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Questions and Answers
In a scenario where a biochemist is investigating a novel enzyme, and they observe that the enzyme's activity sharply declines outside the pH range of 7.1 to 7.3, which buffering system would be most relevant to consider for maintaining optimal activity in vitro?
In a scenario where a biochemist is investigating a novel enzyme, and they observe that the enzyme's activity sharply declines outside the pH range of 7.1 to 7.3, which buffering system would be most relevant to consider for maintaining optimal activity in vitro?
- Citrate buffer, known for its effectiveness at extremely acidic pH levels.
- Phosphate buffer, because its pKa2 is close to physiological pH. (correct)
- Bicarbonate buffer, as it is the primary buffer regulated by the kidneys.
- Protein buffers, due to the broad range of pKa values from various ionizable groups.
A scientist is studying a protein with a critical histidine residue in its active site. They observe that the protein's function is significantly reduced at pH 7.4 compared to pH 6.0. What is the most likely explanation for this pH-dependent activity change?
A scientist is studying a protein with a critical histidine residue in its active site. They observe that the protein's function is significantly reduced at pH 7.4 compared to pH 6.0. What is the most likely explanation for this pH-dependent activity change?
- The active site is optimally configured for substrate binding only at pH 7.4 due to induced conformational changes.
- The overall charge of the protein shifts dramatically at pH 7.4, causing complete denaturation.
- At pH 7.4, the histidine residue is deprotonated, altering its ability to participate in catalysis or ligand binding. (correct)
- At pH 7.4, the histidine residue becomes protonated, leading to steric hindrance within the active site.
In a protein folding experiment, a researcher mutates a glycine residue to proline within an alpha-helix. What direct impact would this mutation likely have on the local protein structure?
In a protein folding experiment, a researcher mutates a glycine residue to proline within an alpha-helix. What direct impact would this mutation likely have on the local protein structure?
- No significant change, as glycine and proline are structurally similar.
- Disruption of the alpha-helix due to the unique conformational constraints imposed by proline's cyclic structure. (correct)
- Enhanced alpha-helical stability due to increased hydrophobic interactions.
- Increased flexibility in the peptide backbone, promoting greater conformational freedom.
During protein synthesis, if a tRNA molecule incorrectly incorporates a D-amino acid instead of an L-amino acid into a growing polypeptide chain, what is the most likely consequence for the protein?
During protein synthesis, if a tRNA molecule incorrectly incorporates a D-amino acid instead of an L-amino acid into a growing polypeptide chain, what is the most likely consequence for the protein?
A researcher is investigating a protein with a known isoelectric point (pI) of 6.5. Under what conditions would the protein migrate towards the cathode (negative electrode) during isoelectric focusing?
A researcher is investigating a protein with a known isoelectric point (pI) of 6.5. Under what conditions would the protein migrate towards the cathode (negative electrode) during isoelectric focusing?
A biochemist is analyzing the amino acid composition of a newly discovered protein and finds an unusually high proportion of phenylalanine and tryptophan residues. What biophysical characteristic is most likely to be prominent in this protein?
A biochemist is analyzing the amino acid composition of a newly discovered protein and finds an unusually high proportion of phenylalanine and tryptophan residues. What biophysical characteristic is most likely to be prominent in this protein?
Consider a scenario where a protein's function is regulated by glycosylation. If a mutation prevents N-linked glycosylation, which amino acid is most likely involved in this mutation?
Consider a scenario where a protein's function is regulated by glycosylation. If a mutation prevents N-linked glycosylation, which amino acid is most likely involved in this mutation?
A scientist aims to design a peptide that remains unstructured in solution. Which amino acid composition would be most likely to achieve this goal?
A scientist aims to design a peptide that remains unstructured in solution. Which amino acid composition would be most likely to achieve this goal?
A mutation in a protein results in the substitution of a glutamate residue with an alanine residue near the protein's surface. What is the most likely consequence of this mutation on the protein's interactions at physiological pH?
