Learning objectives for BIOCHEMISTRY

CHAPTER 4: The Three-dimensional structure of proteins

Explain the 4 different levels of protein structure, for each level of protein structure fully understand and be able to explain:

Specifically what atoms in the amino acids are responsible for making each of these discrete levels of structure,

what forces are involved in forming and stabilizing each structural level, how the structural levels are dependent upon one another (this is expanded

on in bullet point 6 below).

What is a proteins native conformation and describe what chemical forces stabilize this conformation. What is a denatured protein conformation?

Describe the limitations that the peptide bond places on the possible conformations of the peptide backbone and protein.

Define the phi (), psi () & omega () bonds and what bond angles are allowed for these bonds. Which amino acids restrict the rotations of these bonds and which allow the

most rotations?

Understand how the common elements of secondary protein structure are formed (-helix, -sheet & -turn) and how the amino acid sequence can play a role in formation of these structural features be able to draw these out.

Explain why every level of protein structure is dependent on the lower levels of the protein structure. For example: how does the primary structure (or amino acid sequence)

influence the formation of the secondary level of protein structure, etc.?

How is the native conformation of a protein determined explain 2 techniques, the advantages & disadvantages of each

Describe the structural features of the keratin, collegen & globular proteins

Describe what forces drive protein folding and denaturation

What did the experiment involving the denaturation and renaturation of RNase A teach us? Why do not all proteins appear to behave in the same manner as RNase A?

What are the stages that occur during protein folding, what is a molten globule state?

What are the different methods you can use to denature a protein and how does each one of these methods or chemicals work to cause protein denaturation?

Describe how proteins can assist in the folding of other proteins; the function of molecular chaparones, the function of protein disulfide isomerase and peptide prolyl cis-

trans isomerase

Web site: http://www.umass.edu/molvis/tutorials/hemoglobin/pepstruc.htm

gives great description of the peptide backbone and the alpha-helix

Chapter 4 no omissions

Chapter 4 sample problems

1. For each peptide sequence below discuss what type of secondary structural element is

likely to be formed by each, give some explanation to justify your answer for each

sequence:

Sequence A: QKASVEMAVRNSG

Sequence B: SEDNQPGKSILW

Sequence C: MGLIVIVTFVAMH

2. Helices are described by the notation nm, where n is the number of residues per helical

turn and m is the number of atoms including H in the ring that is closed by the hydrogen bond formed in a helical structure. What is the notation for a standard -helix? Compare this helix with the 310 helix, specifically state in what protein is the 310 helix is

likely to be found in and which, an alpha or 310 helix, is steeper (has a greater distance

between amino acids in the helix)?

3. Compare and contrast a globular proteins structure to the structure of a fibrous protein such as keratin.

4. Describe the primary, secondary, tertiary and quaternary structures that are observed

in the protein collagen.

5. How does the 7-residue repeat of -keratin promote formation of the coiled coil?

6. What distinguishes a -bend from an omega () loop?

7. Why is it that ion pairs do not contribute significantly to protein stability?

8. What force or forces are the major contributors to the stability of a proteins native conformation?

9. How does the polarity or charge of an amino acid affect its likely location within a

protein?

10. Explain what the primary structure of a protein is.

11. Explain what a pleated sheet is and how this structure is formed (include in your explanation what atoms in an amino acid help form this structure and how the structure is

stabilized).

12. Explain what is meant by the tertiary structure of a protein, include in this description

what types of interactions and atoms are involved in those interactions in order to

stabilize this structure.

13. The ability to denature and renature a protein outside of the cell led to the conclusion

that which aspect of a peptide is a key feature that determines protein folding?

14. What are the two kinds of molecular chaperones and describe in general how they

work.

15a. What are the two most commonly used methods for determining a proteins native conformation?

15b. What two things do both of these techniques for determining protein structure rely

on the researcher knowing or having?

16a. The fibrous protein collagen has a primary sequence that is mainly repeats of the

sequence: Gly-X-Y (with X usually being proline and Y usually being a modified form of

proline). What does this result in with respect to the formation of the secondary structure of collagen? Give specific details here.

16b. How is this secondary structural conformation in collagen stabilized? Does this

make it like or unlike other secondary structural elements weve discussed?

16c. What is the tertiary structural element that predominates in collagen? How does the

amino acid sequence allow for this to form?

