Quantitative Prediction of Charge Regulation in Oligopeptides

Weak ampholytes are ubiquitous in nature and commonly found in artificial pH-responsive systems. However, our limited understanding of their charge regulation and the lack of predictive capabilities hinder the bottom-up design of such systems. Here, we used a coarse-grained model of a flexible polymer with weakly ionisable monomer units to quantitatively analyse the ionisation behaviour of two oligopeptides. Our model predicts differences in the charge states between oligopeptides and monomeric amino acids, showing that conformational flexibility and electrostatic interactions between weak acid and base side chains play a key role in the charge regulation. By comparing our simulations with experimental results from potentiometric titration, capillary zone electrophoresis and NMR, we demonstrated that our model reliably predicts the charge state of various peptide sequences. Ultimately, our model is the first step towards understanding the charge regualtion in flexible disordered proteins, and towards using predictive bottom-up design of responsive ampholytes to tailor their<br>properties as a function of charge and pH.<br>

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Quantitative Prediction of Charge Regulation in Oligopeptides

10.26434/chemrxiv.12501470.v4 ◽

2020 ◽

Author(s):

Raju Lunkad ◽

Anastasiia Murmiliuk ◽

Pascal Hebbeker ◽

Milan Boublík ◽

Zdeněk Tošner ◽

...

Keyword(s):

Electrostatic Interactions ◽

Weak Acid ◽

Coarse Grained ◽

Disordered Proteins ◽

Quantitative Prediction ◽

Bottom Up ◽

Charge Regulation ◽

Flexible Polymer ◽

Acid And Base ◽

Base Side

Weak ampholytes are ubiquitous in nature and commonly found in artificial pH-responsive systems. However, our limited understanding of their charge regulation and the lack of predictive capabilities hinder the bottom-up design of such systems. Here, we used a coarse-grained model of a flexible polymer with weakly ionisable monomer units to quantitatively analyse the ionisation behaviour of two oligopeptides. Our model predicts differences in the charge states between oligopeptides and monomeric amino acids, showing that conformational flexibility and electrostatic interactions between weak acid and base side chains play a key role in the charge regulation. By comparing our simulations with experimental results from potentiometric titration, capillary zone electrophoresis and NMR, we demonstrated that our model reliably predicts the charge state of various peptide sequences. Ultimately, our model is the first step towards understanding the charge regualtion in flexible disordered proteins, and towards using predictive bottom-up design of responsive ampholytes to tailor their<br>properties as a function of charge and pH.<br>

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Quantitative Prediction of pH-Controlled Ionization of Oligopeptides

10.26434/chemrxiv.12501470.v1 ◽

2020 ◽

Author(s):

Raju Lunkad ◽

Anastasiia Murmiliuk ◽

Pascal Hebbeker ◽

Milan Boublík ◽

Zdeněk Tošner ◽

...

Keyword(s):

Electrostatic Interactions ◽

Weak Acid ◽

Coarse Grained ◽

Quantitative Prediction ◽

Bottom Up ◽

Coarse Grained Model ◽

Flexible Polymer ◽

Acid And Base ◽

Base Side ◽

Capillary Zone

Weak ampholytes are ubiquitous in nature and commonly found in artificial pH-responsive systems. However, our limited understanding of their ionisation response and the lack of predictive capabilities hinder the bottom-up design of such systems. Here, we used a coarse-grained model of a flexible polymer with weakly ionisable monomer units to quantitatively analyse the ionisation behaviour of two oligopeptides. Differences in ionisation response between oligopeptides and monomeric amino acids showed that electrostatic interactions between weak acid and base side chains play a key role in oligopeptide ionisation, as predicted by our model. Moreover, by comparing our simulations with experimental results from potentiometric titration, capillary zone electrophoresis and NMR, we demonstrated that our model reliably predicts the ionisation response and electrophoretic mobilities of various peptide sequences. Ultimately, our model is the first step towards using predictive bottom-up design of responsive ampholytes to tailor their properties as a function of charge and pH.<br>

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Quantitative Prediction of pH-Controlled Ionization of Oligoampholytes

10.26434/chemrxiv.12501470.v2 ◽

2020 ◽

Author(s):

Raju Lunkad ◽

Anastasiia Murmiliuk ◽

Pascal Hebbeker ◽

Milan Boublík ◽

Zdeněk Tošner ◽

...

