The Three Dimensional Structure of Proteins

Question 1

Define Primary Structure

Answer 1

Refers to the amino acid sequence of the protein. It also includes disulfide bonds. It focuses on covalent bonds between the amino acid groups, not the side chains.

Question 2

Define Secondary Structure

Answer 2

It is the local conformations of the polypeptide backbone in regions of a folded protein. It does NOT include the side chains, it is simply H-bonding between the polypeptide backbone. Folds are considered secondary structure. Usually consists of alpha helices and beta sheets. Phi and Psi bonds are all the same; All phi bonds are equal and all psi bonds are equal.

Question 3

Define Tertiary Structure

Answer 3

It is the total 3-D shape of the protein and includes side chains. It is the folded form of the protein and it is determined by the fold of the polypeptide. Information for the tertiary structure is found in the primary structure.

Question 4

Define Quaternary Structure

Answer 4

It is when multiple subunits or tertiary structures are noncovalently attached. It is proteins with multiple subunits.

Question 5

Describe how peptide bond resonance hybridization can account for important properties of this bond such as planarity, polarity, resistance to rotation, and tendency to form hydrogen bonds.

Answer 5

Peptide bond resonance hybridization can account for properties such as planarity, polarity, resistance to rotation and tendency to form hydrogen bonds because of its partial double bond character that is generated. When the peptide bond (C—N) become a double bond, (C=N), the once carbonyl oxygen loses its double bond because it is the one that donated the electrons to the peptide bond. This then gives oxygen a negative bond, allowing it to Hydrogen bond with hydrogens. This double bond character also keeps it relatively planar and rigid (resistant to rotation).

Question 6

What are the characteristics of the peptide bond?

Answer 6

It is planar, has 40% double bond character, there is no rotation around it, it is polar and forms H-bonds, and it is trans (oxygen and hydrogen are trans to one another)

Question 7

Explain how the rotation of covalent single bonds in a polypeptide chain backbone can generate secondary structures.

Answer 7

The rotation around the phi and psi bonds allow the backbone to “rotate” and orient itself in a position that is thus able to generate H-Bonds with other backbone amino acids. Not all angles for the phi and psi bonds are equal though due to steric hindrance. In the secondary structures, all of the phi bonds and all of the psi bonds will be exactly equal in the alpha helix or beta sheet.

Question 8

Indicate whether or not all possible angles of phi and psi are possible.

Answer 8

No they are not due to steric hindrance with other molecules and side chains.

Question 9

Proline differs from other amino acids by having its side chain covalently attached to the amino nitrogen. Describe how this structural feature affects a proline-containing peptide bond and the rotation of around proline’s psi bond.

Answer 9

Proline generates “kinks” in the polypeptide and is known as a “helix breaker”. The phi bond in proline is fixed due to the cyclic structure and the peptide bond is actually much more single-bond in nature so it can rotate slightly. However, the nitrogen in the ring no longer has a hydrogen so it can no longer hydrogen bond. This also eliminates some of the resonance structures that were present previously. Because the phi bond cannot rotate it breaks the helix.

Question 10

Define regular secondary structure.

Answer 10

Regular secondary structures are those that have the same phi and psi angles.

Question 11

Indicate the number of amino acids in each turn of an α-helix.

Answer 11

3.6 residues per turn.

Question 12

Describe the positioning of the H-bonds that stabilize the helix.

Answer 12

The hydrogen bonds are intra-chain hydrogen bonds that stabilize the helix. Each peptide bond contains 2 hydrogen bonds, one between its oxygen and an N-H from an amino acid 4 residues above and one between its N-H and the O from an amino acid 4 residues below.

Question 13

a. Define an “amphipathic” α-helix.

Answer 13

An amphipathic alpha helix has one side that is polar or hydrophilic and the other side is nonpolar or hydrophobic.

Question 14

Describe what happens when an α-helix is followed by a Pro-Gly sequence in a polypeptide chain.

Answer 14

The Proline, Glycine sequence introduces a “kink” in the helix, causing a sharp, hairpin turn in the polypeptide chain. This is due to proline being cyclical and glycine being very flexible. The glycine “flexes” the helix and the proline introduces a “kink”.

