Quiz 3

Question 1

Explain how fitness of a gene product affects mutations which lead to changes in primary structure?

Answer 1

If the mutation is deleterious or lethal in their effects, the mutation does not propagate. • If the mutation has no effect it can remain in the population. • If the mutation improves the fitness of the organism in its environment, it will be selected for and amplified in the population

Question 2

hypervariable residue

Answer 2

Sequence positions that have no functional / structural role are free to mutate without affecting protein structure

Question 3

invariant residue

Answer 3

Critical sequence positions required for function,or to maintain 3D structure, do not change

Question 4

conservative residue

Answer 4

Some other sequence positions can only change to residues with similar chemical properties in order for the protein to remain physically similar

Question 5

Describe the energetics that favour protein folding

Answer 5

∆G_system = ∆H_system -T∆S_system = -T∆S_universe

ΔG_system must be

Folding “orders” the polypeptide, decreasing entropy, leading to ΔS_system

This must be offset by decreasing enthalpy (∆H_system

This adds disorder into the universe, increasing overall entropy (∆S_universe > 0)

Question 6

Protein folding is exothermic or endothermic?

Answer 6

exothermic, water molecules are released out of a locked conformation and have vibrational energy

Question 8

native state of a protein

Answer 8

the fully folded state of that protein

Question 9

how does a misfolded protein escape from a local energy well that is not its native state?

Answer 9

molecular chaperones

Question 10

what proteins can fold on their own

Answer 10

ususally only small, soluble, globular proteins

Question 11

at an extremely high level, what do molecular chaperones do?

Answer 11

Molecular chaperones add energy into the stalled folding process in a effort to push the protein over the activation energy barrier into a lower free energy minimum.

Question 12

what are the two major families of molecular chaperones in eukaryotes?

Answer 12

Hsp70 (DnaJ is the bacterial homologue) and Hsp60

Question 13

How did eukaryotic molecular chaperones get their names?

Answer 13

Hsp60 and Hsp70 (DnaK in bacteria) are “heat shock proteins”, named for the fact they are upregulated when cells briefly experience higher temperatures. Because heat denatures proteins, heat shock proteins attempt to rescue them and refold them correctly.

Question 14

Hsp60

Answer 14

Heat shock protein 60 (Hsp60) is a mitochondrial chaperonin that is typically held responsible for the transportation and refolding of fully synthesized proteins from the cytoplasm into the mitochondrial matrix. In addition to its role as a heat shock protein, Hsp60 functions as a chaperonin to assist in folding fully synthesized linear amino acid chains into their respective three-dimensional structure. Through the extensive study of groEL, Hsp60’s bacterial homolog, Hsp60 has been deemed essential in the synthesis and transportation of essential mitochondrial proteins from the cell’s cytoplasm into the mitochondrial matrix.

Polypeptide initially binds to hydrophobic regions around the rim.

7 subunits, each binds ATP which elicits a conformational change and opens the recess for the rest of the protein to be pulled inside, after which the cap binds. Only one side of the barrel is occupied at any given time.

ATP is hydrolysed and the cap dissociates, the protein is released whether folded or not. Multiple cycles may be required to complete folding.

Question 15

Hsp70

Answer 15

Hsp70 (bacterial homologue DnaK) binds to hydrophobic amino acids about 7 residues in length as the protein emerges from the ribosome they are being synthesized from.

Aided by Hsp40 (bacterial homologue DnaJ)

Uses ATP to ADP + Pi hydrolysis to initiate conformational change that locks Hsp70 tightly to polypeptide.

Hsp40 and ADP dissociate, binding of new ATP reverses conformational change releases folded polypeptide, process repeats.

Question 16

What are the bacterial homologues to Hsp40 and Hsp60 and Hsp70?

Answer 16

• Hsp40* = DnaJ
• Hsp60* = GroEL
• Hsp70* = DnaK

Question 19

Disulfide isomerase

Answer 19

Disulfide isomerases are chaperones that help many proteins form their correct disulfide bonds. Without these enzymes, initial folding may bring two Cys residues together that should not be bonded. Disulfide isomerase interchanges and shuffles these bonds until the correct disulfides are formed.

Question 20

Peptide prolyl cis-trans isomerase

Answer 20

Peptide prolyl cis-trans isomerase (PPI) are chaperones that help in the conversion between trans and cis prolines

Question 21

PPI

Answer 21

Peptide prolyl cis-trans isomerase (PPI) are chaperones that help in the conversion between trans and cis prolines

Question 22

Protease

Answer 22

Proteases cleave off signal sequences, that target proteins to their cellular compartment, prosequences, that prevent a protein being active in the wrong cellular compartment, and secretion signals, that cause proteins to be secreted out of the cell.

Question 23

List five molecular chaperones that aid protein folding

Answer 23

• Hsp60* (barrel and cap that act on fully synthesised proteins, uses ATP)
• Hsp70* (with Hsp40, acts on 7 hydrophobic amino acids as they exit the ribosome during translation, uses ATP)
• Disulfide isomerase* (corrects Cys S-S bonds)
• Peptide prolyl cis-trans isomerase* (PPI) (converts cis and trans prolines)

Proteases (generic term) (cleaves signal sequences, prosequences, and secretion signals)

Question 24

signal sequence

Answer 24

A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short (5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway.[1] These proteins include those that reside either inside certain organelles (the endoplasmic reticulum, golgi or endosomes), secreted from the cell, or inserted into most cellular membranes. Although most type I membrane-bound proteins have signal peptides, the majority of type II and multi-spanning membrane-bound proteins are targeted to the secretory pathway by their first transmembrane domain, which biochemically resembles a signal sequence except that it is not cleaved.

