Chapter 4

Question 1

Make a list of 5 proteins and their general and specific function.

Note the figure and page numbers in your textbook that discuss the protein, note the biological context and note where in the cell the protein is most likely to be found.

Answer 1

There are many proteins in the textbook that you may have chosen. If you did this activity, then you already know about numerous proteins that we will talk about in later lectures.

Question 2

Describe the structural organization of a protein

Answer 2

See answer key

Question 3

What is mRNA? and what does it determine?

Answer 3

• mRNA is a copy of the DNA that encode the protein.

- It determines a proteins sequence of amino acids.

Question 4

What makes alpha helices and beta sheets favorable for folding patterns of proteins?

Answer 4

As the protein is being synthesized, it begins to fold back on itself.

Hydrogen bonds between backbone hydrogen (N-H) and backbone oxygen (C=O) make both alpha helices or beta sheets favorable

Question 5

What does the core of a protein consist of?

Answer 5

Non-polar amino acids.

Question 6

What does the surface of a protein consist of? What does it look like?

Answer 6

-The surface of a protein, which is bumpy and often has grooves or crevices, typically consists of charged or polar amino acids that give the surface a distinct distribution of negative and positive charges.

Question 7

What is a protein domain?

Answer 7

-A domain is a portion of the polypeptide chain that folds independently of the rest of the chain. Domains are structurally distinct regions of a single protein.

Question 8

What is the primary structure?

Answer 8

-The linear sequence of amino acids is referred to as a protein’s primary structure.

Question 9

What are the secondary structures?

Answer 9

-The folding pattern, typically numerous helices and sheets, but also some disordered regions, are referred to as a protein’s secondary structures.

Question 10

What are the tertiary structures?

Answer 10

-The overall shape (conformation) of a protein is referred to as its tertiary structure..

Question 11

In regards to levels of organization, where do domains fall?

Answer 11

Domains are a level of organization between secondary and tertiary structure: a domain has secondary structures and a protein’s tertiary structure often consists of multiple domains.

Question 12

What is the quaternary structure?

Answer 12

If a protein self-assembles (by complementarity) into a multisubunit complex, then the complex is referred to as the proteins’ quaternary structure.

Question 13

What do multisubunit complexes consist of?

Answer 13

Multisubunit complexes may consist of identical subunits (the same protein) or are assembled from different proteins.

Question 14

What do all amino acids share in common?

Answer 14

All amino acids have the same backbone: an amino
group (-NH +) with a covalent bond to a carbon (C ) that has a covalent bond to a 3-α
carboxylic acid (COO ).

-After incorporation into a polypeptide chain, the carboxylic acid group becomes a carbonyl group (C=O) and the amino group becomes an amine (-NH)

Question 15

What is different about each of the 20

amino acids used to build proteins?

Answer 15

-Each type of amino acid differs with respect to the side-chain, which has a covalent bond to the Cα. The side chains vary in size, shape, flexibility, full charge (negative or positive) and polarity or no polarity.

Question 16

What is the chemical reaction of peptide bond formation?

Answer 16

• A condensation reaction involves formation of a covalent bond, between two reactants, with removal of H2O.
• Peptide bond formation involves removal of H2O and so is a condensation reaction.

Question 17

Which end of a protein, the N-terminal or the C-terminal, is synthesized first?

Answer 17

• The N-terminal is synthesized first.

- The C-terminal has the last amino acid added to a polypeptide chain, which is then released from the ribosome.

Question 18

Give three examples of noncovalent bonds that contribute to the structure of a protein?

Answer 18

Hydrogen bonds between backbone hydrogens (N-H) and oxygens (C=O) make alpha helices and beta sheets favorable folds, and hence common secondary structures of proteins.

In the core of a protein, amino acid side chains are often nonpolar and packed tightly together. When pushing up against each other, the nonpolar atoms often form van der Waals attractions. Although each is individually weak, many van der Waals attractions add up to much stability.

