Double mutant of chymotrypsin inhibitor 2 stabilized through increased conformational entropy

2021 ◽  
Author(s):  
Yulian Gavrilov ◽  
Felix Kümmerer ◽  
Simone Orioli ◽  
Andreas Prestel ◽  
Kresten Lindorff-Larsen ◽  
...  
Keyword(s):  
Double Mutant ◽  
Conformational Dynamics ◽  
Nmr Relaxation ◽  
Conformational Heterogeneity ◽  
Native State ◽  
Chymotrypsin Inhibitor 2 ◽  
Relaxation Measurements ◽  
The Stability ◽  
Dynamics Simulations ◽  
Mutant Forms

The conformational heterogeneity of a folded protein can affect both its function but also stability and folding. We recently discovered and characterized a stabilized double mutant (L49I/I57V) of the protein CI2 and showed that state-of-the-art prediction methods could not predict the increased stability relative to the wild-type protein. Here we have examined whether changed native state dynamics, and resulting entropy changes, can explain the stability changes in the double mutant protein, as well as the two single mutant forms. We have combined NMR relaxation measurements of the ps-ns dynamics of amide groups in the backbone and the methyl groups in the side-chains with molecular dynamics simulations to quantify the native state dynamics. The NMR experiments reveal that the mutations have different effects on the conformational flexibility of CI2: A reduction in conformational dynamics (and entropy) of the native state of L49I variant correlates with its decreased stability, while increased dynamics of the I57V and L49I/I57V variants correlates with their increased stability. These findings suggest that explicitly accounting for changes in native state entropy might be needed to improve the predictions of the effect of mutations on protein stability.

scholarly journals Monte Carlo Sampling of Protein Folding by Combining an All-Atom Physics-Based Model with a Native State Bias

2018 ◽  
Author(s):  
Yong Wang ◽  
Pengfei Tian ◽  
Wouter Boomsma ◽  
Kresten Lindorff-Larsen
Keyword(s):  
Monte Carlo ◽  
Protein Folding ◽  
Conformational Changes ◽  
Conformational Dynamics ◽  
Coarse Graining ◽  
Monte Carlo Sampling ◽  
Implicit Solvent ◽  
Great Success ◽  
Native State ◽  
Dynamics Simulations

AbstractEnergy landscape theory suggests that native interactions are a major determinant of the folding mechanism of a protein. Thus, structure-based (Gō) models have, aided by coarse-graining techniques, shown great success in capturing the mechanisms of protein folding and conformational changes. In certain cases, however, non-native interactions and atomic details are also essential to describe the protein dynamics, prompting the development of a variety of structure-based models which include non-native interactions, and differentiate between different types of attractive potentials. Here, we describe an all-protein-atom hybrid model, termed ProfasiGo, that integrates an implicit solvent all-atom physics-based model (called Profasi) and a structure-based Gō potential, and its implementation in two software packages (PHAISTOS and ProFASi) that are developed for Monte Carlo sampling of protein molecules. We apply the ProfasiGo model to study the folding free energy landscapes of four topologically similar proteins, one of which can be folded by the simplified potential Profasi, and two that have been folded by explicit solvent, all-atom molecular dynamics simulations with the CHARMM22∗ force field. Our results reveal that the hybrid ProfasiGo model is able to capture many of the details present in the physics-based potentials, while retaining the advantages of Gō models for sampling and guiding to the native state. We expect that the model will be widely applicable to study the folding of more complex proteins, or to study conformational dynamics and integration with experimental data.

scholarly journals Molecular dynamics ensemble refinement of the heterogeneous native state of NCBD using chemical shifts and NOEs

2018 ◽  
Author(s):  
Elena Papaleo ◽  
Carlo Camilloni ◽  
Kaare Teilum ◽  
Michele Vendruscolo ◽  
Kresten Lindorff-Larsen
Keyword(s):  
Force Field ◽  
Chemical Shifts ◽  
Molten Globule ◽  
Conformational Dynamics ◽  
Nmr Relaxation ◽  
Structural Heterogeneity ◽  
Order Parameters ◽  
Protein Domain ◽  
Conformational Ensemble ◽  
Native State

