Definition and testing of the GROMOS force-field versions 54A7 and 54B7

New parameter sets of the GROMOS biomolecular force field, 54A7 and 54B7, are introduced. These parameter sets summarise some previously published force field modifications: The 53A6 helical propensities are corrected through new φ/ψ torsional angle terms and a modification of the N-H, C=O repulsion, a new atom type for a charged −CH3 in the choline moiety is added, the Na+ and Cl− ions are modified to reproduce the free energy of hydration, and additional improper torsional angle types for free energy calculations involving a chirality change are introduced. The new helical propensity modification is tested using the benchmark proteins hen egg-white lysozyme, fox1 RNA binding domain, chorismate mutase and the GCN4-p1 peptide. The stability of the proteins is improved in comparison with the 53A6 force field, and good agreement with a range of primary experimental data is obtaine

Experimental and computational analysis of the structure and dynamics of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) are abundant in cells and have central roles in protein-protein interaction networks. Many are involved in cancer, aging and neurodegenerative diseases. The structure and dynamics of IDPs is intimately related to their interactions with binding partners. Because IDPs are inherently flexible and do not have a single conformation, conventional methods and conditions for determining structure and dynamics of globular proteins may not be directly applicable. Nuclear magnetic resonance (NMR) spectroscopy is one of the primary techniques characterizing the structures and dynamics of IDPs, but one cannot rely solely on NMR data. A primary aim of this work was to use Molecular Dynamics (MD) simulations in conjunction with NMR and other biophysical techniques to achieve a deeper understanding of the structure and dynamics of IDPs. To establish suitable parameters and force field choice for simulating IDPs, extensive MD simulations were performed and the results were compared to experimental data. Using computational and experimental techniques, the interactions between peptides from 9 disordered proteins with a common target were interrogated. The findings allowed us to determine key factors in modulating the affinities of the various interactions and highlighted the importance of molecular recognition fragments (MoRFs) in IDP target recognition and binding. IDP binding was also investigated from the perspective of the binding partner. The backbone resonances of the ~32 kDa target were assigned and the binding interface was mapped in the presence of a peptide from a disordered binding partner. Chemical shift changes distant from the interaction site indicated that IDP binding is a complex process, which should be studied from the perspectives of the partner and target. Because IDPs are highly sensitive to environmental conditions, the effects of molecular crowding on the dynamics of IDPs were also investigated. I found that crowding might have differential effects on the conformational propensities of distinct regions of some IDPs. This information will help to understand the behavior of IDPs in cellular environments and to determine suitable conditions for accurately studying them. This work has helped to improve the understanding of how IDP structure and dynamics relate to target binding

Doctor of Philosophy

dissertationThe coiled-coil is a common protein tertiary structural motif that is composed of two or more alpha helices intertwined together to formed a supercoil. In biological systems, the coiledcoil motif often forms the oligomerization domain of various proteins including DNA binding proteins, structural and transport proteins, and cellular transport and fusion proteins. It was first described by Crick in the 1950s while describing the structure of α-keratin and has since that time been the subject of numerous engineering and mutation studies. This versatile motif has been adapted to a number of nonbiological applications including environmentally responsive hydrogels, crosslinking agents, the construction of self-assembling fibers for tissue engineering, and biosensor surfaces. In this dissertation, we test the applicability of computational methods to understand the underlying energetics in coiled-coils as we apply molecular modeling approaches in the development of pharmaceutics. Two studies are described which test the limits of modern molecular dynamic force fields to understand the structural dynamics of the motif and to use energy calculation methodologies to predict favorable mutations for heterodimer formation and specificity. The first study considers the increasingly common use of fluorinated residues in protein pharmaceutics with regard to their incorporation in coiled-coils. Many studies find that fluorinated residues in the hydrophobic core increase protein stability against chemical and thermal denaturants. Often their incorporation fails to consider structural, energetic, and geometrical differences between these fluorinated residues and their nonfluorinated counterparts. To consider these differences, several variants of Hodges' very stable parallel heterodimer coiledcoil were constructed to examine the effect of salt bridge lengths and geometries with mixed fluorinated and nonfluorinated packed hydrophobic cores. In the second study, we collaborated with an experimental laboratory in the development of a mutant Bcr monomer with designed mutations to increase specificity and binding to the oncoprotein Bcr-Abl for use as an apoptosis inducing agent in chronic myelogenous leukemia (CML) cells. The final chapters of this dissertation discuss challenges and limitations that were encountered using force fields and energetic methods in our attempts to use computational chemistry to model this protein motif