Question 25

prosequence

Answer 25

A protein precursor, also called a pro-protein or pro-peptide, is an inactive protein (or peptide) that can be turned into an active form by posttranslational modification. The name of the precursor for a protein is often prefixed by pro. Examples include proinsulin and proopiomelanocortin.

Protein precursors are often used by an organism when the subsequent protein is potentially harmful, but needs to be available on short notice and/or in large quantities. Enzyme precursors are called zymogens or proenzymes. Examples are enzymes of the digestive tract in humans.

Question 26

secretion signal

Answer 26

A form of signal sequence that targets a protein for excretion: A signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide) is a short (5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway.[1] These proteins include those that reside either inside certain organelles (the endoplasmic reticulum, golgi or endosomes), secreted from the cell, or inserted into most cellular membranes. Although most type I membrane-bound proteins have signal peptides, the majority of type II and multi-spanning membrane-bound proteins are targeted to the secretory pathway by their first transmembrane domain, which biochemically resembles a signal sequence except that it is not cleaved.

Question 27

cofactor

Answer 27

an additional atom or molecule essential for protein structure/function

A cofactor is a non-protein chemical compound that is required for the protein’s biological activity. These proteins are commonly enzymes, and cofactors can be considered “helper molecules” that assist in biochemical transformations.

Cofactors can be subdivided into either one or more inorganic ions, or a complex organic or metalloorganic molecule called a coenzyme; most of which are derived from vitamins and from required organic nutrients in small amounts. A cofactor that is tightly or even covalently bound is termed a prosthetic group. Some sources also limit the use of the term “cofactor” to inorganic substances. An inactive enzyme without the cofactor is called an apoenzyme, while the complete enzyme with cofactor is called a holoenzyme.

Some enzymes or enzyme complexes require several cofactors. For example, the multienzyme complex pyruvate dehydrogenase at the junction of glycolysis and the citric acid cycle requires five organic cofactors and one metal ion: loosely bound thiamine pyrophosphate (TPP), covalently bound lipoamide and flavin adenine dinucleotide (FAD), and the cosubstrates nicotinamide adenine dinucleotide (NAD+) and coenzyme A (CoA), and a metal ion (Mg2+).

Organic cofactors are often vitamins or are made from vitamins. Many contain the nucleotide adenosine monophosphate(AMP) as part of their structures, such as ATP, coenzyme A, FAD, and NAD+. This common structure may reflect a common evolutionary origin as part of ribozymes in an ancient RNA world. It has been suggested that the AMP part of the molecule can be considered a kind of “handle” by which the enzyme can “grasp” the coenzyme to switch it between different catalytic centers.

Question 28

metal cofactor

Answer 28

the simplest cofactors, metal ions that bind directly to the protein.

can be involved in catalysis, in binding ligands, in electron transfer, can have structural role, or help neutralise highly charged biomolecules

different metals have different preferred protein ligands (generally N, O, or S) and adopt different geometries. Water (or hydroxide) generally occupies at least one ligand position

metals must come from diet

Question 29

what roles can metal cofactors fufill?

Answer 29

can be involved in catalysis, in binding ligands, in electron transfer, can have structural role, or help neutralise highly charged biomolecul

Question 30

what are the most common structural metal cofactors and why?

Answer 30

calcium (Ca²⁺), magnesium (Mg²⁺), zinc (Zn²⁺)

they are charge neutralising metals

(shown: X, Y are negative residues like D or E, stabilised in close proximity by Ca²⁺, which prevents charge repulsion between the residues)

Question 31

organometallic cofactor

Answer 31

in-class example: Haeme group

cofactors in many proteins, most important of which are haemoglobin, myoglobin, cytochrome

consists of a porphyrin ring which may or may not be covalently bonded to the protein backbone.

synthesised enzymatically, disease results from dysfunction in its synthesis.

Question 32

vitamin

Answer 32

All the water soluble vitamins are either coenzymes or are the raw material for the synthesis of coenzymes.

Question 33

what is the difference between ligands and cofactors?

Answer 33

Ligand is a more general term refering to a compound that binds to a given enzyme. It could be a substrate, but also have regulatory roles, for instance. A cofactor refers specifically to a compounds that are part of an active enzyme.

Question 34

what are the most common metal cofactors for catalysis and why?

Answer 34

Fe²⁺ and Fe³⁺

Cu²⁺ and Cu⁺

generally cofactors that can change their state easily

Although Zn²⁺ cannot change state, it is a good Lewis Acid (accepts electron pair) and plays a catalytic role in some enzymes

Question 35

what are the most common metal cofactors for electron transfer and why?

Answer 35

Fe²⁺ and Fe³⁺

Cu²⁺ and Cu⁺

generally cofactors that can change their state easily