Keyword(s):

Electrostatic Interactions ◽

Weak Acid ◽

Coarse Grained ◽

Quantitative Prediction ◽

Bottom Up ◽

Coarse Grained Model ◽

Flexible Polymer ◽

Acid And Base ◽

Base Side ◽

Capillary Zone

Weak ampholytes are ubiquitous in nature and commonly found in artificial pH-responsive systems. However, our limited understanding of their ionisation response and the lack of predictive capabilities hinder the bottom-up design of such systems. Here, we used a coarse-grained model of a flexible polymer with weakly ionisable monomer units to quantitatively analyse the ionisation behaviour of two oligopeptides. Differences in ionisation response between oligopeptides and monomeric amino acids showed that electrostatic interactions between weak acid and base side chains play a key role in oligopeptide ionisation, as predicted by our model. Moreover, by comparing our simulations with experimental results from potentiometric titration, capillary zone electrophoresis and NMR, we demonstrated that our model reliably predicts the ionisation response and electrophoretic mobilities of various peptide sequences. Ultimately, our model is the first step towards using predictive bottom-up design of responsive ampholytes to tailor their properties as a function of charge and pH.<br>

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Role of pKA in Charge Regulation and Conformation of Various Peptide Sequences

Polymers ◽

10.3390/polym13020214 ◽

2021 ◽

Vol 13 (2) ◽

pp. 214

Author(s):

Raju Lunkad ◽

Anastasiia Murmiliuk ◽

Zdeněk Tošner ◽

Miroslav Štěpánek ◽

Peter Košovan

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Coarse Grained ◽

Disordered Proteins ◽

Total Charge ◽

Side Chains ◽

Intrinsically Disordered ◽

Acid And Base ◽

Base Side

Peptides containing amino acids with ionisable side chains represent a typical example of weak ampholytes, that is, molecules with multiple titratable acid and base groups, which generally exhibit charge regulating properties upon changes in pH. Charged groups on an ampholyte interact electrostatically with each other, and their interaction is coupled to conformation of the (macro)molecule, resulting in a complex feedback loop. Their charge-regulating properties are primarily determined by the pKA of individual ionisable side-chains, modulated by electrostatic interactions between the charged groups. The latter is determined by the amino acid sequence in the peptide chain. In our previous work we introduced a simple coarse-grained model of a flexible peptide. We validated it against experiments, demonstrating its ability to quantitatively predict charge on various peptides in a broad range of pH. In the current work, we investigated two types of peptide sequences: diblock and alternating, each of them consisting of an equal number of amino acids with acid and base side-chains. We showed that changing the sequence while keeping the same overall composition has a profound effect on the conformation, whereas it practically does not affect total charge on the peptide. Nevertheless, the sequence significantly affects the charge state of individual groups, showing that the zero net effect on the total charge is a consequence of unexpected cancellation of effects. Furthermore, we investigated how the difference between the pKA of acid and base side chains affects the charge and conformation of the peptide, showing that it is possible to tune the charge-regulating properties by following simple guiding principles based on the pKA and on the amino acid sequence. Our current results provide a theoretical basis for understanding of the complex coupling between the ionisation and conformation in flexible polyampholytes, including synthetic polymers, biomimetic materials and biological molecules, such as intrinsically disordered proteins, whose function can be regulated by changes in the pH.

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Bottom-Up Coarse-Grained Models for Intrinsically Disordered Proteins

Biophysical Journal ◽

10.1016/j.bpj.2020.11.433 ◽

2021 ◽

Vol 120 (3) ◽

pp. 29a

Author(s):

Thomas P. Dannenhoffer-Lafage ◽

Robert B. Best

Keyword(s):

Intrinsically Disordered Proteins ◽

Coarse Grained ◽

Disordered Proteins ◽

Bottom Up ◽

Intrinsically Disordered

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Comparative roles of charge,π, and hydrophobic interactions in sequence-dependent phase separation of intrinsically disordered proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2008122117 ◽

2020 ◽

Vol 117 (46) ◽

pp. 28795-28805

Author(s):

Suman Das ◽

Yi-Hsuan Lin ◽

Robert M. Vernon ◽

Julie D. Forman-Kay ◽

Hue Sun Chan

Keyword(s):

