Deck 20: Designer Proteins and Protein Folding

Question 1

If you would synthesize a 15-amino acid long peptide, and each cycle of the solid-phase synthesis had a 99% yield, what would be your overall yield?

A) 99%
B) 75%
C) 80%
D) 86%
E) 95%

Answer 1

86%

Question 2

The conserved folds found in proteins prove that these structures are selected by ________.

A) evolution
B) scientists
C) random sampling
D) mutation
E) default

Answer 2

evolution

Question 3

Protein engineering is possible because of advances in biochemistry and molecular biology.
Various _______ in a protein sequence are _____ in order to determine the role they played in the protein.

A) side chains; activated
B) amino acids; mutated
C) amino acids; activated
D) side chains; truncated
E) functional groups; replaced

Answer 3

amino acids; mutated

Question 4

An _______ is a _________ monomer used in peptide synthesis. A peptide is a ____ polymer and contains _____ bonds.

A) acid; single; homogeneous; covalent
B) amino acid; heterogeneous; condensation; hydrogen
C) amino acid; bifunctional; condensation; amide
D) ?-hydroxy acid; bifunctional; homogeneous; amide
E) none of the above

Question 5

Using a resin in solid-phase protein synthesis allows for _______.
I. efficient washing steps to remove the side products
II. keeping the growing peptide in solid phase
III. obtaining higher product yields
IV. using lower reaction temperatures

A) I and IV
B) II and III
C) I, II and III
D) II and IV
E) II, III and IV

Question 6

A solid-phase protein synthesis reaction cycle includes_______________.

A) washing steps
B) deprotection of the peptide amino terminus using piperidine
C) activation of the carboxy group on the amino acid monomer to be added to the growing peptide
D) coupling of the activated amino acid with the amino group of the growing peptide polymer
E) all of the above

Question 7

During solid-phase protein synthesis (SPPS) it is common to link the first amino acid to the resin by a(n) ___________.

A) hydrogen bond
B) ionic bond
C) ester bond
D) amide bond
E) none of the above

Question 8

The amine group of the amino acid monomers used in SPPS can be protected by a ____________.
I. phosphate group
II. tert-butoxycarbonyl group (t-Boc)
III. fluoprenylmethyloxycarbonyl group (Fmoc)
IV. sulfonamide group

A) I
B) II
C) III
D) II and III
E) III and IV

Question 9

Which amino acid side chains would need to be protected in SPPS?
I. hydroxyl groups of tyrosine and serine
II. carboxyl groups of glutamic acid and aspartic acid
III. amine group of lysine
IV. methyl group of alanine
V. phenyl group of phenylalanine

A) I
B) II
C) III and V
D) I, II and III
E) II, III and IV

Question 10

Protecting groups used for side chains of amino acids are different than the protecting group used on the terminal amine because ___________.

A) they can ionize.
B) of chemical interference.
C) of more activity.
D) they should not be deprotected during the deprotection reaction of the terminal amine
E) none of the above

Question 11

The purity of a peptide synthesized by SPPS is analyzed by ____________.

A) UV-VIS spectrophotometry
B) reverse phase HPLC
C) circular dichroism
D) GC-MS
E) IR

Question 12

What are the disadvantages of studying proteins in their purified form?

A) The natural regulators and molecular interactors are missing from the assay, which can lead to missing out on the actual function of the protein.
B) The protein may not be soluble.
C) The protein may not be stable.
D) The protein may aggregate.
E) none of the above

Question 13

Proteins can be synthesized in the lab by using ____________.
I. solid-phase peptide synthesis (SPPS)
II. protein overexpression in biological systems (cells)
III. esterification reactions
IV. polymerization reactions
V. hydrolysis reactions

A) I
B) II
C) III
D) I and II
E) IV and V

Question 14

Which statement is true about SPPS:

A) In SPPS the carboxy terminus of the peptide is covalently linked to the resin.
B) The next amino acid is coupled to the N- terminus of the growing peptide.
C) The carboxylic group of the amino acid to be added to the growing peptide is activated by using dicyclohexylcarbodiimide (DCC)
D) SPPS is used to synthesize up to 100-amino acid long peptides.
E) all of the above

Question 15

Engineered proteins can be studied in their natural environment in the cell. Natural proteins are engineered to contain a tag or a fusion protein in order to ____________.
I. improve protein stability at high temperatures
II. detect the localization of the natural protein in the cell
III. analyze the level of expression in the cell
IV. detect interactions with potential protein ligands in the cell
V. improve stability of the protein in the cell

A) I and II
B) II, III and IV
C) I, III and V
D) II, III and V
E) I and IV

Question 16

Expression vectors are engineered fragments of DNA that contain the necessary information, such as ______, for the cell to be able to express the protein at a high level.
I. cloning sites tailored to various restriction enzymes
II. promoters designed to work in the individual cell lines
III. an origin of replication
IV. visualization sites
V. antibiotics resistance marker gene

A) I
B) II
C) III
D) II, III, and V
E) I, II, and III

Question 17

The origin of replication enables the plasmid to ______________.

A) fuse with the call wall
B) replicate within the cell
C) fold properly
D) react to antibiotics
E) all of the above

Question 18

Plasmids are _________. They can be delivered into the cell by ______ or _______.

A) circular pieces of DNA; transfection; electroporation
B) pieces of DNA; translocation; transfusion
C) pieces of RNA; translocation; transfusion
D) pieces of RNA; transfection; elimination
E) none of the above

Question 19

Bacterial expression systems are used __________________.

A) when single chain proteins are expressed
B) to produce large quantities of protein
C) with a variety of expression vectors
D) with a variety of promoters to control the expression level of the protein
E) all of the above

Question 20

Mammalian protein expression systems __________.
I. sometimes use the strong viral promoter CMV
II. are used when the expressed proteins require post-translational modifications
III. require expensive cell culture reagents
IV. are easy to handle
V. are used for producing insulin analogs

A) I
B) II
C) III
D) I, II and III
E) II, IV and V

Question 21

An insect expression system uses a baculovirus that is engineered to encode the protein of interest. The virus is used to infect ovarian cells from Spodoptera frugiperda, the fall armyworm. The advantages of using this system to produce engineered proteins are _________.

A) correct post-translational modifications on the protein of interest
B) cells grow in suspension in a simple broth
C) cells lyse at the end of the viral life cycle releasing the protein of interest
D) high expression level of the engineered protein
E) all of the above

Question 22

The advantages of using a yeast expression system are due to _______.

A) a fast grow rate of yeast
B) the proteins being secreted into the growth media
C) the ability of producing large quantities of engineered protein
D) the lower cost than the mammalian expression systems
E) all of the above

Question 23

Proteins expressed in bacterial cells ________.
I. fold properly if they contain several domains
II. have to be released from the cells during a cell lysis step
III. do not go through post-translational modifications
IV. are secreted into the growth media

A) I
B) II
C) III
D) II and III
E) III and IV

Question 24

Engineered proteins can be expressed in a variety of host cells (mammalian, bacterial, etc). Choosing the host cell depends on ___________.

A) how fast the cells grow
B) the post-translational modifications that are required for the expressed protein to be biologically active
C) the type of single-domain or multi-domain protein that is expressed
D) amount of protein that needs to be produced
E) all of the above

Question 25

Using site-directed mutagenesis, the mutation of a serine residue to an alanine residue might be used to _________.
I. decrease the solubility of a protein
II. eliminate the possibility of phosphorylation at that location
III. observe the impact of a non-polar side chain at that location
IV. prove that serine had a catalytic function in the native protein
V. promote proper folding of the protein

A) I and II
B) I and III
C) II, III and IV
D) II, IV and V
E) I, II and V

Question 26

It is common, when making mutations in a cDNA sequence ___________________.

