Section 4 Flashcards
(85 cards)
1
Q
What does the primary structure of a protein refer to? How is this sequence read?
A
The linear sequence of AA. It is read from the amino (N) terminus to the carboxyl (C) terminus.
2
Q
A ___________ reaction occurs to form a peptide bond, linking the _________ group to the _________ group of the next amino acid.
A
condensation reaction in order to form a peptide bond linking the carboxyl group to the amino group of the next amino acid
3
Q
The alpha-helix arises due twisting of a section of the primary structure into a ______-handed screw.
a) Right
b) Left
A
a) right-handed screw
4
Q
What is one major difference between the alpha-helix and the DNA helix?
A
R groups project outwards and perpendicular in the alpha-helix. It is also one stranded.
The DNA double helix has it’s R groups projecting inwards.
5
Q
What are some bonds in the alpha helix?
A
Imino groups (NH) of each AA residue forms H-bonds to the carbonyl (CO) of another amino acid in the adjacent turn of the helix
6
Q
Are hydrogen bonds further apart in a-helices or b-sheets?
A
In b-sheets they are a lot further apart than a-helices. This is because instead of forming a coiled structure, the backbone of the polypeptide is expanded outward.
7
Q
Describe the difference in the orientation of H-bonds in antiparallel vs parallel beta-sheets.
A
Antiparallel Beta-Sheet:
- Adjacent beta strands run in opposite directions, and the hydrogen bonding occurs between the oxygen from carbonyl group of one amino acid and the H from the amide in the neighboring strand.
- The atoms involved in the hydrogen bond are approximately in a straight line, allowing for STRONGER hydrogen bonding interactions.
Parallel Beta-Sheet:
- Adjacent beta strands run in the same direction, and the hydrogen bonding also occurs between the carbonyl oxygen of one amino acid residue and the amide hydrogen of a residue in the neighboring strand.
- The atoms involved in the hydrogen bond are not in line; they are at an angle to each other, which results in WEAKER hydrogen bonding interactions.
8
Q
How are secondary structures linked?
A
By reverse turns in order to reverse direction and fold into tertiary structures.
Beta-turns are small, precise reverse turns, stabilized by H-bonds between the imino (NH) group and carbonyl (CO) of nearby amino acids that form the turn
9
Q
Which one is more favourable in linking secondary structures:
a) beta-turns
b) gamma-turns
A
a)
10
Q
Why are secondary structures like alpha helices and beta sheets not always stable?
A
Secondary structures can transiently form and break apart until sufficient stabilizing interactions (e.g., hydrogen bonds, van der Waals forces) are established.
11
Q
What happens in that location when amino acids within a protein resist or hinder the formation of preferred secondary structures?
A
Amino acids within a protein may not always encourage the formation of preferred secondary structures, potentially resisting or hindering these structures. this is where we can find a turn in the alpha helices.
12
Q
What are “turns” in the context of alpha helices, and what role do they play?
A
“Turns” or “bends” in alpha helices represent regions where the helical structure changes direction. They are essential for flexibility but can disrupt the helical pattern when excessive.
13
Q
What can happen when deviations from the ideal secondary structure accumulate in a protein?
A
Accumulation of deviations can lead to the formation of non-functional or misfolded proteins, which may have the correct shape but lack proper functionality.
14
Q
True or false: There is no partial double bond character in a peptide bond.
A
False. There is a sharing of electrons between the carboxyl oxygen and the amide nitrogen, creating partial double bond character.
15
Q
True or false: the N-C(alpha) and the C(alpha)-C bons are free to rotate.
A
True. But the angles between these bonds are constrained. These angles are called torsion angles (or dihedral angles).
16
Q
Match the torsion angles with the bonds.
Phi for ______________
Psi for ______________
A
Phi for the N-C(alpha) bond
Psi for the C(alpha)-C bond
17
Q
Why are psi and phi angles limited?
A
Due to “steric clash”between an amino acid side chain and neighbouring atoms. The size of the bulky side chain may prevent a close approach to nearby atoms in the polypeptide chain.