A mutation in a protein results in the substitution of a glutamate residue with an alanine residue near the protein's surface. What is the most likely consequence of this mutation on the protein's interactions at physiological pH?
A researcher is investigating how changes in a protein's torsion angles (phi and psi) affect its stability. What experimental technique would provide the most direct information about the distribution of these angles in the protein structure?
A researcher is investigating how changes in a protein's torsion angles (phi and psi) affect its stability. What experimental technique would provide the most direct information about the distribution of these angles in the protein structure?
Which of the following is NOT a characteristic of peptide bonds that contributes to the secondary structure of proteins?
Which of the following is NOT a characteristic of peptide bonds that contributes to the secondary structure of proteins?
A protein sample is known to have a high concentration of histidine residues. Which of the following conditions would require careful pH control to accurately assess the protein's activity due to the buffering capacity of histidine?
A protein sample is known to have a high concentration of histidine residues. Which of the following conditions would require careful pH control to accurately assess the protein's activity due to the buffering capacity of histidine?
How does the Henderson-Hasselbalch equation apply to understanding the buffering capacity of blood?
How does the Henderson-Hasselbalch equation apply to understanding the buffering capacity of blood?
In a scenario involving a protein that undergoes significant conformational change upon ligand binding, what best describes the role of amino acids in this process?
In a scenario involving a protein that undergoes significant conformational change upon ligand binding, what best describes the role of amino acids in this process?
If an enzyme's catalytic activity is known to depend on a specific lysine residue being protonated, what would be the predicted effect of performing the reaction at a pH significantly above the lysine's typical pKa?
If an enzyme's catalytic activity is known to depend on a specific lysine residue being protonated, what would be the predicted effect of performing the reaction at a pH significantly above the lysine's typical pKa?
Suppose a researcher discovers a new amino acid in an extremophile bacterium. This amino acid has a unique side chain with a pKa of 9.0. How would this amino acid's charge state change as the pH is increased from 7.0 to 11.0?
Suppose a researcher discovers a new amino acid in an extremophile bacterium. This amino acid has a unique side chain with a pKa of 9.0. How would this amino acid's charge state change as the pH is increased from 7.0 to 11.0?
A research team is engineering a protein to have increased stability at high temperatures. Which of the following strategies targeting specific amino acid properties would likely be most effective?
A research team is engineering a protein to have increased stability at high temperatures. Which of the following strategies targeting specific amino acid properties would likely be most effective?
A biochemist is studying the effect of salt concentration on protein-protein interactions. Why is it important to consider the isoelectric points (pIs) of the proteins involved?
A biochemist is studying the effect of salt concentration on protein-protein interactions. Why is it important to consider the isoelectric points (pIs) of the proteins involved?
Consider the classification of amino acids. Which set of amino acids is most likely to be found in the interior of a water-soluble globular protein?
Consider the classification of amino acids. Which set of amino acids is most likely to be found in the interior of a water-soluble globular protein?
Which statement best describes the consequence of altering the ratio of weak acid to conjugate base in a buffer system that is significantly outside of its effective buffering range?
Which statement best describes the consequence of altering the ratio of weak acid to conjugate base in a buffer system that is significantly outside of its effective buffering range?
In what way could a mutation causing a shift in the pKa of a critical amino acid side chain within an enzyme's active site affect the enzyme's catalytic activity, assuming the residue participates directly in catalysis?
In what way could a mutation causing a shift in the pKa of a critical amino acid side chain within an enzyme's active site affect the enzyme's catalytic activity, assuming the residue participates directly in catalysis?
A scientist is studying a protein that requires a specific copper ion to maintain its active conformation. Which amino acid side chains would be most likely to coordinate the copper ion?
A scientist is studying a protein that requires a specific copper ion to maintain its active conformation. Which amino acid side chains would be most likely to coordinate the copper ion?
What is the primary reason why the peptide bond is generally considered to be planar?
What is the primary reason why the peptide bond is generally considered to be planar?
