Effect of Additional Terminal Residues on the Folding and Unfolding Dynamics of Cold Shock Protein

Enhancing protein stability through N‐ and C‐terminal modifications presents a safe and cost‐effective strategy. We investigated this by combining single‐molecule magnetic tweezers experiments and molecular dynamics simulations to study the folding and unfolding dynamics of CSP with various appended residues (LE‐CSP‐GS, KL‐CSP‐GS, KL‐CSP‐LE). The LE‐CSP‐GS variant showed significantly faster unfolding and slower folding, leading to a substantial decrease in stability (∼5 kBT). Simulations revealed that stability differences originated from additional hydrogen bonds formed by residues K6 and E56. This work demonstrates that terminal modifications are a powerful tool for tuning protein stability and dynamics. Abstract Enhancing protein stability through modifications to the N‐ and C‐termini of natural proteins offers the distinct advantages of safety and cost‐effectiveness when compared to the denovo design of proteins. To explore the effect of additional residues at the termini on protein stability, single‐molecule magnetic tweezers are employed to examine the folding and unfolding dynamics of Cold Shock Protein (CSP) with various appended residues (LE‐CSP‐GS, KL‐CSP‐GS, KL‐CSP‐LE). The unfolding rate constant of the LE‐CSP‐GS is an order of magnitude faster than the others, while its folding rate constant decreased by more than an order of magnitude, resulting in upto ≈5 kBT bigger in folding free energy. Molecular dynamics (MD) simulations revealed that the stability differences are due to additional hydrogen bonds formed by residues K6 and E56. The combination of single‐molecule experiments and MD simulations indicates that additional residues at the termini can significantly affect protein stability and dynamics. Enhancing protein stability through N- and C-terminal modifications presents a safe and cost-effective strategy. We investigated this by combining single-molecule magnetic tweezers experiments and molecular dynamics simulations to study the folding and unfolding dynamics of CSP with various appended residues (LE-CSP-GS, KL-CSP-GS, KL-CSP-LE). The LE-CSP-GS variant showed significantly faster unfolding and slower folding, leading to a substantial decrease in stability (∼5 kBT). Simulations revealed that stability differences originated from additional hydrogen bonds formed by residues K6 and E56. This work demonstrates that terminal modifications are a powerful tool for tuning protein stability and dynamics. Abstract Enhancing protein stability through modifications to the N- and C-termini of natural proteins offers the distinct advantages of safety and cost-effectiveness when compared to the denovo design of proteins. To explore the effect of additional residues at the termini on protein stability, single-molecule magnetic tweezers are employed to examine the folding and unfolding dynamics of Cold Shock Protein (CSP) with various appended residues (LE-CSP-GS, KL-CSP-GS, KL-CSP-LE). The unfolding rate constant of the LE-CSP-GS is an order of magnitude faster than the others, while its folding rate constant decreased by more than an order of magnitude, resulting in upto ≈5 k B T bigger in folding free energy. Molecular dynamics (MD) simulations revealed that the stability differences are due to additional hydrogen bonds formed by residues K6 and E56. The combination of single-molecule experiments and MD simulations indicates that additional residues at the termini can significantly affect protein stability and dynamics. Advanced Science, Volume 12, Issue 48, December 29, 2025.