18
Q
What is a ramachandran plot?
A
A way to graphically represent the allowed values of psi and phi for each amino acid.
Darker areas are easily allowed conformations. Lighter areas represent conformations that are allowed if some flexibility is permitted in the torsion angles. Unshaded regions indicated conformations that are not allowed or are ‘disfavoured’.
19
Q
True or false: A typical protein contains about one-third alpha-helix and 1/3 b-sheet
A
True.
20
Q
In the alpha helix do the R groups protrude outward or inward?
A
Outward
21
Q
Is the alpha helix a left or right handed helix?
A
Right-handed helix
22
Q
What is one helical turn of an alpha helix?
A
The hydrogen on the amide nitrogen forms a hydrogen bond with the carbonyl oxygen of the fourth residue toward the N-terminus, which makes about one helical turn.
23
Q
Can carbonyl (CO) and imino groups (NH) on the same peptide chain hydrogen bond?
A
Yes. Favourable psi and phi angles accommodate regular pattern of hydrogen bonding between carbonyl and imino groups on the same peptide chain (strand).
24
Q
What is the most and second most stable arrangement of the polypeptide backbone?
A
Alpha helix is #1
Beta sheet is #2
25
How is the beta-sheet formed?
By hydrogen bonds between the backbone amide and carbonyl groups
Forms from multiple beta-strands forming hydrogen bonds together
26
How are interactions of R groups of adjacent residues prevented in beta-sheets?
The R groups of adjacent amino acid residues in a beta-strand lie on opposite sides of the sheet, and this alternating geometry prevents the interaction of R groups of adjacent residues.
This makes a zigzag pattern with pleats involving 4-6 strands, therefore the beta-sheet is referred to as a "Beta-pleated sheet"
27
What is a parallel vs antiparallel beta sheet?
ANTIPARALLEL: When beta-strands are oriented in the opposite N- to C-terminal directions
PARALLEL: When they run in the same direction
The sheets can also be composed of a mixture of both
28
True or false: the beta-sheet can readily accommodate large aromatic residues.
True, such as Tyr, Trp and Phe residues
29
Which one is proline unfavoured in:
a) alpha-helix
b) beta-sheets
a) alpha-helix
It is often found in beta-sheets, especially in the "edge" strands
30
Can a Ramachandran plot identify the types of secondary structure within a specific protein?
Ye
31
what are the small and precise turns of secondary structures of polypeptide chains called?
beta-turns
32
How does a beta turn work?
- Complete Reversal of Direction: A beta turn is a structural motif within a protein where the polypeptide chain changes direction. In other words, it makes a U-turn or a complete reversal of its path.
- Four Residues: This structural feature involves four consecutive amino acid residues in the protein's primary sequence.
- Hydrogen Bond Formation: hydrogen bonds between specific atoms in these four residues. Specifically, the backbone carbonyl oxygen of the first residue forms a hydrogen bond with the amide hydrogen of the fourth residue
- Lack of Inter-Residue Hydrogen Bonds: In contrast to the strong hydrogen bond between residues 1 and 4, the second (position 2) and third (position 3) residues do not typically form hydrogen bonds with each other or with other residues in the beta turn.
Location on the Protein Surface: Beta turns are often found on the surface of proteins because the structure of the beta turn allows the backbone atoms to be exposed to the surrounding water molecules. This exposure makes it possible for the backbone atoms to form hydrogen bonds with water, which helps stabilize the protein's overall structure.
Common Amino Acid Residues: It's common to find a Proline (abbreviated as Pro) residue at position 2 and a Glycine (abbreviated as Gly) residue at position 3.Proline's unique structure allows it to fit well in the tight turns (due to the imino nitrogen readily assuming a cis configuration) of the beta turn, and glycine's small side chain helps accommodate the bend in the protein chain.
33
What is the gamma turn?