If a protein's activity is dependent on the precise positioning of several amino acid side chains in its active site, which level of protein structure is most directly responsible for ensuring this precise arrangement?
If a protein's activity is dependent on the precise positioning of several amino acid side chains in its active site, which level of protein structure is most directly responsible for ensuring this precise arrangement?
A researcher is investigating the effects of temperature on the stability of a specific alpha-helix within a protein. What type of interaction would be most critical to monitor to understand the temperature-dependent stability of this helix?
A researcher is investigating the effects of temperature on the stability of a specific alpha-helix within a protein. What type of interaction would be most critical to monitor to understand the temperature-dependent stability of this helix?
Suppose a novel drug binds tightly to a protein, altering its conformation in such a way that a critical salt bridge is disrupted. What is the most likely energetic consequence of disrupting this salt bridge?
Suppose a novel drug binds tightly to a protein, altering its conformation in such a way that a critical salt bridge is disrupted. What is the most likely energetic consequence of disrupting this salt bridge?
A researcher is designing a protein with enhanced stability in a low-pH environment. Which amino acid substitution would be most effective at preventing denaturation caused by protonation effects?
A researcher is designing a protein with enhanced stability in a low-pH environment. Which amino acid substitution would be most effective at preventing denaturation caused by protonation effects?
A sample of pure alanine is titrated from a pH well below its pKa1 to a pH well above its pKa2. What would be observed at the isoelectric point (pI)?
A sample of pure alanine is titrated from a pH well below its pKa1 to a pH well above its pKa2. What would be observed at the isoelectric point (pI)?
During the proper folding of a protein, which of the following plays the most significant role in directing a protein into its correct three-dimensional structure?
During the proper folding of a protein, which of the following plays the most significant role in directing a protein into its correct three-dimensional structure?
A protein's function depends on the precise formation of a specific disulfide bond. How would performing site-directed mutagenesis to replace one of the cysteine residues involved in the disulfide bond with an alanine residue affect the protein?
A protein's function depends on the precise formation of a specific disulfide bond. How would performing site-directed mutagenesis to replace one of the cysteine residues involved in the disulfide bond with an alanine residue affect the protein?
In an experiment designed to determine the buffering capacity of a novel solution, a researcher adds a strong acid and observes a smaller than expected pH change. What might explain this unexpected result?
In an experiment designed to determine the buffering capacity of a novel solution, a researcher adds a strong acid and observes a smaller than expected pH change. What might explain this unexpected result?
Under which of the following conditions would a buffer be LEAST effective?
Under which of the following conditions would a buffer be LEAST effective?
If a protein is required to have a permanently positive charged region in its structure, which residue(s) would be best?
If a protein is required to have a permanently positive charged region in its structure, which residue(s) would be best?
Which amino acid is achiral?
Which amino acid is achiral?
If the buffer has more conjugate base than weak acid, then:
If the buffer has more conjugate base than weak acid, then:
Hydrogen bonds are most directly responsible for maintaining which level of protein structure?
Hydrogen bonds are most directly responsible for maintaining which level of protein structure?
Which amino acid has the highest propensity to be present in loops?
Which amino acid has the highest propensity to be present in loops?
Flashcards
Acids (Bronsted-Lowry)
Acids (Bronsted-Lowry)
Proton donors in chemical reactions.
Bases (Bronsted-Lowry)
Bases (Bronsted-Lowry)
Proton acceptors in chemical reactions.
Acids and Bases (Lewis)
Acids and Bases (Lewis)
Molecules or ions that can either donate or accept an electron pair.
pH Definition
pH Definition
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Buffers Definition
Buffers Definition
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Henderson-Hasselbalch
Henderson-Hasselbalch
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Effective Buffer Range
Effective Buffer Range
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pKa Definition
pKa Definition
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Major buffer systems
Major buffer systems
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Amino Acid Structure
Amino Acid Structure
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Chiral Center
Chiral Center
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Zwitterions Definition
Zwitterions Definition
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Isoelectric Point (pI)
Isoelectric Point (pI)
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Nonpolar Amino Acids
Nonpolar Amino Acids
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Polar Amino Acids
Polar Amino Acids
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Negatively Charged AA
Negatively Charged AA
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Positively Charged AA
Positively Charged AA
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Ligand Binding Sites
Ligand Binding Sites
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Primary Structure
Primary Structure
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Chain Direction Definition
Chain Direction Definition
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Peptide Bond Definition
Peptide Bond Definition
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Atom mainchain sequence
Atom mainchain sequence
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Peptide bonds character.