Phase Separation ◽

Electrostatic Interactions ◽

Intrinsically Disordered Proteins ◽

Protein Structures ◽

Coarse Grained ◽

Disordered Proteins ◽

Chain Model ◽

Statistical Potentials ◽

Intrinsically Disordered ◽

Sequence Dependent

Endeavoring toward a transferable, predictive coarse-grained explicit-chain model for biomolecular condensates underlain by liquid–liquid phase separation (LLPS) of proteins, we conducted multiple-chain simulations of the N-terminal intrinsically disordered region (IDR) of DEAD-box helicase Ddx4, as a test case, to assess roles of electrostatic, hydrophobic, cation–π, and aromatic interactions in amino acid sequence-dependent LLPS. We evaluated three different residue–residue interaction schemes with a shared electrostatic potential. Neither a common hydrophobicity scheme nor one augmented with arginine/lysine-aromatic cation–π interactions consistently accounted for available experimental LLPS data on the wild-type, a charge-scrambled, a phenylalanine-to-alanine (FtoA), and an arginine-to-lysine (RtoK) mutant of Ddx4 IDR. In contrast, interactions based on contact statistics among folded globular protein structures reproduce the overall experimental trend, including that the RtoK mutant has a much diminished LLPS propensity. Consistency between simulation and experiment was also found for RtoK mutants of P-granule protein LAF-1, underscoring that, to a degree, important LLPS-driving π-related interactions are embodied in classical statistical potentials. Further elucidation is necessary, however, especially of phenylalanine’s role in condensate assembly because experiments on FtoA and tyrosine-to-phenylalanine mutants suggest that LLPS-driving phenylalanine interactions are significantly weaker than posited by common statistical potentials. Protein–protein electrostatic interactions are modulated by relative permittivity, which in general depends on aqueous protein concentration. Analytical theory suggests that this dependence entails enhanced interprotein interactions in the condensed phase but more favorable protein–solvent interactions in the dilute phase. The opposing trends lead to only a modest overall impact on LLPS.

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Sequence Specificity Despite Intrinsic Disorder: How a Disease-Associated Val/Met Polymorphism Shifts Tertiary Interactions in a Long Disordered Protein

10.26434/chemrxiv.8135777.v1 ◽

2019 ◽

Author(s):

Ruchi Lohia ◽

Reza Salari ◽

Grace Brannigan

Keyword(s):

Secondary Structure ◽

Electrostatic Interactions ◽

Intrinsically Disordered Proteins ◽

Disordered Proteins ◽

Sequence Specificity ◽

Intrinsically Disordered ◽

Specific Effects ◽

Heterogeneous Polymer ◽

Non Local ◽

Dynamics Simulations

<div>The role of electrostatic interactions and mutations that change charge states in intrinsically disordered proteins (IDPs) is well-established, but many disease-associated mutations in IDPs are charge-neutral. The Val66Met single nucleotide polymorphism (SNP) encodes a hydrophobic-to-hydrophobic mutation at the midpoint of the prodomain of precursor brain-derived neurotrophic factor (BDNF), one of the earliest SNPs to be associated with neuropsychiatric disorders, for which the underlying molecular mechanism is unknown. Here we report on over 250 μs of fully-atomistic, explicit solvent, temperature replica exchange molecular dynamics simulations of the 91 residue BDNF prodomain, for both the V66 and M66 sequence.</div><div>The simulations were able to correctly reproduce the location of both local and non-local secondary changes due to the Val66Met mutation when compared with NMR spectroscopy. We find that the local structure change is mediated via entropic and sequence specific effects. We show that the highly disordered prodomain can be meaningfully divided into domains based on sequence alone. Monte Carlo simulations of a self-excluding heterogeneous polymer, with monomers representing each domain, suggest the sequence would be effectively segmented by the long, highly disordered polyampholyte near the sequence midpoint. This is qualitatively consistent with observed interdomain contacts within the BDNF prodomain, although contacts between the two segments are enriched relative to the self-excluding polymer. The Val66Met mutation increases interactions across the boundary between the two segments, due in part to a specific Met-Met interaction with a Methionine in the other segment. This effect propagates to cause the non-local change in secondary structure around the second methionine, previously observed in NMR. The effect is not mediated simply via changes in inter-domain contacts but is also dependent on secondary structure formation around residue 66, indicating a mechanism for secondary structure coupling in disordered proteins. </div>

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