A) to remove a nucleotide
B) to use a codon with the smallest change in the nucleotides
C) to delete a large cDNA sequence
D) to exchange nucleic acids
E) to replace phosphate groups

Question 27

Adding restriction sites when constructing plasmids will help with _______.

A) extraction
B) purification
C) cloning
D) solubility
E) electrophoresis

Question 28

When using cassette mutagenesis, in order to insert an engineered cDNA sequence into a plasmid, a __________ is used to cut out the sequence that is no longer needed, and a __________ is used to insert the new mutant cassette into the plasmid.

A) restriction enzyme; ligase
B) clone; enzyme
C) catalase; polymerase
D) reductase; polymerase
E) all of the above

Question 29

PCR-based mutagenesis is a widely used technique due to the mutation being introduced in _________.

Question 30

Mutation of a serine residue to a(n) _____ residue is a good modeling tool for a phosphorylated serine.

A) alanine
B) methionine
C) lysine
D) aspartate
E) glutamate

Question 31

__________ suppression is used to incorporate unnatural amino acids into a protein using cellular expression.

Question 32

Subtilisin is a bacterial serine protease that is commonly used as an additive in laundry detergent. In its native state, it has a methionine that becomes oxidized, surprisingly leading to enzyme inactivation. To prevent inactivation, subtilisin could be engineered by mutating methionine residues to ___________.
I. isoleucine
II. alanine
III. cysteine
IV. phenylalanine

A) I
B) II
C) III
D) II, III and IV
E) I, II and IV

Question 33

The green fluorescent protein (GFP) ___________.
I. is a large protein, MW larger than 60 kDa
II. is used in chimeric protein constructs
III. is extremely useful in tagging proteins inside the cell, to visualize the localization of the expressed proteins
IV. was first isolated from the jellyfish Aequorea victoria
V. needs a substrate to exhibit fluorescence

A) II, III, and IV
B) II and V
C) III and IV
D) I and II
E) II and IV

Question 34

Proteins can be engineered to express a tag that is beneficial in __________ of the protein by _______ chromatography.

Question 35

Choose fusion proteins from the list that could be used in biochemical or cellular assays:
I. GFP-protein constructs
II. extra cellular matrix proteins
III. anti-His6 tag-antibody conjugated to horseradish peroxidase
IV. maltose binding proteins
V. subtilisin

A) I
B) II
C) III
D) I, III and IV
E) II, III and V

Question 36

His6-tagged proteins are often overexpressed in E. coli cells and purified by _______ chromatography.

A) ion exchange
B) size exclusion
C) nickel-affinity
D) high pressure
E) reverse phase

Question 37

In the cell, most proteins fold without assistance and within milliseconds. The information needed to fold a protein properly is found in the ______ sequence of the protein.

A) primary
B) secondary
C) tertiary
D) quaternary
E) complementary

Question 38

Protein folding is the process by which proteins ____________.
I. are stored
II. gain their secondary structure
III. gain their tertiary structure
IV. lose activity
V. burry hydrophobic side chains and expose hydrophilic ones

A) I
B) II
C) III and IV
D) II and V
E) II, III and V

Question 39

Levinthal's conclusion was that protein folding _______, because it would take way too long to complete the folding if all randomly available rotations around the main chain bonds would be sampled to achieve the most stable structure.

Question 40

During folding the entropy of the protein _______.

A) decreases
B) stays the same
C) converts
D) increases
E) none of the above

Question 41

Proteins unfold (denature) under various experimental conditions, such as heat or presence of _______ in the buffer.

A) very low or very high pH
B) $\beta$ -mercaptoethanol
C) urea
D) guanidinium hydrochloride
E) all of the above

Question 42

Unfolded (denatured) proteins can be re-folded by removing the chemicals in a process called _________.

A) chromatography
B) dialysis
C) precipitation
D) nucleation
E) condensation

Question 43

Proteins can be denatured using $\beta$ -mercaptoethanol, since this reagent will _____________.

