Double Mutant of Chymotrypsin Inhibitor 2 Stabilized through Increased Conformational Entropy

Gavrilov, Yulian; Kümmerer, Felix; Orioli, Simone; Prestel, Andreas; Lindorff‐Larsen, Kresten; Teilum, Kaare

doi:10.1021/acs.biochem.1c00749

Cited by 7 publications

(32 citation statements)

References 75 publications

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“…Protein flexibility was investigated with biochemical experiments and related to protein function . The influence of mutations on conformational entropy is another line of research . Conformational entropy in proteins was related to intrinsic structural disorder, folding, and binding .…”

Section: Introductionmentioning

confidence: 99%

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Microsecond MD Simulations of the Plexin-B1 RBD: N–H Probability Density as Descriptor of Structural Dynamics, Dimerization-Related Conformational Entropy, and Transient Dimer Asymmetry

Pshetitsky

Mendelman

et al. 2022

J. Phys. Chem. B

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Amide-bond equilibrium probability density, P eq = exp(−u) (u, local potential), and associated conformational entropy, S k = −∫P eq (ln P eq) d Ω ln ∫dΩ, are derived for the Rho GTPase binding domain of Plexin-B1 (RBD) as monomer and dimer from 1 μs MD simulations. The objective is to elucidate the effect of dimerization on the dynamic structure of the RBD. Dispersed (peaked) P eq functions indicate “flexibility” (“rigidity”; the respective concepts are used below in this context). The L1 and L3 loops are throughout highly flexible, the L2 loop and the secondary structure elements are generally rigid, and the L4 loop is flexible in the monomer and rigid in the dimer. Overall, many residues are more flexible in the dimer. These features, and their implications, are discussed. Unexpectedly, we find that monomer unit 1 of the dimer (in short, d1) is unusually flexible, whereas monomer unit 2 (in short, d2) is as rigid as the RBD monomer. This is revealed due to their engagement in slow-to-intermediate conformational exchange detected previously by 15N relaxation experiments. Such motions occur with rates on the order of 103–104 s–1; hence, they cannot be completely sampled over the course of 1 μs simulation. However, the extent to which rigid d2 is affected is small enough to enable physically relevant analysis. The entropy difference between d2 and the monomer yields an entropic contribution of −7 ± 0.7 kJ/mol to the free energy of RBD dimerization. In previous work aimed at similar objectives we used 50–100 ns MD simulations. Those results and the present result differ considerably. In summary, bond-vector P eq functions derived directly from long MD simulations are useful descriptors of protein structural dynamics and provide accurate conformational entropy. Within the scope of slow conformational exchange, they can be useful, even in the presence of incomplete sampling.

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Section: Introductionmentioning

confidence: 99%

“…27 The influence of mutations on conformational entropy is another line of research. 28 Conformational entropy in proteins was related to intrinsic structural disorder, folding, and binding. 29 Deliberations on conformational entropy, and protein dynamics in general, being slaved to water appear in refs 30 and 31.…”

Section: Introductionmentioning

confidence: 99%

Microsecond MD Simulations of the Plexin-B1 RBD: N–H Probability Density as Descriptor of Structural Dynamics, Dimerization-Related Conformational Entropy, and Transient Dimer Asymmetry

Pshetitsky

Mendelman

et al. 2022

J. Phys. Chem. B

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“…The most affected regions are clustered at the N-terminal end of the two main b-strands and in the inhibitory loop. These areas of CI2 already stood out as exchange outliers in our recent analysis of the fast dynamics in CI2 10 . The present analysis provides more details about the thermodynamics, and kinetics of this slow process, as well as the structures of the involved states.…”

Section: Discussionmentioning

confidence: 92%

“…CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 22, 2022. ; https://doi.org/10.1101/2022.12.21.521530 doi: bioRxiv preprint Our analysis of the fast sub-nanosecond dynamics revealed that CI2 also experiences conformational dynamics on the ms time scale, but from that work we could not extract any details about the slow process 10 . In the present work, we asked what the characteristics of the slow conformational process are and if this might help rationalize some of the stabilizing effect in the L49I/I57V variant of CI2.…”