17. What is the amide plane and how is it that the amide plan is formed?

18. Explain the difference between a protein in its native conformation and its denatured

state. Give specific details about the structural elements that the protein in each form

would possess.

19. Draw a stereochemically correct tripeptide (use R to indicate the side chains) and

clearly label all phi, psi and omega bonds.

Chapter 4 sample problems answers

1. For each peptide sequence below discuss what type of secondary structural element is

likely to be formed by each, give some explanation to justify your answer for each

sequence:

Sequence A: QKASVEMAVRNSG

Sequence B: SEDNQPGKSILW

Sequence C: MGLIVIVTFVAMH

Seq A : has QKASVEMAVRNSG The bold font amino acids are all charged and are

regularly spaced so that in an alpha helix they would form a charged face to the helix

Seq B: beta turn because of the PG in the sequence and the surrounding hydrophilic

amino acids around the PG sequence

Seq C: very V and I rich these are mostly found in a beta sheet conformation.

2. Helices are described by the notation nm, where n is the number of residues per helical

turn and m is the number of atoms including H in the ring that is closed by the hydrogen bond formed in a helical structure. What is the notation for a standard -helix? Compare this helix with the 310 helix, specifically state in what proteins is the 310 helix is

likely to be found in and which, an alpha or 310 helix, is steeper (has a greater distance

between amino acids in the helix)?

The standard -helix is described by a 3.613 notation. The 310 helix is found in fibrous proteins like collagen (collagen has 3.3 turns in its helical secondary structure). The 310

helix is steeper between amino acids there is a 0.2 nm rise (distance between amino

acids) and the -helix has a 0.15 nm rise.

3. Compare and contrast a globular proteins structure to the structure of a fibrous protein such as keratin.

Keratin has a sequence that is highly repetitive (every fourth amino acid is

nonpolar).

Globular proteins are usually made up of more than one type of secondary

structural element but keratin is only made up of an -helix element. Fibrous proteins are elongated in overall shape where as most globular proteins

are more spherical in overall shape.

Both types of proteins have secondary structures stabilized by H-bonds, tertiary

and quaternary structures are stabilized by hydrophobic interactions, disulfide bond

formation between cysteine side chains,

4. Describe the primary, secondary, tertiary and quaternary structures that are observed

in the protein collagen.

Primary structure of collagen is the repeating sequence: gly-X-Y where X often is pro

and Y is often hydroxyproline.

Secondary structure is a left-handed helix with 3.3 residues per turn formed by repulsion

of prolines. Often collagen is considered to have no secondary structural elements since

there is no H-bonding interactions stabilizing this helical arrangement of the amino acids.

Tertiary structure is a triple helix (tropocollagen molecule) where 3 secondary structural

elements wrap around each other in a right-handed twist. This is stabilized by H-bonds

between the individual helices.

Quaternary structure is the alignment of tropocollagen molecules in a side-by-side

staggered fashion. This is stabilized by covalent bonds or crosslinks formed between

lysine side chains.

5. How does the 7-residue repeat of -keratin promote formation of the coiled coil?

The first and fourth amino acid residues of -keratins 7-residue repeat are primarily non-polar residues that form a hydrophobic strip along one side of the helix. When the

hydrophobic strips of the 2 keratin helices interact the helices incline toward one another

slightly causing the helices to coil around one another.

6. What distinguishes a -bend from an omega () loop?

The -bend involves only 4 amino acids and results in the reversal of direction of the peptide chain. The loop involved 6 16 amino acids and is a large structure often located on the exterior of the protein.

7. Why is it that ion pairs do not contribute significantly to protein stability?

Electrostatic interactions contribute little to the stability of tertiary structures because

water interacts similarly with the native and denatured forms of the proteins. These

electrostatic interactions play a larger role in aligning residues in specific secondary

structures and domains.

8. What force or forces are the major contributors to the stability of a proteins native conformation?

Hydrophobic interactions are the major stabilizing force for a proteins structure.

9. How does the polarity or charge of an amino acid affect its likely location within a protein?

Charged or polar amino acids are commonly on the outside of the protein molecule, where these

are in contact with water. Nonpolar amino acids are in the interior region of the protein, out of

contact with water due to the hydrophobic effect.

10. Explain what the primary structure of a protein is.

This is the order (or sequence) of amino acids in the protein, the amino acids are held

together by a covalent peptide (or amide) bonds.