A) melt the protein
B) oxidize the amine groups
C) reduce the disulfide bonds
D) precipitate the protein
E) all of the above

Question 44

Proteins containing ________ residues may not re-fold properly.

A) few cysteine
B) multiple serine
C) few serine
D) numerous cysteine
E) multiple lysine

Question 45

Given a peptide:
Salt bridges will form in this peptide in a buffer at pH 7 between:
I. the amino terminus -NH₃⁺ and the carboxy terminus -COO^-
II. the side chain of Asp6 and the amino terminus -NH₃⁺
III. the side chains of Ser6 and Gln9
IV. the side chain of His13 and the carboxy terminus -COO^-
V. the side chains of Gln9 and His13

A) I
B) II
C) III
D) I, II and IV
E) III, IV, and V

Question 46

Given a peptide: Disulfide bonds will form between the side chains of ______ in a buffer at pH 7 (under oxidizing conditions).

A) Met1 and Cys7
B) Met1 and Cys12
C) Cys 7 and Cys12
D) Ser3 and Cys12
E) none of the above

Question 47

Given a peptide:
Hydrogen bonds will form in this peptide in a buffer at pH 7 between:

A) the amino terminus -NH₃⁺ and Ser3
B) the side chain of Asp6 and Ser3
C) the side chains of Ser6 and His13
D) the side chains of Gln9 and His13
E) all of the above

Question 48

Several neurodegenerative diseases (Alzheimer's, Parkinson's, etc.) present __________ structures at the molecular level.

Question 49

The 3D solution structure of a 20mer peptide could be determined by_________.

A) electron microscopy
B) x-ray crystallography
C) multidimensional NMR
D) circular dichroism
E) none of the above

Question 50

The thermodynamic stabilization of the protein in folded state is partly due to the hydrophobic effect that accompanies protein folding. The hydrophobic effect in proteins is manifested in ________ and _________.

A) burial of the hydrophilic side chains; release of the water molecules
B) burial of the exposed hydrophobic side chains; release of the solvating water molecules
C) release of the water molecules; solvation of cations
D) release of the water molecules; formation of salt bridges
E) none of the above

Question 51

Forming bonds _______ energy. Thus, formation of numerous _______ during protein folding contributes to the stability of the folded structure.

A) increases; ionic bonds
B) increases; Van der Waals interactions
C) releases; metallic bonds
D) releases; hydrogen bonds
E) none of the above

Question 52

During protein folding the entropy of the solvent molecules _______.

A) does not change
B) decreases
C) increases
D) is zero
E) none of the above

Question 53

The most stable 3D protein structure has the ______ free energy, and is therefore assumed to be the native conformation of the protein.

A) highest
B) lowest
C) widest
D) most defined
E) least defined

Question 54

The ___________ sequence is needed to predict the 3D structure of a relatively small protein using de novo structure prediction software.

A) base
B) amino acid
C) side chain
D) ionization
E) propagation

Question 55

_____ of protein structures is a useful tool in building 3D structures for proteins that do not have a solved x-ray structure. It uses the x-ray structure of a related protein as a template and the amino acid sequence of the protein of interest.

A) Solvation
B) Structural dynamics
C) Molecular dynamics
D) Homology modeling
E) none of the above

Question 56

Molecular dynamics simulations can be used to model the sequence in which the _____ structure of a protein forms during protein folding.

Question 57

Protein folding can be described in terms of energy, and visualized by an energy funnel diagram .The width of the funnel is given by the________.

A) size of the funnel
B) number of conformational states of the protein
C) shape of the molecule
D) motion of the molecule
E) all of the above

Question 58

Depositing falsified protein structure data into the Protein Data Bank, PDB, is a huge disservice to the scientific community because it leads to ___________.

A) misleading protein-drug interactions when used in drug discovery
B) wasted financial resources
C) wasted time using erroneous structures
D) retracted publications
E) all of the above

Question 59

Whistleblowers are in general protected from being fired from the job by __________.

A) federal and state laws
B) regulations
C) internal guidelines
D) company policy
E) all of the above