Peptide bonds character.
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Study Notes
- Proteins are agents of biological function and were recognized in the early 19th century.
- Cellular processes depend on proteins.
- Including catalysis, transport, structure, motion, and communication.
- Proteins bind to ligands specifically prior to any action.
- Proteins are composed of amino acids.
Review of Acid/Base Chemistry
- Amino acids are weak acids.
- Brønsted-Lowry theory defines acids as proton donors and bases as proton acceptors via proton exchange.
- Lewis theory defines acids as electron pair acceptors and bases as electron pair donors.
- The Brønsted-Lowry definition is more commonly used in Biochemistry to focus on protons.
Water Dissociation
- Water dissociates into a hydronium ion (H3O+) and a hydroxyl ion (OH-).
- Simplified, HOH dissociates into H+ + OH-.
- Ion products of water (H+, OH-) are stoichiometric.
- The ion product constant for water (Kw) equals 1 x 10-14.
- In water, the concentration of dissociated protons and hydroxyl groups is 1 x 10-7 M for both.
- pH is expressed as pH = -log10[H+].
- The pH of water is 7.0, and at this pH, there is no net charge, which defines a charge-neutral state.
- There is a 10-fold change in [H+] for every unit change in pH.
- The pH scale spans 14 units, with [H+] ranging from 1 M to 10-14 M.
pH of Body Fluids
- Biochemical activities are restricted to a narrow pH range between 7.2 and 7.45.
- pH values in different compartments:
- Gastric acid: 1
- Lysosomes: 4.5
- Granules of chromaffin cell: 5.5
- Human skin: 5.5
- Urine: 6.0
- Cytosol: 7.2
- Cerebrospinal fluid (CSF): 7.3
- Mitochondrial matrix: 7.8
- Pancreas secretions: 8.1
- Blood: 7.35-7.45
- [H+] in blood = 35 – 45 nM (~ 40 nM).
Buffers
- Buffers in aqueous compartments maintain required pH.
- Buffers are mixtures of a weak acid/conjugate base (HA/A-) that function as a reservoir of proton-accepting (A-) and proton-donating (H+) species.
- Buffers neutralize acidic (H+) and basic (B) products of metabolism.
- Henderson-Hasselbalch equation relates pH to proton affinity.
- Ka = [H+][A-]/[HA], where pKa = -log Ka.
- pH = pKa + log([A-]/[HA]).
Titration
- At the origin of the X axis, no strong base solution has been added, and the weak acid is fully protonated.
- At 50% titration:
- Protonated and deprotonated forms are at the same concentration, and pH = pKa.
- Buffer capacity:
- Weak acid/conjugate base pairs are most effective as buffers at the pH equal to the acid pKa value.
- Buffers are effective over the pH range of pKa ± 1, with [A-]/[HA] ratios between 10 and 0.1.
- The pKa of HAc is 4.76, which means that the buffering capacity of HAc is maximal at pH 4.76.
- The buffering region extends 1 pH unit on either side to cover the pH range between 3.76 and 5.76.
- The protonated state is in excess at pH values less than the pKa.
- The deprotonated state is in excess at pH values greater than the pKa.
Major Buffer Systems
- Major buffer systems in the human body: Proteins, Phosphate, CO2/bicarbonate.
- Proteins contain ionizable groups with a broad pH range.
- Phosphate is triprotic with three pKa values: 2.1, 6.8, and 12.3.
- The buffering capacity of phosphate is maximal at pH = 2.1, pH = 6.8, and pH = 12.3.
