Conformational Stability Study of a Therapeutic Peptide Plectasin Using Molecular Dynamics Simulations in Combination with NMR

Indrakumar, Sowmya; Zalar, Matja; Pohl, Christin; Nørgaard, Allan; Streicher, Werner; Harris, Pernille; Golovanov, Alexander P.; Peters, Günther H.J.

doi:10.1021/acs.jpcb.9b02370

Cited by 14 publications

(23 citation statements)

References 47 publications

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“…MD simulation were performed in Amber as all-atom classical constant pH molecular dynamics (MD) simulations 33 in explicid solvent. The MD simulation setup is explained in detail in our previous work 34 . They were carried out in Amber 16 implementing the amber force field ff99SB 35 with water represented using the TIP3P water model 36 .…”

Section: Constant Ph MD Simulationsmentioning

confidence: 99%

The Effect of Point Mutations on the Biophysical Properties of an Antimicrobial Peptide: Development of a Screening Protocol for Peptide Stability Screening

et al. 2020

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Therapeutic peptides and proteins show an enormous potential on the pharmaceutical market, but high costs in discovery and development are limiting factors so far. Single or multiple point mutations are commonly introduced in protein drugs to increase their binding affinity or selectivity. They can also induce adverse properties, which might be overlooked in a functional screen, such as a decreased colloidal or thermal stability, leading to problems in later stages of the development. In this study, we address the effect of point mutations on the stability of the 4.4 kDa anti-microbial peptide plectasin, as a case study. We combined a systematic high throughput biophysical screen of the peptide thermal and colloidal stability using dynamic light scattering and differential scanning calorimetry in combination with structure-based methods including small angle X-ray scattering, analytical ultracentrifugation and nuclear magnetic resonance spectroscopy. Additionally, we applied molecular dynamics simulations to link obtained protein stability parameters to the protein's molecular structure. Despite their predicted structural similarities, all four plectasin variants showed a substantially different behavior in solution. We observed an increasing propensity of plectasin to aggregate at higher pH, and the introduced mutations influenced the type of aggregation. Our strategy for systematically assessing the stability and aggregation of protein drugs is generally applicable and is of particular relevance given the increasing number of protein drugs in development.

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Section: Constant Ph MD Simulationsmentioning

confidence: 99%

The Effect of Point Mutations on the Biophysical Properties of an Antimicrobial Peptide: Development of a Screening Protocol for Peptide Stability Screening

et al. 2020

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“…Each system was neutralized with either sodium or chloride depending on the overall charge of the protein. The complete protocol used to setup MD simulations is described in our previous work 27 . www.nature.com/scientificreports www.nature.com/scientificreports/ HSA-NEP interaction interface is defined as follows: consider HSA, residues belonging to NEP within 5 Å of HSA's protein surface, and vice versa is defined as interface residues for HSA-NEP interaction.…”

Section: Methodsmentioning

confidence: 99%

“…Parameter file for the tris molecule was prepared using the antechamber 36 module in Amber 16 at pH 8.5 and applying the AM1-BCC 37 charge method. Furthermore, an interaction score per residue (P(I score )) 27 was calculated to estimate the binding capacity of tris to the protein surface. The amount of tris accumulating at the HSA-NEP interaction surface was calculated by summing the number of contacts formed between the compact 3 interface residues and tris molecules, further normalized by the number of tris molecules in the simulated system, which is defined as average normalized contact score.…”

Section: Methodsmentioning

confidence: 99%

Albumin-neprilysin fusion protein: understanding stability using small angle X-ray scattering and molecular dynamic simulations

Kulakova

Indrakumar

Tuelung

et al. 2020

Sci Rep

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fusion technology is widely used in protein-drug development to increase activity, stability, and bioavailability of protein therapeutics. fusion proteins, like any other type of biopharmaceuticals, need to remain stable during production and storage. Due to the high complexity and additional intramolecular interactions, it is not possible to predict the behavior of fusion proteins based on the behavior the individual proteins. therefore, understanding the stability of fusion proteins on the molecular level is crucial for the development of biopharmaceuticals. the current study on the albuminneprilysin (HSA-nep) fusion protein uses a combination of thermal and chemical unfolding with small angle X-ray scattering and molecular dynamics simulations to show a correlation between decreasing stability and increasing repulsive interactions, which is unusual for most biopharmaceuticals. it is also seen that HSA-NEP is not fully flexible: it is present in both compact and extended conformations. Additionally, the volume fraction of each conformation changes with pH. finally, the presence of nacl and arginine increases stability at pH 6.5, but decreases stability at pH 5.0.

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“…The structure was initially prepared at pH 5.0 and pH 6.5 using the H++ server (http://biophysics.cs.vt.edu/H++) (27) which accounts for the protonation state of the titratable residues. Full details of the setup of the MD simlations has been described previously (28).The excipients acetate, phosphate, arginine, histidine, sodium chloride were included in the study. Structures were obtained from PubChem (29) and Zinc Database (30).…”

Section: Molecular Dynamics Simulationmentioning

confidence: 99%

“…Preferential interaction coefficient (PIC) for the specific simulated system was calculated using the method described previously (28). Furthermore, an interaction score per ( ) was calculated to estimate binding capacity of co-solute to residues on protein surface as described.…”

Section: Molecular Dynamics Simulationmentioning

confidence: 99%

Structural-stability studies on recombinant human transferrin

Kulakova

Indrakumar

Sønderby

et al. 2019

Preprint

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Transferrin is an attractive candidate for drug delivery due to its ability to cross the blood brain barrier. However, in order to be able to use it for therapeutic purposes, it is important to investigate how its stability depends on different formulation conditions. Combining high-throughput thermal and chemical denaturation studies with small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, it was possible to connect the stability of transferrin with its conformational changes. The release of iron induces opening of transferrin, which results in a negative effect on its stability. Presence of NaCl, arginine, and histidine leads to opening of the transferrin N-lobe and has a negative impact on the overall protein stability. Statement of significanceProtein-based therapeutics have become an essential part of medical treatment. They are highly specific, have high affinity and fewer off-target effects. However, stabilization of proteins is critical, timeconsuming, and expensive, and it is not yet possible to predict the behavior of proteins under different conditions. The current work is focused on a molecular understanding of the stability of human serum transferrin; a protein which is abundant in blood serum, may pass the blood brain barrier and therefore with high potential in drug delivery. Combination of high throughput unfolding techniques and structural studies, using small angle X-ray scattering and molecular dynamic simulation, allows us to understand the behavior of transferrin on a molecular level.

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Conformational Stability Study of a Therapeutic Peptide Plectasin Using Molecular Dynamics Simulations in Combination with NMR

Cited by 14 publications

References 47 publications

The Effect of Point Mutations on the Biophysical Properties of an Antimicrobial Peptide: Development of a Screening Protocol for Peptide Stability Screening

The Effect of Point Mutations on the Biophysical Properties of an Antimicrobial Peptide: Development of a Screening Protocol for Peptide Stability Screening

Albumin-neprilysin fusion protein: understanding stability using small angle X-ray scattering and molecular dynamic simulations

Structural-stability studies on recombinant human transferrin

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