Acidic and basic amino acids that are oriented towards each other form ionic bonds that contribute to conformation stability.

In addition to hydrogen bonds between backbone hydrogens and oxygens, hydrogen bonds can also form between two polar amino acid side chains.

Question 19

How are multi-protein complexes assembled?

Answer 19

Multiprotein complexes are self-assembled.

The protein subunits, after being synthesized, randomly diffuse about, until bumping into each other.

The protein subunits each have a surface that has complementarity to each other so that many noncovalent bonds form between subunits.

The noncovalent bonds hold the subunits together as a multprotein complex.

Question 20

Are disulfide bonds important to the structure of proteins, particularly extracellular proteins? Explain your answer

Answer 20

Yes, disulfide bonds can protect a protein from unfolding.

• Disulfide bonds happen between two cysteines. Disulfide bonds are covalent so require much more energy to break relative to noncovalent bonds.
• The covalent bond between two cysteine side chains adds much stability to a protein’s conformation.
• Extracellular conditions are more likely to cause a protein to unfold, so to maintain function, extracellular proteins often have disulfide bonds to prevent unfolding.

Question 21

What is an allosteric protein?

Answer 21

An allosteric protein has at least two binding sites, whose surface details depend on whether the other site is in a bound or unbound state. The two sites are coupled by conformational change. Binding at one site causes a change in conformation that alters the surface details at the other site.

Question 22

Why would the work ‘coupler’ be used to describe an allosteric protein?

Answer 22

-An allosteric protein is a coupler protein; the protein couples two events. For example, in feedback regulation, an allosteric enzyme couples the concentration of an end product (output) to the catalysis of an input substrate.

Question 23

What is the mechanism that couples two events? What does it refer to?

Answer 23

• The mechanism that couples two events is allostery (or ‘coupling’),
• It refers to a protein changing shape (changing conformation). For example, binding of an end product to an allosteric enzyme causes the enzyme to change shape so that its catalytic surface can no longer bind to, or catalyze, the input substrate or chemical reaction.

Question 24

In general terms what are a proteins 3 intrinsic functions?

Answer 24

proteins have one or more of 3 intrinsic functions:
-Selective binding to a partner(s)

• Catalyzing a chemical reaction.
• Coupling two or more events.

Question 25

How does a proteins structure influence its function of selective binding?

Answer 25

• The surface details of a protein, its topology and charge distribution, determine which partner, out of thousands of different possible partners, the protein will bind to.
• A protein will only bind to molecules that have matching geometry and distribution of charge so that noncovalent bonds can form.
• The more noncovalent bonds that form, the greater the binding affinity and the longer the two partners stay together. Matching geometry and charge is called complementarity, and results in selective binding.

Question 26

How does a proteins structure influence its function of catalysis?

Answer 26

• The surface amino acids in the binding site (active site) of an enzyme determine which of the substrate’s covalent bonds will break, or which atoms of two substrates will form a covalent bond.
• The surface amino acids also participate in lowering the activation energy so that the rate of the chemical reaction is increased, a process called catalysis.

Question 27

How does a proteins structure influence its function of coupling?

Answer 27

An allosteric protein’s structure influences the coupling between two binding sites. Binding of one partner at one binding site causes the protein to change shape (change conformation) so that the surface details at the second binding site are altered, which can influence binding affinity or catalysis of the second partner. Conformational change involves breaking of one set of noncovalent bonds, often movement of a helix, and forming of a new set of noncovalent bonds.

Question 28

What are 3 ways that protein activity is regulated.

Answer 28

A protein can be regulated by:
-Covalent modification

• Allostery that involves nucleotides
• Allostery that doesn’t involve nucleotides

Question 29

How is protein activity regulated by covalent modification?

Answer 29

Protein kinases activate other proteins by transferring a phosphate onto the protein, a process called phosphorylation. Protein kinases function in signal transduction pathways.