- Phosphate pKa2 (6.8) is within the effective range for buffering at physiological pH (~7.2).
- CO2/bicarbonate is regulated by physiological processes, with CO2 levels regulated by the lungs and HCO3- levels regulated by the kidney.
Amino acids
- Amino acids are weak acids of the general formula: CN(H)2HRCOOH.
- 20 different common amino acids are proteinogenic.
- Structures and components include:
- C Alpha-carbon (anchor for 4 substituents)
- NH2 Amino group
- H Hydrogen atom
- R Side-chain* (unique identifier)
- COOH Carboxyl group
- The a-carbon is a chiral center in all amino acids except glycine.
- The physiologically relevant form of chiral amino acids is the L-stereoisomer.
Classification of Amino Acids
- The common amino acids are divided into nonpolar and polar groups.
- Nine amino acids have nonpolar/hydrophobic R groups: Alanine, Glycine, Isoleucine, Leucine, Methionine, Phenylalanine, Proline, Tryptophan, Valine.
- Phe and Trp have R groups with aromatic rings (visible by UV spectroscopy).
- Six of the eleven polar amino acids are uncharged at neutral pH: Asparagine, Cysteine, Glutamine, Serine, Threonine, Tyrosine.
- Ser, Thr, and Tyr have a hydroxyl group in their side chain, which are potential sites for protein phosphorylation.
- Ser and Thr hydroxyls are the targets of O-linked glycosylation.
- Asn amide nitrogen is the target of N-linked glycosylation.
- Tyr and Cys have ionizable R groups, with Tyr being also aromatic.
- The remaining 5 polar amino acids are electrically charged at pH 7.2.
- Asp and Glu carry a full negative charge.
- Arg and Lys carry a full positive charge.
- Histidine carries a partial positive charge.
Ionization of Amino Acids
- The pKa of the α-carboxyl group of an amino acid ranges between 2.2-2.8.
- The pKa of the α-amino group ranges between 8.5-9.5.
- Amino acids are zwitterions at physiological pH (7.2-7.4).
- The pKa value of an amino acid side chain measured in water is not an accurate indication of what the pKa value is when that amino acid is part of a protein.
- Histidine is commonly found in the ligand binding site of proteins.
- The pKa values of histidine side-chains depend on the chemical environment generated by the protein structure and can vary from about 4 to 9.
Isoelectric Point
- The isoelectric point (pI) is the pH at which an amino acid has a net charge of zero.
- At the pI, an amino acid is least soluble in water and does not migrate in an electric field.
- For amino acids without ionizable side chains, take the average of the two pKa's.
- pI = (pK1 + pK2)/2
Protein Primary Structure
- Linear chain of amino acids arranged in the order specified by the encoding gene.
- The chain is directional, with the first residue having a free α-amino group (N-terminus) and the last residue having a free α-carboxylate group (C-terminus).
- The 1° structure is formed on ribosomes by energy-requiring, dehydration reactions in which covalent amide bonds (OC-NH) are created from the α-carboxylate carbon of one amino acid to the adjacent molecule's α-amino nitrogen.
- These linkages are typically referred to as peptide bonds, with a single protein chain commonly called a polypeptide.
- The atom sequence in the main chain is NCCNCCNCC.
- Peptide bonds are actually hybrid structures of two resonance forms and exhibit the characteristics of a partial double bond: planar structure, chemically inert, and inflexible.
Impact on Protein Structure
- The double bond character of peptide bonds underscores their impact on protein structure.
- The acquisition of secondary structure is dictated by two torsion angles, phi (Φ) and psi (Ψ), which are set by the limited flexibility of bonds to the α-carbon.
- A Ramachandran Plot of Ψ vs. Φ reports on the compatibility of all possible combinations of the two torsion angles with respect to protein structure.
- Steric hindrance caused by the side chains (R groups) of vicinal amino acids limits the combinations of tolerated Φ and Ψ angles, to those that give rise to either an α-helix or a β-sheet.
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