Section: Introductionmentioning

confidence: 89%

“…Our analysis of the fast sub-nanosecond dynamics revealed that CI2 also experiences conformational dynamics on the ms time scale, but from that work we could not extract any details about the slow process 10 . In the present work, we asked what the characteristics of the slow conformational process are and if this might help rationalize some of the stabilizing effect in the L49I/I57V variant of CI2.…”

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confidence: 87%

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Slow conformational changes in the rigid and highly stable chymotrypsin inhibitor 2

Gavrilov

Prestel

Lindorff‐Larsen

et al. 2022

Preprint

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Slow conformational changes are often directly linked to protein function. It is however less clear how such processes may perturb the overall folding stability of a protein. We previously found that the stabilizing double mutant L49I/I57V in the small protein chymotrypsin inhibitor 2 from barley led to distributed increased nano second and faster dynamics. Here we asked what effect this mutant and the two individual mutants L49I and I57V have on the slow conformational dynamics of CI2. We used 15N CPMG spin relaxation dispersion experiments to measure the kinetics, thermodynamics and structural changes associated with slow conformational change in CI2. These changes result in an excited state that is populated to 4.3% at 1 °C. As the temperature is increased the population of the excited state decreases. Structural changes in the transition to the excited state are associated with residues that interact with water molecules that have well defined positions and are found at these positions in all crystal structures of CI2. The mutations in CI2 have only little effect on the structure of the excited state whereas the stability of the excited state to some extent follows the stability of the main state. The minor state is thus most populated for the most stable CI2 variant and least populated for the least stable variant. We hypothesize that the interactions between the mutated residues and the well-ordered water molecules links subtle structural changes around the mutated residues to the region in the protein that experience slow conformational changes.

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Slow conformational changes in the rigid and highly stable chymotrypsin inhibitor 2

et al. 2023

Self Cite

View full text Add to dashboard Cite

Slow conformational changes are often directly linked to protein function. It is however less clear how such processes may perturb the overall folding stability of a protein. We previously found that the stabilizing double mutant L49I/I57V in the small protein chymotrypsin inhibitor 2 from barley led to distributed increased nanosecond and faster dynamics. Here we asked what effects the L49I and I57V substitutions, either individually or together, have on the slow conformational dynamics of CI2. We used 15N CPMG spin relaxation dispersion experiments to measure the kinetics, thermodynamics, and structural changes associated with slow conformational change in CI2. These changes result in an excited state that is populated to 4.3% at 1°C. As the temperature is increased the population of the excited state decreases. Structural changes in the excited state are associated with residues that interact with water molecules that have well defined positions and are found at these positions in all crystal structures of CI2. The substitutions in CI2 have only little effect on the structure of the excited state whereas the stability of the excited state to some extent follows the stability of the main state. The minor state is thus most populated for the most stable CI2 variant and least populated for the least stable variant. We hypothesize that the interactions between the substituted residues and the well‐ordered water molecules links subtle structural changes around the substituted residues to the region in the protein that experience slow conformational changes.

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Double Mutant of Chymotrypsin Inhibitor 2 Stabilized through Increased Conformational Entropy

Cited by 7 publications

References 75 publications

Microsecond MD Simulations of the Plexin-B1 RBD: N–H Probability Density as Descriptor of Structural Dynamics, Dimerization-Related Conformational Entropy, and Transient Dimer Asymmetry

Microsecond MD Simulations of the Plexin-B1 RBD: N–H Probability Density as Descriptor of Structural Dynamics, Dimerization-Related Conformational Entropy, and Transient Dimer Asymmetry

Slow conformational changes in the rigid and highly stable chymotrypsin inhibitor 2

Slow conformational changes in the rigid and highly stable chymotrypsin inhibitor 2

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