ABSTRACTMany proteins display complex dynamical properties that are often intimately linked to their biological functions. As the native state of a protein is best described as an ensemble of confor-mations, it is important to be able to generate models of native state ensembles with high accuracy. Due to limitations in sampling efficiency and force field accuracy it is, however, challenging to obtain accurate ensembles of protein conformations by the use of molecular simulations alone. Here we show that dynamic ensemble refinement, which combines an accurate atomistic force field with commonly available nuclear magnetic resonance (NMR) chemical shifts and NOEs, can provide a detailed and accurate description of the conformational ensemble of the native state of a highly dynamic protein. As both NOEs and chemical shifts are averaged on timescales up to milliseconds, the resulting ensembles reflect the structural heterogeneity that goes beyond that probed e.g. by NMR relaxation order parameters. We selected the small protein domain NCBD as object of our study since this protein, which has been characterized experimentally in substantial detail, displays a rich and complex dynamical behaviour. In particular, the protein has been described as having a molten-globule like structure, but with a relatively rigid core. Our approach allowed us to describe the conformational dynamics of NCBD in solution, and to probe the structural heterogeneity resulting from both short- and long-time-scale dynamics by the calculation of order parameters on different time scales. These results illustrate the usefulness of our approach since they show that NCBD is rather rigid on the nanosecond timescale, but interconverts within a broader ensemble on longer timescales, thus enabling the derivation of a coherent set of conclusions from various NMR experiments on this protein, which could otherwise appear in contradiction with each other.

Enhanced dynamics of conformationally heterogeneous T7 bacteriophage lysozyme native state attenuates its stability and activity

2019 ◽  
Vol 476 (3) ◽  
pp. 613-628 ◽  
Author(s):  
Meenakshi Sharma ◽  
Nancy Jaiswal ◽  
Dinesh Kumar ◽  
Krishna Mohan Poluri
Keyword(s):  
Dynamic Properties ◽  
Nmr Relaxation ◽  
Dynamic Features ◽  
Native State ◽  
Structure Dynamics ◽  
Functional Dynamics ◽  
State Ensemble ◽  
The Stability ◽  
Ph Range ◽  
Relationship Of

Abstract Proteins are dynamic in nature and exist in a set of equilibrium conformations on various timescale motions. The flexibility of proteins governs various biological functions, and therefore elucidation of such functional dynamics is essential. In this context, we have studied the structure–dynamics–stability–activity relationship of bacteriophage T7 lysozyme/endolysin (T7L) native-state ensemble in the pH range of 6–8. Our studies established that T7L native state is conformationally heterogeneous, as several residues of its C-terminal half are present in two conformations (major and minor) in the slow exchange time scale of nuclear magnetic resonance (NMR). Structural and dynamic studies suggested that the residues belonging to minor conformations do exhibit native-like structural and dynamic features. Furthermore, the NMR relaxation experiments unraveled that the native state is highly dynamic and the dynamic behavior is regulated by the pH, as the pH 6 conformation exhibited enhanced dynamics compared with pH 7 and 8. The stability measurements and cell-based activity studies on T7L indicated that the native protein at pH 6 is ∼2 kcal less stable and is ∼50% less active than those of pH 7 and 8. A comprehensive analysis of the T7L active site, unfolding initiation sites and the residues with altered dynamics outlined that the attenuation of stability and activity is a resultant of its enhanced dynamic properties, which, in turn, can be attributed to the protonation/deprotonation of its partially buried His residues. Our study on T7L structure–dynamics–activity paradigm could assist in engineering novel amidase-based endolysins with enhanced activity and stability over a broad pH range.

scholarly journals Deciphering the Mechanism of Gilteritinib Overcoming Lorlatinib Resistance to the Double Mutant I1171N/F1174I in Anaplastic Lymphoma Kinase

2021 ◽  
Vol 9 ◽  
Author(s):  
Shuai Liang ◽  
Qing Wang ◽  
Xuesen Qi ◽  
Yudi Liu ◽  
Guozhen Li ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Targeted Therapies ◽  
Anaplastic Lymphoma Kinase ◽  
Double Mutant ◽  
Conformational Dynamics ◽  
Bound State ◽  
Kinase Domain ◽  
Alk Inhibitors ◽  
Anaplastic Lymphoma ◽  
Dynamics Simulations