A molecular view on the organizational complexity of proteins in membranes

Alle levende wezens bestaan uit cellen, en elke cel is omgeven door een celmembraan. Tot voorkort dachten we dat de celmembraan niks meer was dan een vliesje dat de inhoud van de cel afscheid van alles daarbuiten, maar meer en meer ontdekken we dat dit vliesje, dat voornamelijk bestaat uit lipiden, cholesterol en eiwitten, nog vele andere functies heeft. In mijn onderzoek heb ik computer simulaties gebruikt om de wisselwerking tussen lipiden en eiwitten in de membraan te bestuderen. Door het kleine formaat van celmembranen en de hele korte tijdschaal waarin processen plaatsvinden, zijn de wetenschappelijke vraagstukken lastig met experimentele technieken te bestuderen. Met behulp van computer modellen, zoals het in Groningen ontwikkelde “Martini” model, kunnen we deze processen op bijna atomistische resolutie observeren. Het onderzoek richtte zich op twee aspecten. Ten eerste is het Martini krachtenveld verder getest en ontwikkeld, wat de mogelijkheid geeft tot nog betrouwbaardere simulaties. Het tweede aspect is het simuleren van kleine membraaneiwitten in een (lipide)membraan met behulp van het Martini krachtenveld. Deze simulaties hebben meer inzicht verschaft in hoe de eiwitten zich bewegen tussen verschillende domeinen binnen het membraan en de belangrijke rol die een specifiek lipide hierin speelt: het GM1 ganglioside lipide

Coarse-grained peptide models: conformational sampling, peptide association and dynamical properties for peptides