11. Explain what a pleated sheet is and how this structure is formed (include in your explanation what atoms in an amino acid help form this structure and how the structure is

stabilized).

The pleated sheet is a common element of secondary structure formed by H-bonding between the Carbonyl oxygen and amino H atoms in the peptide backbone. The H-bonding here occurs

between amino acids that are more than 4 amino acids away from one another in the primary

protein sequence. Overall, 2 or more sections of the polypeptide chain interact through H-

bonding and the peptide chain takes on a pleated appearance such that the amino acid side chains

are located at the bends or points of the pleats. The pleating shape helps maximize the H-bonding

interactions between strands.

12. Explain what is meant by the tertiary structure of a protein, include in this description what

types of interactions and atoms are involved in those interactions in order to stabilize this

structure.

The tertiary structure of a protein is formed by interactions between secondary structural

elements. These interactions are stabilized by hydrophobic interactions, H-bonding, ionic

interactions, various types of chemical crosslinking (like Zinc finger formation) and disulfide

bridge formation mainly between atoms in the amino acid side chains.

13. The ability to denature and renature a protein outside of the cell led to the conclusion that

which aspect of a peptide is a key feature that determines protein folding?

The proteins primary sequence contains most or all of the information needed for a protein to fold.

14. What are the two kinds of molecular chaperones and describe in general how they work.

The two kinds are: 1. heat shock proteins 2. Chaperonins

Molecular chaperones make hydrophobic interactions with unfolded proteins and this prevents

hydrophobic regions of different proteins from aggregating (forming large protein complexes that

are dead-end protein products). The chaperones will hydrolyze ATP (use energy) and this will change the structure of the chaperone and the chaperone-unfolded protein interaction allowing for

the protein to fold.

15a. What are the two most commonly used methods for determining a proteins native conformation?

1. NMR

2. X-ray crystallography

15b. What two things do both of these techniques for determining protein structure rely

on the researcher knowing or having?

1. pure protein

2. knowing the amino acid sequence of the protein

3. having a water soluble protein or a protein that will crystallize

16a. The fibrous protein collagen has a primary sequence that is mainly repeats of the

sequence: Gly-X-Y (with X usually being proline and Y usually being a modified form of

proline). What does this result in with respect to the formation of the secondary structure of collagen? Give specific details here.

An extended helix is formed with 3 amino acids per turn of the helix. This is NOT an

alpha helix.

16b. How is this secondary structural conformation in collagen stabilized? Does this

make it like or unlike other secondary structural elements weve discussed?

By repulsion of the proline side chains and the additional constraints on the peptide back

bone (fewer bonds have free rotation because of the constraints put onto the peptide

backbone by proline). This makes it unlike the other secondary structural elements weve discussed since those are all stablilized by hydrogen bonding interactions between atoms

in the peptide backbone, there is no hydrogen bonding in this secondary structural

element.

16c. What is the tertiary structural element that predominates in collagen? How does the

amino acid sequence allow for this to form?

A triple helix where 3 extended helices wrap around one another and are stabilized by

hydrogen bonding between atoms in the peptide backbone on the different helix strands

that are in the triple helix. The presence of glycine every third amino acid allows these

peptides to come into close contact with one another to form the triple helix. This is a

direct result of glycine having the smallest side chain.

17. What is the amide plane and how is it that the amide plan is formed?

The amide plane is the carbonyl oxygen, amide H, and the two alpha Cs attached to the amide N and carbonyl C all lie in the same plane (the atoms outlined by the blue

rectangle).

C N

C

O

H

C This is caused by the ability of the carbonyl C and amide N to form a double bond with

one another through resonance, shown below. This means that the amide C-N bond has

double bond characteristics specifically no free rotation of that bond.

C N

C

O

H

C

18. Explain the difference between a protein in its native conformation and its denatured

state. Give specific details about the structural elements that the protein in each form

would possess.

A native protein is a protein in its biologically active conformation and the protein here

would have all structural elements: primary, secondary, tertiary and if needed quaternary

elements present. A denatured protein doe not have any biological activity and has only

the primary protein structure present; none of the other structural features are formed.

19. Draw a stereochemically correct tripeptide (use R to indicate the side chains) and

clearly label all phi, psi and omega bonds.

H3NC

CN

CC

NC

C

RH

O HR

O

O

O

H

H

RH

The purple arrows point to the phi bonds, the blue arrows point to the psi bonds and the

short black arrows point to the omega bonds.