Anaplastic lymphoma kinase (ALK) is validated as a therapeutic molecular target in multiple malignancies, such as non-small cell lung cancer (NSCLC). However, the feasibility of targeted therapies exerted by ALK inhibitors is inevitably hindered owing to drug resistance. The emergence of clinically acquired drug mutations has become a major challenge to targeted therapies and personalized medicines. Thus, elucidating the mechanism of resistance to ALK inhibitors is helpful for providing new therapeutic strategies for the design of next-generation drug. Here, we used molecular docking and multiple molecular dynamics simulations combined with correlated and energetical analyses to explore the mechanism of how gilteritinib overcomes lorlatinib resistance to the double mutant ALK I1171N/F1174I. We found that the conformational dynamics of the ALK kinase domain was reduced by the double mutations I1171N/F1174I. Moreover, energetical and structural analyses implied that the double mutations largely disturbed the conserved hydrogen bonding interactions from the hinge residues Glu1197 and Met1199 in the lorlatinib-bound state, whereas they had no discernible adverse impact on the binding affinity and stability of gilteritinib-bound state. These discrepancies created the capacity of the double mutant ALK I1171N/F1174I to confer drug resistance to lorlatinib. Our result anticipates to provide a mechanistic insight into the mechanism of drug resistance induced by ALK I1171N/F1174I that are resistant to lorlatinib treatment in NSCLC.

scholarly journals Synergistic stabilization of a double mutant in chymotrypsin inhibitor 2 from a library screen in E. coli

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Louise Hamborg ◽  
Daniele Granata ◽  
Johan G. Olsen ◽  
Jennifer Virginia Roche ◽  
Lasse Ebdrup Pedersen ◽  
...  
Keyword(s):  
Double Mutant ◽  
Single Point ◽  
Point Mutations ◽  
Multiple Point ◽  
Chymotrypsin Inhibitor ◽  
E Coli ◽  
Chymotrypsin Inhibitor 2 ◽  
Folded Proteins ◽  
The Stability ◽  
The Individual

AbstractMost single point mutations destabilize folded proteins. Mutations that stabilize a protein typically only have a small effect and multiple mutations are often needed to substantially increase the stability. Multiple point mutations may act synergistically on the stability, and it is often not straightforward to predict their combined effect from the individual contributions. Here, we have applied an efficient in-cell assay in E. coli to select variants of the barley chymotrypsin inhibitor 2 with increased stability. We find two variants that are more than 3.8 kJ mol−1 more stable than the wild-type. In one case, the increased stability is the effect of the single substitution D55G. The other case is a double mutant, L49I/I57V, which is 5.1 kJ mol−1 more stable than the sum of the effects of the individual mutations. In addition to demonstrating the strength of our selection system for finding stabilizing mutations, our work also demonstrate how subtle conformational effects may modulate stability.

Silver nitrate in aqueous solution and as molten salt: A molecular dynamics simulation and NMR relaxation study

1994 ◽  
Vol 72 (11) ◽  
pp. 2278-2285 ◽  
Author(s):  
Aatto Laaksonen ◽  
Helena Kovacs
Keyword(s):  
Molecular Dynamics ◽  
Aqueous Solution ◽  
Silver Nitrate ◽  
Nmr Relaxation ◽  
Md Simulations ◽  
Dynamics Simulation ◽  
Correlation Times ◽  
Nitrate Ion ◽  
Relaxation Measurements ◽  
Dynamics Simulations

Using molecular dynamics simulations, the motion and intermolecular interactions of the ions of silver nitrate are studied in aqueous solution and compared to the results obtained from simulations of molten AgNO3. The particularly interesting and experimentally frequently studied modes of reorientational motion (in-plane and end-over-end) of the planar nitrate ion have been determined from the simulation results. In accordance with earlier experimental results, the correlation times for the end-over-end rotation in aqueous solution are longer than those for the in-plane rotation, while the opposite is found to hold in the melt. In addition, the rotational motion of the nitrate ion in aqueous solution is experimentally studied using 14N relaxation measurements. Good agreement is found between the reorientational correlation times obtained from MD simulations and from NMR relaxation measurements.