Question 30

How is protein activity regulated by Allostery that involves nucleotides?

Answer 30

A protein that binds to and hydrolyses a nucleotide (ATP or GTP) can change between one or more shapes (conformations), with each shape having a different activity. For example, motor proteins use the binding and hydrolysis of ATP to walk in one direction along a microtubule. Another example is a G-protein using the binding and hydrolysis of GTP to switch between ‘on’ and ‘off’ conformations. Some G-proteins are signal transduction proteins.

Question 31

How is protein activity regulated by Allostery that does not involves nucleotides?

Answer 31

Proteins can be regulated by allostery that does not involve a nucleotide. For example, a product in a metabolic pathway can bind to an enzyme that catalyzes an earlier step in the pathway. Binding of the product causes the enzyme to change conformation so that it binds its substrate with more (positive feedback) or less affinity (negative feedback).

Question 32

Describe how enzymes work. Use the terms active site, activation energy, high- energy transition state and rate of reaction.

Answer 32

Enzymes have a catalytic, active site that selectively binds a substrate. When binding happens, the covalent bond to be broken is often distorted or strained, which puts the bond in a high-energy transition state. The high-energy state is a step that lowers the activation energy. Functional groups of amino acids in the active site (such as a carboxylic acid of glutamic acid) are oriented towards the substrate so that electrons move (are pushed or pulled) in the bond to be broken. This also lowers the activation energy and so increases the rate of the reaction. Sometimes amino acid side chains participate in the reaction by forming temporary covalent bonds with the substrate.

If there are two substrates and a bond is to form between them, selective binding aligns the substrates for correct reaction geometry: the atoms involved in the breaking and forming of covalent bonds are oriented next to each other. Orienting the two substrates helps lower the activation energy.

Question 33

List several small molecules that, when bound tightly to a protein, provide extra function to the protein

Answer 33

A heme group, Retinal (vitamin), Fe-S clusters, Copper Biotin (vitamin)

Question 34

Draw the structure of 4 different amino acids: one that has a basic side chain, one that has an acidic side chain, one that has an uncharged polar side chain and one that has a nonpolar side chain

Answer 34

Look at panel 2-5

Question 35

4-8: Explain how phosphorylation and the binding of a nucleotide can both be used to regulate protein activity.

Answer 35

Phosphorylation is when a protein kinase transfers a phosphate (from ATP hydrolysis) onto a protein substrate. The phosphate is covalently bonded to a serine, threonine or tyrosine (all three side chains have a -OH, which becomes -O- Phosphate). Phosphorylation can cause the protein to change shape thereby altering the protein’s catalytic activity or selective binding to partners. Even without changing shape, the phosphate adds a bump that can be selectively recognized by other proteins.
Binding of a nucleotide, and hydrolysis of the nucleotide, can cause a protein to change shape. Each shape can have a different catalytic activity or selective binding to partners.

Question 36

What do you suppose are advantages of either form of regulation?

Answer 36

Phosphorylation requires that the protein substrate bind to a protein kinase. In addition, returning the protein substrate to its original conformation requires interacting with another protein, a phosphatase, which removes the phosphate. Two proteins finding each other in the crowded cytosol takes more time then a small molecule, such as a nucleotide, finding a protein. So nucleotide binding has a kinetic advantage: faster regulation.
Regulation by a nucleotide usually involves binding, hydrolysis, and then release of the products (GDP + Pi). Binding, hydrolysis and release can cause a cycle of conformation
change, with the protein returning to its original state after a cycle. In contrast, a protein can be phosphorylated multiple times, with each combination (one phosphate, two phosphates, three . . .) causing the protein to have a different activity. So phosphorylation has an advantage of a protein having multiple, possible active conformations.

Question 37

T or F

a. The active site of an enzyme usually occupies only a small fraction of its surface.

Answer 37

True. Proteins are quite large relative to their substrates, so the active site where the substrate binds is only a fraction of its entire surface.