- less common reverse turn
- consists of only three amino acids
- backbone carbonyl and amide groups of the first and third amino acid residues form a hydrogen bond, and the second (middle) amino acid is not involved in inter-residue hydrogen bonding
34
what are globular proteins? provide an example
- water soluble
- spherical in shape
- often found in aqueous environments of cells
- ex. hemoglobin
35
what are fibrous proteins? provide an example
- elongated shapes
- structural role
- e.g collagen and keratin
36
What do collagen and keratin do in the body, briefly?
Both fibrous proteins
Collagen is involved in supportive connective tissue network
Keratin forms a protective layer on the skin, and structures like nails and hair
37
Do proteins fold spotaneously?
Yes, under physiological conditions they fold spontaneously as they are biosynthesized.
38
Which structure is more conserved? Tertiary or primary?
The tertiary structure of proteins is more conserved than their primary structure (both across species and within protein families)
39
What is the quaternary structure of a protein?
Connections between two or more polypeptide chains
e.g the association of two identical subunits - a protein consisting of two polypeptide subunits is called a dimer
40
Is it more favourable to form large complexes or a single large protein?
More favourable to form large complexes
41
What is one issue with protein folding, and what resolves this?
the folding of many different domains in a single, very large polypeptide chain can be problematic.
- if one domain doesn't fold properly the ENTIRE protein would lack function so energy in making it would be completely wasted
- a multisubunit composition avoids this problem; if a protein subunit misfolds, it will not be included in the oligomer but at least only cellular resources to make just this one domain were wasted and not energy. A similar thing can happen to an entire subunit if it misfolds, it can be replaced by another subunit
42
Differentiate between the words oligomer, protomer, multimer, and subunit
A protein composed of multiple polypeptide chains is referred to as an oligomer or multimer.
The individual polypeptide chains are referred to as subunits or protomers.
43
What is a homoligomer?
An oligomer with identical subunits
44
What is a hetero-oligomer?
An oligomer with non-identical subunits
45
Define the difference between primary, secondary, tertiary, and quaternary structure.
Primary - the order of AAs
Secondary - due to interactions of backbone
Tertiary - due to side chain interactions
Quaternary - two or more polypeptide chains interacting together
46
What is proteolysis?
A technique that cleaves the polypeptide backbone.
This can separate a protein into its domains
47
Give examples of a 1, 2, 3, and 4 protein domain
1 domain: myoglobin, which is a small, oxygen-binding protein
2 domains: y crystallin, which is a component in the lens of the eye
3 domains: DnaA, which is a bacterial protein involved in the initiation of DNA synthesis
4 domains: fragment of E.coli DNA polymerase I, which is an enzyme that synthesizes new DNA strands. the four domains have two seperate enzymatic activities.
48
True or false: Once a protein is correctly folded, it's atoms and structural elements are still.
False: Even when correctly folded, a protein is constantly in flux, and all of its atoms and structural elements vibrate rapidly.
49
What are chaperones and chaperonins, and how do they relate to protein folding?
Chaperones and chaperonins are molecular helpers that assist partially folded protein states in achieving the lower-energy conformation of the native protein during the folding process.
50
What is the significance of the "wells" in the energy landscape of protein folding intermediates?
These "wells" represent relatively stable intermediate states with lower free energy, allowing partially folded proteins to persist and eventually reach their native conformation.
51
Why is misfolding of proteins a concern, and what can it lead to?
Misfolding of proteins can lead to irreversible aggregations, potentially resulting in the development of amyloid fibrils or plaques. These aggregations are associated with many neurological diseases.
52
Which of the following is NOT a function of chaperones and chaperonins in protein folding?
A) Assisting partially folded protein states
B) Promoting irreversible aggregations
C) Aiding in reaching the native protein conformation
D) Ensuring correct protein folding
B) Promoting irreversible aggregations
53
What are repeating structural units within a protein complex called?
Protomers
54
What are the four main steps of protein purification?
Cell lysis, centrifugation, fractionation and protein detection.
55
What is the main goal of cell lysis, and what are the four most common methods of it?