Question 15

Describe the basic structure of a β-strand and indicate how the adjacent amino acid carbonyl oxygen atoms are pointed relative to each other.

Answer 15

The beta-strand is structured in a pleated sheet. The sheets can be parallel in which both strands have their N-terminus and C-terminus lined up (N to N, C to C). They can also be antiparallel in which the strands run opposite of one another. The adjacent amino acid carbonyl oxygen atoms are alternating relative to one another in order to produce the most energetically stable product.

Question 16

Describe how H-bonds are oriented in a β-sheet.

Answer 16

The hydrogen bonds in the beta sheet are made with every other amino acid residue compared to the alpha helix in which H-bonds occurred with every amino acid. This alternating pattern, however, only occurs in the beta-strands that are on the “outside” of the sheet. A sheet with two adjacent beta-strands on it would have hydrogen bonding on every amino acid.

Question 17

Indicate how amino acid side chains are oriented in a β-sheet.

Answer 17

Amino acid side chains are oriented such that they also alternate direction every other amino acid. However, in this case, one is up and the other is down.

Question 18

Define parallel and antiparallel β-sheets.

Answer 18

The sheets can be parallel in which both strands have their N-terminus and C-terminus lined up (N to N, C to C). They can also be antiparallel in which the strands run opposite of one another.

Question 19

What is the function of loops and turns?

Answer 19

Loops and turns link the regular secondary structure elements, α-helices and β-strands

Question 20

Indicate whether or not loops and turns can be categorized as regular secondary structures.

Answer 20

Loops and turns cannot be categorized as regular secondary structures because by definition, regular secondary structures have the same phi and psi bond angles. In loops and turns, the phi and psi bond angles differ.

Question 21

Predict where most hydrophobic amino acids are located within a protein structure.

Answer 21

Most hydrophobic amino acids are located within the inside of the protein. They are then surrounded by the exterior hydrophilic amino acids.

Question 22

Define a domain.

Answer 22

Domains are building blocks that nature uses to construct larger proteins. A domain is a small globular portion of a protein that is on average about 100 amino acids long (

Question 23

Indicate the average size of a domain.

Answer 23

They are on average 100 amino acids long but less than 200 amino acids.

Question 24

Indicate how domains are covalently linked in a multi-domain protein.

Answer 24

They are connected by linking polypeptides. Keep in mind, domains are part of the same polypeptide chain, thus some of the polypeptide chain is coding for these “linkers”. The linker protein can be an alpha helix or other structure.

Question 25

Explain why domains are referred to as building blocks or modules.

Answer 25

They are referred to as building blocks or modules because they can independently function on their own, however, that independent function is only a piece of the overall function of the protein. Therefore, they are specific building blocks that are necessary for the overall protein to carry out its necessary function.

Question 26

Define a “fold.”

Answer 26

A fold is the secondary structure of a domain. It is the specific arrangement of secondary structure elements of a domain. There is a large number of different folds.

Question 27

Define a “superfold.”

Answer 27

Superfolds are the overall common “folds” that reoccur in different proteins even that have different functions. They are folds that occur even in proteins that are unrelated by function, sequence, or evolution. They must be thermodynamically stable and easy to form.

Question 28

What determines the folding of a protein?

Answer 28

The amino acid sequence or primary structure

Question 29

Describe homology modeling as a technique for predicting the tertiary structure of a protein from the amino acid sequence.

Answer 29

Homology modeling involves using computational methods to predict a structure based on amino acid sequence alone for some small proteins.

It uses the following steps:
1. Search x-ray and NMR structure databases for a protein that has a similar amino acid sequence (the template)

2. Perform a sequence alignment between the protein and the “template protein,” optimizing for identical or similar amino acids.
3. Use computer programs to “thread” the protein sequence through to the x-ray structure of the template.

Question 30

Define “identity” as it applies to sequence alignment of two proteins.

Answer 30

Identity: Identity refers to the amino acids that are exactly the same as the template protein. One would tend to see the greatest identity between two similar enzymes in the amino acids in the active site.