Question 38

T or F

Allosteric enzymes have two or more binding sites.

Answer 38

True. An allosteric protein couples two sites by conformational change. Binding of substrate or ligand at one site causes change in conformation that alters the properties at the second site.

Question 39

T or F

Noncovalent bonds are too weak to influence the three-dimensional structure of macromolecules

Answer 39

False. Although weak individually, many, many noncovalent bonds contribute to the folding and overall structure of a protein. The structure of nucleic acids is also held together by many, many hydrogen bonds between complementary base pairs.

Question 40

T or F
Catalysis by some enzymes involves the formation of a covalent bond between an amino acid side chain and a substrate molecule.

Answer 40

True. Temporary covalent bonds do sometimes form between enzyme and substrate.

Question 41

What common feature of α-sheets and β-sheets makes them universal building blocks for proteins?

Answer 41

Both α-helices and β-sheets are stabilized by hydrogen bonds that form between backbone amine (NH) and carbonyl (C=O) groups. Most amino acid sequences can form these two regular folding patterns because side chains are not involved in stabilizing the structures.
The exception is the amino acid proline, which causes the backbone to bend and thus prevents helices or sheets from forming

Question 42

Consider the following protein sequence as an α-helix:
Leu-Lys-Arg-Ile-Val-Asp-Ile-Leu-Ser-Arg-Leu-Phe-Lys-Val

How many turns does this helix make?

Answer 42

14/3.6 ≈ 4 (an α-helix has ≈ 3.6 amino acids per turn)

Question 43

Consider the following protein sequence as an α-helix:
Leu-Lys-Arg-Ile-Val-Asp-Ile-Leu-Ser-Arg-Leu-Phe-Lys-Val

Do you find anything remarkable about the arrangement of the amino acids in this sequence when folded into an α-helix? (Consult the properties of the amino acid side- chains)

Answer 43

Nonpolar and polar side chains alternate and result in a helix with a hydrophobic face and a polar face. Such a helix could be part of an ion channel or a coiled-coil domain of a protein or the polar face could be oriented to the surface of a protein, with the hydrophobic face oriented to the hydrophobic interior of the protein.

Question 44

Simple enzyme reactions often conform to the equation:

E+S ES –> EP E+P

A. What does ES represent in this equation?

Answer 44

ES represents the enzyme with its substrate bound.

Question 45

Simple enzyme reactions often conform to the equation:

E+S ES –> EP E+P

Why is the first step shown with bidirectional arrows and the second step as a unidirectional arrow?

Answer 45

After a substrate binds (associate), there is a chance the substrate will fall off (disassociate) before a reaction happens. After the chemical reaction happens, it is unlikely that the reverse reaction will happen, because the reverse reaction typically requires a greater input of energy (activation energy).

Question 46

Simple enzyme reactions often conform to the equation:

E+S ES –> EP E+P

Why does E appear at both ends of the equation?

Answer 46

E represents the enzyme. An enzyme is not chemically altered when it catalyzes a reaction and then releases the product, so the enzyme can bind another substrate.

Question 47

Simple enzyme reactions often conform to the equation:

E+S ES –> EP E+P

One often finds that high concentrations of P inhibit the enzyme. Suggest why this might occur.

Answer 47

Notice that the third step has a bidirectional arrow. The product often has complementarity to the active site, binding pocket, so the product can bind to the enzyme. If there is more product than substrate, then there will be more EP than ES.

Question 48

Simple enzyme reactions often conform to the equation:

E+S ES –> EP E+P

How is it possible for a change in a single amino acid in a protein of 1000 amino acids to destroy its function, even when that amino acid is far away from any binding site?

Answer 48

A change in a single amino acid can disrupt the proper folding of a protein. A protein that does not fold correctly will have altered surface details, including the precise geometry and charge distribution necessary for its binding site to selectively bind its substrate or ligand.