The main goal is to open the cells to get access to the proteins.
The four most common methods are:
- Detergent (compromise the integrity of cell membranes, facilitating lysis of cells and extraction of soluble protein)
- Shear force (physically disrupting cells by applying high frequency sonic waves to agitate and break the cell membrane, or by rapidly shaking the sample (e.x with a blender)
- Low ionic salt (causing cells to osmotically absorb water and pop easily)
- Changes in pressure (high amounts of pressure causing cells to break)
56
What is the principle behind centrifugation, and what is the result of this process in a test tube?
Centrifugation relies on the principle of sedimentation, where particles are exposed to radial acceleration. Denser particles settle at the bottom of the test tube, forming a solid mass called the "pellet," while less dense substances remain suspended in the liquid above, known as the "supernatant."
57
What is differential centrifugation, and how does it work to separate components in a sample?
Differential centrifugation is a specialized technique that separates components in a sample based on their density differences. It involves multiple rounds of centrifugation at varying rotational speeds to gradually separate less dense material from denser particles within the sample.
58
Describe the step by step process of differential centrifugation.
1. Low-speed centrifugation (1,000 g, 10 min): In this initial step, the sample, which may contain a mixture of cells and cellular components, is subjected to relatively low centrifugal force. The result is the formation of a pellet at the bottom of the tube, which contains whole cells, nuclei, cytoskeletons, and plasma membranes. These components are relatively dense and settle quickly.
2. Medium-speed centrifugation (20,000 g, 20 min): The supernatant (liquid above the pellet) from the first centrifugation is transferred to a new tube and subjected to higher centrifugal force. This step separates out mitochondria, lysosomes, and peroxisomes, which are less dense than the components that settled in the first step.
3. High-speed centrifugation (80,000 g, 1 h): The supernatant from the second centrifugation is again transferred to a new tube and subjected to even higher centrifugal force. This process results in the formation of a pellet containing microsomes, which are fragments of the endoplasmic reticulum (ER) and small vesicles. Microsomes are less dense than the components separated in previous steps.
4. Very high-speed centrifugation (150,000 g, 3 h): In the final step, the remaining supernatant is subjected to extremely high centrifugal force. This step yields a pellet containing ribosomes and large macromolecules. Ribosomes are smaller and less dense than the components separated in earlier steps.
59
What is column chromatography and describe the process.
Column chromatography is a fundamental laboratory technique used to separate a mixture of compounds into its individual components. The process relies on the interaction between two phases: the mobile phase and the stationary phase.
1. Mobile Phase: The mixture to be separated is first dissolved in a liquid called the "mobile phase." This mobile phase serves as a carrier for the sample and allows it to flow through the column.
2. Stationary Phase: Within the column, there is a material referred to as the "stationary phase." This material is typically a solid or a gel-like substance packed into the column. The stationary phase does not move; it remains fixed within the column.
3. Separation Mechanism: Separation occurs as the mobile phase flows through the stationary phase. Different components of the mixture interact with the stationary phase to varying degrees based on their physical and chemical properties. Factors such as the size, charge, and chemical affinity of the components influence how they interact with the stationary phase.
4. Different Speeds: As the mobile phase travels through the column, the various components within the mixture move through at different speeds. This differential migration is due to the interactions between the components and the stationary phase. Components that interact more strongly with the stationary phase move more slowly through the column, while those with weaker interactions move faster.
In the context of protein purification, column chromatography is often used to separate the protein of interest from a complex mixture like a cell lysate. After initial steps like centrifugation have removed larger cellular debris, the remaining lysate can be applied to a column containing a stationary phase that selectively interacts with the protein of interest. By controlling the conditions of the chromatography, such as the choice of stationary phase and mobile phase composition, scientists can effectively separate and isolate the desired protein from the rest of the mixture
60
Describe the steps in column chromatography
1. A protein mixture is applied to a column containing a resin, or matrix (generally some kind of polymer), that interacts differently with the various proteins.