In dieser Arbeit wird ein vergröbertes (engl. coarse-grained, CG) Simulationsmodell für Peptide in wässriger Lösung entwickelt. In einem CG Verfahren reduziert man die Anzahl der Freiheitsgrade des Systems, so dass manrngrössere Systeme auf längeren Zeitskalen untersuchen kann. Die Wechselwirkungspotentiale des CG Modells sind so aufgebaut, dass die Peptid Konformationen eines höher aufgelösten (atomistischen) Modells reproduziert werden.rnIn dieser Arbeit wird der Einfluss unterschiedlicher bindender Wechsel-rnwirkungspotentiale in der CG Simulation untersucht, insbesondere daraufhin,rnin wie weit das Konformationsgleichgewicht der atomistischen Simulation reproduziert werden kann. Im CG Verfahren verliert man per Konstruktionrnmikroskopische strukturelle Details des Peptids, zum Beispiel, Korrelationen zwischen Freiheitsgraden entlang der Peptidkette. In der Dissertationrnwird gezeigt, dass diese “verlorenen” Eigenschaften in einem Rückabbildungsverfahren wiederhergestellt werden können, in dem die atomistischen Freiheitsgrade wieder in die CG-Strukturen eingefügt werden. Dies gelingt, solange die Konformationen des CG Modells grundsätzlich gut mit der atomistischen Ebene übereinstimmen. Die erwähnten Korrelationen spielen einerngrosse Rolle bei der Bildung von Sekundärstrukturen und sind somit vonrnentscheidender Bedeutung für ein realistisches Ensemble von Peptidkonformationen. Es wird gezeigt, dass für eine gute Übereinstimmung zwischen CG und atomistischen Kettenkonformationen spezielle bindende Wechselwirkungen wie zum Beispiel 1-5 Bindungs- und 1,3,5-Winkelpotentiale erforderlich sind. Die intramolekularen Parameter (d.h. Bindungen, Winkel, Torsionen), die für kurze Oligopeptide parametrisiert wurden, sind übertragbarrnauf längere Peptidsequenzen. Allerdings können diese gebundenen Wechselwirkungen nur in Kombination mit solchen nichtbindenden Wechselwirkungspotentialen kombiniert werden, die bei der Parametrisierung verwendet werden, sind also zum Beispiel nicht ohne weiteres mit einem andere Wasser-Modell kombinierbar. Da die Energielandschaft in CG-Simulationen glatter ist als im atomistischen Modell, gibt es eine Beschleunigung in der Dynamik. Diese Beschleunigung ist unterschiedlich für verschiedene dynamische Prozesse, zum Beispiel für verschiedene Arten von Bewegungen (Rotation und Translation). Dies ist ein wichtiger Aspekt bei der Untersuchung der Kinetik von Strukturbildungsprozessen, zum Beispiel Peptid Aggregation.rnA bottom-up coarse-graining (CG) procedure for peptides in aqueous solutionrnis studied in this thesis. The coarse-graining procedure reduces the numberrnof degrees of freedom of the system, enabling us to investigate larger systemsrnand due to the smoother energy landscape one can get faster a better sam-rnpling of the system. The interaction potentials in our coarse-grained modelrnare constructed in a such way, that the coarse-grained peptide reproducesrnconformations according to a high-resolution (atomistic) model.rnIn this work the influence of differently constructed bonded potentials onrnthe reproduction of atomistic characteristics in structure-based CG simula-rntion was investigated. In the coarse-graining procedure one loses by constuc-rntion microscopic structural details of the peptide. This can be for examplerncorrelations between degrees of freedom. In the thesis it is presented thatrnthose “lost” properties can be recovered in a backmapping procedure whichrnreintroduces atomistic degrees of freedom into CG structures – as long asrnthe overall conformational sampling of the molecule is correctly representedrnin the CG level of resolution. These correlations play an important role inrnsecondary structure formation. Therefore they are crucial for a realistic con-rnformational ensemble of the peptide. It is shown that for an exact agreementrnof the CG conformations with the atomistic reference additional bonded po-rntentials are required such as 1-5 bond and 1,3,5-angle potentials.rnIt is shown that the intramolecular parameters (i.e. bonds, angles, tor-rnsions) determined for short oligopeptides are transferable to longer peptidernsequences. But one has to be aware that bonded potentials should be usedrnonly in combination with those nonbonded interaction potentials, with whichrnthey were parametrized. So, they cannot necessarily be combined with otherrnnonbonded interactions, for example a different water model.rnSince the energy landscape is smoother in CG simulations, there is thernacceleration in time and in principle the CG time does not corresponds onernto one to the atomistic time any more. The dynamical properties of thernpeptide in water on the atomistic and CG levels were investigated in order tornget an estimate for the speed-up of the dynamics in the CG model comparedrnto the atomistic one. We found that these scaling factors are different forrndifferent dynamical properties and concluded that there is different “speed-rnup” for different types of motions (for example rotation and translation).rnThis is an important observation for the kinetics of processes such as peptidernaggregation.r

Next-generation coarse-grained models for molecular dynamics simulations of fluid phase equilibria and protein biophysics using the relative entropy