Question 31

Define “similarity” as it applies to sequence alignment of two proteins.

Answer 31

Similarity: Similarity is defined as those that are identical PLUS those in which the difference is conservative. By being conservative it refers to the amino acids that are “similar” or, in other words, the mutation of an amino acid replacing a polar amino acid, for example, with another polar amino acid.

Question 32

Indicate whether or not mutations in a protein automatically lead to loss of protein function.

Answer 32

This statement is not necessarily true. Mutations in a protein DO NOT automatically lead to loss of protein function. For example, the amino acid change may not alter the conformation of the protein and not be involved in the active site and thus not have any true effect. Furthermore, the mutation could just be a mutation in the hydrophilic amino acids on the outside of the protein that still just need to be able to come in contact with water.

Question 33

Indicate what is most commonly affected by disease-associated mutations.

Answer 33

Mutations of amino acids involved in enzymatic activity or basic structure of the protein are most likely to be deleterious.

Question 34

Define a silent mutation

Answer 34

That which has no effect on the protein

Question 35

List the forces that stabilize a folded protein and describe how they work.

Answer 35

The stability of proteins are dependent on non-covalent bonds.

Hydrogen Bonds: Hydrogen bonds between the side chains help stabilize the overall tertiary structure of the protein. It is when a hydrogen atom is shared by two electronegative atoms, usually oxygen and nitrogen.

Ionic Bonds: Also referred to as salt bridges, are between a positive and negative amino acid of the protein just not on the surface since they would easily be disrupted by water.

Van der Waals Interactions: These are very weak but there are a lot of them. These are temporary attractions between two partial charges created by the electron cloud not “evenly” dispersing. They are distance-dependent forces meaning that an attractive force occurs at a certain distance. But, as the molecules move closer together you can start to get a repulsive force.

Hydrophobic Interactions: These are also very weak, but add up. They are interactions between two hydrophobic amino acid residues. Hydrophobic amino acids tend to aggregate with one another forming these interactions. These are not “true bonding.” When there are two hydrophobic molecules with water between them, the water is “unhappy” and thus it is squeezed out, creating more entropy. It is entropy driven. By increasing entropy it is favorable.

Question 36

Define “denaturation.”

Answer 36

Changes in temperature, pH, or other parameters can cause “denaturation”. Denaturation refers to a disruption in the protein folding or unfolding altogether. It is destabilizing these non-covalent interactions.

Question 37

Indicate whether an X-ray structure can always reveal the structure of the active protein.

Answer 37

No, an x-ray structure cannot always reveal the structure of the active protein because it is simply an average of the protein. Proteins are always moving on the order of picoseconds and due to the molecules constantly vibrating within it.

Question 38

Describe large “conformational changes” using hexokinase as an example.

Answer 38

Large conformational changes refer to the structure of the enzyme changing drastically upon binding of a substrate. In this example, hexokinase binds glucose and undergoes a drastic conformational change in order to get the correct aligning for the reaction to take place in the active site.

Question 39

Describe the structure of collagen.

Answer 39

Collagen is a three-chain superhelix (triple helix). It is 3 polyproline type II helices and each are ~1,000 amino acids long. It is NOT an alpha helix! The helices contain a large number of glycine, proline and hydroxyproline residues with glycine appearing every third residue. Gly-X-X-Gly-X-X-Gly…. Proline is typically found next to Glycine, followed by hydroxyproline.

Question 40

Indicate why glycine occurs in the amino acid sequence every third residue.

Answer 40

The glycine occurs in the amino acid sequence every third residue because it is where the three helices join one another. Therefore, as one looks at the structure of glycine, it can be noted that is the smallest amino acid residue containing only a hydrogen atom as the “R-group” or simply not having a side-chain.

Question 41

Predict the consequence of mutating a glycine to another amino acid in collagen.

Answer 41

By mutating the glycine to another amino acid, it will disrupt the tight “contact point” that previously existed between all of the glycines. Thus, one would expect that the triple helix would be disrupted, having a bump in the helix and causing it to be more susceptible to degradation.