scholarly journals Molecular dynamics ensemble refinement of the heterogeneous native state of NCBD using chemical shifts and NOEs

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5125 ◽  
Author(s):  
Elena Papaleo ◽  
Carlo Camilloni ◽  
Kaare Teilum ◽  
Michele Vendruscolo ◽  
Kresten Lindorff-Larsen
Keyword(s):  
Force Field ◽  
Chemical Shifts ◽  
Molten Globule ◽  
Conformational Dynamics ◽  
Nmr Relaxation ◽  
Structural Heterogeneity ◽  
Order Parameters ◽  
Protein Domain ◽  
Conformational Ensemble ◽  
Native State

Many proteins display complex dynamical properties that are often intimately linked to their biological functions. As the native state of a protein is best described as an ensemble of conformations, it is important to be able to generate models of native state ensembles with high accuracy. Due to limitations in sampling efficiency and force field accuracy it is, however, challenging to obtain accurate ensembles of protein conformations by the use of molecular simulations alone. Here we show that dynamic ensemble refinement, which combines an accurate atomistic force field with commonly available nuclear magnetic resonance (NMR) chemical shifts and NOEs, can provide a detailed and accurate description of the conformational ensemble of the native state of a highly dynamic protein. As both NOEs and chemical shifts are averaged on timescales up to milliseconds, the resulting ensembles reflect the structural heterogeneity that goes beyond that probed, e.g., by NMR relaxation order parameters. We selected the small protein domain NCBD as object of our study since this protein, which has been characterized experimentally in substantial detail, displays a rich and complex dynamical behaviour. In particular, the protein has been described as having a molten-globule like structure, but with a relatively rigid core. Our approach allowed us to describe the conformational dynamics of NCBD in solution, and to probe the structural heterogeneity resulting from both short- and long-timescale dynamics by the calculation of order parameters on different time scales. These results illustrate the usefulness of our approach since they show that NCBD is rather rigid on the nanosecond timescale, but interconverts within a broader ensemble on longer timescales, thus enabling the derivation of a coherent set of conclusions from various NMR experiments on this protein, which could otherwise appear in contradiction with each other.

scholarly journals Stability and Mobility of Lid Lipmnk in Acetonitrile by Molecular Dynamics Simulations Approach

2018 ◽  
Vol 15 (2) ◽  
pp. 295-299
Author(s):  
Dian Herasari ◽  
Rukman Hertadi ◽  
Fida M. Warganegara ◽  
Akhmaloka Akhmaloka
Keyword(s):  
Molecular Dynamics ◽  
Conformational Dynamics ◽  
Solvent Polarity ◽  
Thermophilic Bacterium ◽  
Solvent Mixtures ◽  
The Stability ◽  
Dynamics Simulations ◽  
Opening And Closing ◽  
Acetonitrile Concentration ◽  
Detailed Molecular Analysis

Manuk lipase (lipMNK) from the thermophilic bacterium Geobacillus sp is a double lid lipase containing short and long lid segments. A few studies demonstrated that catalytic action of lipase involved the movement of lid segments from closed to open conformation upon the substrate binding. One factor that affects conformational dynamics of the lid segments is solvent polarity. The presence of acetonitrile in certain concentration has showed to enhance lipase activity. In this study, the effect of acetonitrile to the stability and activity of lipMNK was studied at the atomic level by molecular dynamics (MD) simulation. MD was carried out by NPT ensemble at 358 K for 100 nano seconds in various ratio of acetonitrile:water solvent mixtures. The results showed that the conformation of lipMNK was stable up to 70%. However, the effect of lid movement was significantly observed since the concentration at 20% acetonitrile. Detailed molecular analysis at this acetonitrile concentration revealed that the two lids moved in different modes upon opening and closing movement. In the opening movement, the two lids appeared to move in almost simultaneously, while during the closing movement, it was observed sequentially, started by short segment followed by long segment lid.

Sign in / Sign up

Export Citation Format

Share Document