively charged & if the pH of a solution is below the pI of a protein, that protein is positively charged

Which protein is positively charged? Which protein is negatively charged? Which protein elutes first?

Relevant Info

• CMC is negatively charged

• solution pH = 7

• protein 1 pI = 6.5

• protein 2 pI = 10.5

• protein 2 is positively charged

• protein 1 is negatively charged

• protein 1 elutes first

What is affinity chromatography based on?

specific ligand interactions of protein

Describe how affinity chromatography works.

ligand is covalently bound to matrix where target protein should have optimal interaction w/ ligand & therefore bind to column while other proteins flow through

How do you release the target protein after it binds to the column via the ligand?

the addition of free competing ligands that the target protein then binds to