Stability-based approaches in protein engineering MCQs With Answer

Introduction: Stability-based approaches in protein engineering are central to designing therapeutic proteins and biopharmaceutical formulations that maintain activity, safety, and shelf-life. This blog offers targeted multiple-choice questions for M.Pharm students focusing on strategies to enhance protein stability—thermal, chemical, and colloidal—through rational design, directed evolution, surface engineering, and formulation techniques. Questions cover molecular determinants of stability (hydrophobic core packing, disulfide bonds, salt bridges), computational prediction tools, experimental assays (DSC, DSF, CD), and formulation excipients that reduce aggregation. These MCQs will test conceptual understanding and practical application of stability optimization for protein drugs, preparing students for both exams and real-world formulation challenges.

Q1. Which of the following mutations is most commonly introduced to increase protein thermostability by restricting backbone flexibility?

• Substitution of glycine to proline in loop regions
• Substitution of alanine to valine in the hydrophobic core
• Introduction of a surface lysine to increase solubility
• Replacement of serine with threonine to add a hydroxyl group

Correct Answer: Substitution of glycine to proline in loop regions

Q2. What does an increase in the melting temperature (Tm) measured by differential scanning calorimetry indicate for a protein?

• Higher conformational flexibility at physiological temperature
• Increased resistance to thermal unfolding
• Greater susceptibility to proteolytic degradation
• Reduced aggregation propensity under stress

Correct Answer: Increased resistance to thermal unfolding

Q3. Which stability-based engineering approach relies on creating combinatorial libraries followed by screening under selective conditions?

• Rational design using Rosetta energy calculations
• Directed evolution
• Consensus design from multiple sequence alignments
• Site-directed mutagenesis of predicted hotspots

Correct Answer: Directed evolution

Q4. Consensus design improves protein stability primarily by which principle?

• Choosing the most frequent amino acid at each position across homologs
• Introducing disulfide bonds between non-conserved cysteines
• Replacing all surface residues with charged amino acids
• Mimicking thermostable extremophile sequences in full length

Correct Answer: Choosing the most frequent amino acid at each position across homologs

Q5. Which computational tool is widely used for predicting the energetic impact of point mutations on protein stability?

• BLAST for sequence alignment
• FoldX for ΔΔG calculations
• Clustal Omega for consensus design
• AutoDock for ligand docking

Correct Answer: FoldX for ΔΔG calculations

Q6. Formation of which intramolecular bond is often engineered to rigidify proteins and improve stability?

• Peptide bond
• Disulfide bond between cysteines
• Glycosidic bond through glycosylation
• Phosphodiester bond on serine residues

Correct Answer: Disulfide bond between cysteines

Q7. Which assay rapidly screens many variants for relative thermal stability using a fluorescent dye that binds to exposed hydrophobic regions?

• Differential scanning calorimetry (DSC)
• Differential scanning fluorimetry (DSF) or Thermofluor
• Circular dichroism spectroscopy (CD)
• Size-exclusion chromatography (SEC)

Correct Answer: Differential scanning fluorimetry (DSF) or Thermofluor

Q8. In protein formulation, which excipient primarily stabilizes proteins by preferential exclusion and increasing the chemical potential of the unfolded state?

• Sodium dodecyl sulfate (SDS)
• Polyethylene glycol (PEG)
• Glycerol or trehalose (osmolytes)
• EDTA as a chelator

Correct Answer: Glycerol or trehalose (osmolytes)

Q9. Which modification often reduces immunogenicity and increases serum half-life while also sometimes improving colloidal stability?

• Deamidation of asparagine residues
• PEGylation of the protein surface
• Oxidation of methionine residues
• Cleavage of N-terminal residue

Correct Answer: PEGylation of the protein surface

Q10. What is the main cause of colloidal instability leading to protein aggregation in concentrated formulations?

• High intrinsic thermal stability (high Tm)
• Attractive intermolecular interactions and decreased repulsion
• Excessive glycosylation improving solubility
• Optimized pH for structural stability

Correct Answer: Attractive intermolecular interactions and decreased repulsion

Q11. Which design strategy uses structural models to identify and fill internal cavities in the hydrophobic core to enhance stability?

• Surface charge reversal
• Core packing optimization via mutation to bulkier residues
• Adding flexible glycine-rich loops
• Replacing buried polar residues with charged ones

Correct Answer: Core packing optimization via mutation to bulkier residues

Q12. Introducing salt bridges into a protein structure often stabilizes it. Which pair of residues typically form a stabilizing salt bridge?

• Arginine and glutamate (or aspartate)
• Leucine and isoleucine
• Serine and threonine
• Tyrosine and phenylalanine

Correct Answer: Arginine and glutamate (or aspartate)

Q13. Which experimental technique provides information about secondary structure content and can monitor unfolding transitions?

• Circular dichroism spectroscopy (CD)
• Mass spectrometry (MS)
• Dynamic light scattering (DLS)
• Fourier-transform infrared microscopy

Correct Answer: Circular dichroism spectroscopy (CD)

Q14. Which chemical modification can be intentionally engineered to increase thermal stability by introducing additional sugar moieties?

• PEGylation of lysines
• Site-specific glycosylation
• Acetylation of N-terminus
• Oxidation of cysteines

Correct Answer: Site-specific glycosylation

Q15. What is the trade-off commonly observed when stabilizing enzymes for higher thermostability?

• Increased thermostability always increases catalytic rate
• Higher stability often reduces catalytic activity or flexibility at active site
• Stabilization eliminates the need for formulation excipients
• Stabilized enzymes become more immunogenic

Correct Answer: Higher stability often reduces catalytic activity or flexibility at active site

Q16. Which protein engineering approach integrates structural modeling with laboratory mutagenesis to target stabilizing substitutions rationally?

• Random mutagenesis without screening
• Rational design guided by computational energy calculations
• Phage display with no sequence information
• Consensus sequence swapping only

Correct Answer: Rational design guided by computational energy calculations

Q17. Which property measured by dynamic light scattering (DLS) is most relevant to assessing formulation stability?

• Secondary structure content
• Hydrodynamic radius and polydispersity indicating aggregation
• Thermal transition midpoint (Tm)
• Enzyme turnover number (kcat)

Correct Answer: Hydrodynamic radius and polydispersity indicating aggregation

Q18. Which strategy reduces chemical degradation such as deamidation and oxidation in protein formulations?

• Storing at elevated temperatures to speed reactions
• Optimizing pH, adding antioxidants and chelators, and lowering storage temperature
• Increasing ionic strength to extreme levels
• Removing all stabilizing excipients to avoid interactions

Correct Answer: Optimizing pH, adding antioxidants and chelators, and lowering storage temperature

Q19. Which metric represents the free energy difference between folded and unfolded states and is directly related to protein thermodynamic stability?

• kcat
• ΔG of unfolding (ΔG°)
• Isoelectric point (pI)
• Hydropathy index

Correct Answer: ΔG of unfolding (ΔG°)

Q20. What is the benefit of using molecular dynamics (MD) simulations in stability-guided protein engineering?

• MD provides definitive ΔG values without experiments
• MD helps explore conformational flexibility, identify unstable regions and predict effects of mutations over time
• MD eliminates the need for any laboratory validation
• MD always accurately predicts immunogenic epitopes

Correct Answer: MD helps explore conformational flexibility, identify unstable regions and predict effects of mutations over time

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