2. A buffer is passed through the column to thoroughly wash away any proteins that do not bind to the matrix.
3. Another buffer is applied that causes bound proteins to dissociate from the matrix and carry them out in the buffer flow, in a process referred to as elution, at different times, depending on how they interact with the resin. The column matrix and “elution buffer” are carefully chosen so that different proteins dissociate from the matrix at different times.
4. The eluted proteins are collected in a fraction collector, which gradually moves test tubes under the column, thus keeping the proteins that elute at different times separate from one another.
61
What are the three types of column chromatography we should know for this class?
1. Ion-exclusion chromatography
2. Size-exclusion chromatography
3. Affinity chromatography
62
Describe ion-exchange chromatography (what types are there and how does it work)?
- Proteins are separated by charge
Can be either:
1. Anion exchange resin, which is positively charged (coated with cations) and BINDS ANIONS
2. Cation exchange resin, which is negatively charged (coated with anions) and BINDS CATIONS
Proteins are usually eluted from the column with an increasing concentrations of salt solution, and
their release depends on the nature of charged amino acid residues on their surface. Charged proteins
will bind to an oppositely charged solid matrix
63
Describe the relationship of pH and pI.
When the pH of the solution surrounding the protein is lower than the pI, the protein has an increased cationic character with a net positive charge.
The reverse is also true about pH above the pI. This property can help bind the protein-of-interest to the resin.
64
What happens when you add counter-ions to the bound proteins on an ion-exchange column?
By adding counter-ions (NaCl) to the bound proteins on the column, these compete for the ionic interactions the proteins make with the oppositely charged matrix in the column, leading to their elution.
better explanation: As a result of this competition, some of the bound proteins will be displaced from the column's charged groups by the excess counter-ions from NaCl. These displaced proteins are said to be "eluted" from the column. The extent to which they are displaced and, therefore, the order in which they are eluted, depends on the strength of the protein's electrostatic interactions with the column and the concentration of counter-ions in the solution.
65
Describe size-exclusion chromatography.
This is where the column matrix contains beads that have pores of specific size, which allows for any protein smaller than the pore to enter.
The smaller proteins that enter the pores will thus take longer to migrate through the column, whereas large proteins elute the fastest.
66
What is affinity chromatography?
Takes advantage of the fact that many proteins specifically bind other molecules, or ligands, as part of their function.
A column is constructed containing the ligand covalently attached to a matrix.
67
What does it mean when we "engineer" proteins?
Helping purify proteins by adding short amino acid sequences, known as tags, to either the beginning or end of the target protein.
These tags are typically introduced through molecular cloning methods.
68
Give an example of an engineered protein.
Glutathione-S-transferase (GST) is a naturally occurring protein that has some useful properties for protein purification:
Rapid Folding: GST is known to fold into a stable and highly soluble protein structure quickly after translation. This property makes it easier to produce recombinant proteins fused with the GST tag, as they are more likely to fold correctly and remain soluble.
Solubility Promotion: The inclusion of the GST tag often promotes greater expression and solubility of recombinant proteins compared to expressing the target protein without the tag. This is particularly beneficial because insoluble proteins can be challenging to work with.
Enzyme Activity: GST is also an enzyme that has the ability to bind to its specific substrate, which is glutathione (GSH). Glutathione is a tripeptide consisting of the amino acids Glutamate (Glu), Cysteine (Cys), and Glycine (Gly).
Affinity-Based Purification: The key application of the GST tag is in affinity-based protein purification. In this method, you take a protein that has been fused with a GST tag and run it over a chromatography column that has glutathione immobilized to the resin.
Binding Reaction: Due to the specific binding interaction between GST and its substrate, glutathione (GSH), the GST-tagged protein will bind to the glutathione that is immobilized on the column resin. This binding is highly selective because GST binds specifically to GSH.
Capturing GST-Tagged Proteins: As the GST-tagged protein passes through the column, it will selectively bind to the glutathione on the resin, while other non-tagged proteins and impurities will flow through or bind less strongly. This allows you to capture and isolate the GST-tagged protein of interest.
