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 cistrans 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 nonpolar 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 Hbonding 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).

H
C

N
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.

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.

H3N

HR

HR

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.