Coarse grained models for molecular dynamic simulations of liquid structure and protein folding and self-assembly have been the subject of decades of research efforts. Although such models enable probing into longer length and time scales, they are still limited in accuracy and scale poorly to thermodynamic conditions or chemical entities beyond the ones at which they are developed. In this work, I leverage a powerful coarse graining framework based on an information theoretic metric known as the relative entropy to design novel coarse grained models for equilibrium phase behavior in liquid mixtures that correctly address the relevant manybody physics critically involved in phase behavior and thus, can provide structurally accurate descriptions of macroscopic phase separation across a large range of mixture compositions. I also develop protein models that are “next- generation” in the sense that they are systematically extendable in complexity while depending minimally on experimental data. These protein models offer remarkable predictive accuracy for folding of single proteins and insights into large scale protein self-assembly commonly seen in neurodegenerative pathologies such as Alzheimer’s and prion diseases. This dissertation develops and applies powerful coarse-graining techniques to meet longstanding challenges in the field and elevate coarse grained models from being “toy” systems to accurate, “production-level” tools for the development of biotechnologies and advanced materials

The interaction of materials and biology: simulations of peptides, surfaces, and biomaterials

Biomaterials were originally designed to augment or replace damaged tissue in the body, but now encompass a wider range of applications including drug delivery, cancer vaccines, electronic sensor devices, and non-fouling coatings for ship hulls. At the heart of all of these applications is the interface between synthetic materials and biology. Modern techniques for studying this interface are limited to the macro and micro scales. With the advent of high performance computing clusters, molecular simulation is now capable of simulating the interface at the nano-scale. This thesis demonstrates how simulation adds important insights to the understanding of biomaterials. It begins with a comprehensive outline of the theoretical aspects of simulating the interface between water and solid surfaces. After this, small surface-bound biological molecules are modelled to explain experiments showing that they can capture cells on the surface. Finally, a new and practical, scalable technique for controlling biological molecules at the surface is developed. This work advances the field of biomaterials by explaining important processes that occur at the interface of biology and technology

Potential and Free Energy Surfaces of Adsorbed Peptides

Peptide adsorption on solid surfaces is a common process that occurs in nanotechnology and biology, with applications in the formation of nanomaterials, biosensing and drug delivery, amongst many others. Peptide adsorption involves complex processes that are difficult to characterise experimentally. Computational approaches such as molecular dynamics (MD) are often employed to better understand biomolecular systems. However, the computationally demanding nature of such systems combined with the long characteristic timescales of peptide adsorption means MD is not well suited to its study with current computing capacities. An alternative computational approach to characterising the behaviour of atoms and molecules is mapping the potential energy surface (PES) – the molecular energy as a function of the positions of all atoms – by determining its local energy minima and saddle points, which represent stable configurations and transition states that lie between them. These minima and saddle points may be located using optimisation algorithms. Harmonic approximations yield information about transition rates between minima via saddle points as well as the free energy surface (FES). This methodology – which is referred to as ‘energy landscape mapping’ (ELM) hereafter – is able to characterise fast and slow processes equally, only being limited by the size and complexity of the system studied, and the applicability of the potential energy models used. In the past, it has largely been applied to atomic and molecular clusters, and to biomolecules. It has never been applied to adsorption of peptides or any other biomolecule. In three journal papers included in this thesis, this approach is for the first time applied to adsorbed peptides. Firstly, ELM was applied to polyalanine adsorbed on surfaces of varying interactions strengths. Results obtained were comparable results to those obtained in a prior study of the same system using an evolutionary algorithm. In the second paper, ELM was applied to met-enkephalin at a gas/graphite interface, and compared with a molecular simulation technique designed for accelerating the simulation of slow processes, replica exchange molecular dynamics (REMD). In the final paper, ELM was applied to two met-enkephalin molecules at a gas/graphite interface, introducing an additional level of complexity and a step towards practical application, given real peptide adsorption processes often occur en masse. In all of these studies, information about transitions between conformations, energy barriers, rates, and the nature of the overall PES and FES, all of which were previously unknown for the systems studied, was obtained by ELM. The work conducted here has demonstrated the applicability of ELM to peptide/surface systems. Future work may consist of applying ELM to other similar processes of practical importance, developing and validating potential energy models suitable for modelling interfacial systems, including the effect of solvents, and continual development of the methodology to accelerate calculations.Thesis (Ph.D.) -- University of Adelaide, School of Chemical Engineering and Advanced Materials, 202