69
What does SDS-PAGE stand for, and what is its primary purpose in biochemistry?
SDS-PAGE stands for "Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis." Its primary purpose in biochemistry is to separate proteins based on their size.
70
In SDS-PAGE, what is the role of acrylamide concentration in the polyacrylamide gel, and how does it affect protein separation?
The acrylamide concentration in the gel is crucial because it determines the size range of proteins that can be separated effectively. "High percent" gels with lower acrylamide concentrations are better at resolving small proteins, while "low percent" gels with higher acrylamide concentrations are better for larger proteins.
71
What is the function of SDS (Sodium Dodecyl Sulfate) in SDS-PAGE, and how does it affect protein behavior?
SDS is a negatively charged detergent used in SDS-PAGE. Its function is to denature proteins, giving them a consistent linear shape and a uniform negative charge. SDS binds to proteins in a manner proportional to their size, ensuring that all proteins have a similar charge-to-mass ratio.
72
How does SDS-PAGE separate proteins during electrophoresis
In SDS-PAGE, when an electric current is applied, proteins move through the polyacrylamide gel. Since SDS imparts a consistent charge-to-mass ratio, the primary factor affecting protein migration is their size. Smaller proteins migrate more quickly through the gel, while larger proteins migrate more slowly.
73
Explain the purpose of the GST (Glutathione-S-transferase) tag in protein engineering and purification.
The GST tag is added to target proteins during molecular cloning to facilitate their purification. It promotes greater expression and solubility of recombinant proteins. Additionally, GST is an enzyme that can bind specifically to its substrate, glutathione (GSH), which allows GST-tagged proteins to be captured via an enzyme-substrate binding reaction, simplifying their purification.
74
What happens in the first step of SDS-PAGE?
In the first step, treated protein samples are loaded into wells at the top of the gel, and an electric field is applied to the gel.
75
What drives the movement of proteins during SDS-PAGE?
Proteins are separated based on their relative molecular mass (size), with smaller proteins migrating faster towards the bottom of the gel and larger proteins remaining closer to the top.
76
What is the purpose of fixing proteins in SDS-PAGE?
Fixing proteins prevents them from diffusing out of the gel and helps maintain their positions during subsequent staining and visualization steps.
77
What type of dye is commonly used to stain proteins in SDS-PAGE?
Coomassie Blue is a common dye used to selectively bind to proteins during staining in SDS-PAGE.
78
Why are protein samples treated before loading them onto an SDS-PAGE gel?
Protein samples are treated to denature the proteins, impart a uniform charge, and break down their tertiary structure, ensuring separation based on size alone.
79
How does SDS-PAGE separate proteins based on size?
SDS-PAGE separates proteins based on their molecular mass; smaller proteins migrate faster through the gel, while larger proteins migrate more slowly.
80
What is the role of the electric field in SDS-PAGE?
The electric field is applied to the gel to create a driving force that moves the charged proteins through the gel matrix towards the positively charged cathode.
81
What is the purpose of soaking the gel in an acidic buffer in SDS-PAGE?
Soaking the gel in an acidic buffer "fixes" the proteins, preventing them from diffusing out of the gel during subsequent staining steps.
82
Why is staining with Coomassie Blue or a similar dye important in SDS-PAGE?
Staining with Coomassie Blue selectively binds to proteins, making them visible as distinct bands on the gel, allowing for protein visualization and quantification.
83
How does the percentage of acrylamide in the gel affect protein separation in SDS-PAGE?
The percentage of acrylamide in the gel affects the size range of proteins that can be separated effectively; "high percent" gels are better for smaller proteins, while "low percent" gels are better for larger proteins.
84
What is the significance of proteins having a similar charge-to-mass ratio in SDS-PAGE?
Proteins having a similar charge-to-mass ratio due to SDS ensures that their migration through the gel is primarily determined by their size, facilitating size-based separation.
85
