Die Verbindung von Kleinwinkelstreuung und in silico-Methodik zur Aufkl��rung von Konformationswechseln gro��er Proteinkomplexe

In dieser Dissertation wurden die Methoden Homologiemodellierung und Molekulardynamik genutzt, um die Struktur und das Verhalten von Proteinen in L��sung zu beschreiben. Mit Hilfe der R��ntgenkleinwinkelstreuung wurden die mit den Computermethoden erzeugten Vorhersagen verifiziert. F��r das alpha-H��molysin, ein Toxin von Staphylococcus aureus, das eine heptamere Pore formen kann, wurde erstmalig die monomere Struktur des Protein in L��sung beschrieben. Homologiemodellierung auf Basis verwandter Proteine, deren monomere Struktur bekannt war, wurde verwendet, um die monomere Struktur des Toxins vorherzusagen. Flexibilit��t von Strukturelementen in einer Molekulardynamiksimulation konnte mit der Funktionalit��t des Proteines korreliert werden: Intrinsische Flexibilit��t versetzt das Protein in die Lage den Konformationswechsel zur Pore nach Assemblierung zu vollziehen. R��ntgenkleinwinkelstreuung bewies die Unterschiede der monomeren Struktur zu den Strukturen der verwandten Proteine und belegt den eigenen Vorschlag zur Struktur. ��berdies konnten Arbeiten an einer Mutante, die in einer sogenannten Pr��porenkonformation arretiert und nicht in der Lage ist eine Pore zu formen, zeigen, dass dieser ��bergangszustand mit der Rotationsachse senkrecht zur Membran gelagert ist. Eine geometrische Analyse beweist, dass es sterisch m��glich ist ausgehend von dieser Konformation die Konformation der Pore zu erreichen. Eine energetische und kinetische Analyse dieses Konformationswechsels steht noch aus. Ein weiterer Teil der Arbeit befasst sich mit den Konformationswechseln von H��mocyaninen. Diese wurden experimentell mittels R��ntgenkleinwinkelstreuung verfolgt. Konformationswechsel im Zusammenhang mit der Oxygenierung konnten f��r die 24meren H��mocyanine von Eurypelma californicum und Pandinus imperator beschrieben werden. F��r eine Reihe von H��mocyaninen ist nachgewiesen, dass sie unter Einfluss des Agenz SDS Tyrosinaseaktivit��t entfalten k��nnen. Der Konformationswechsel der H��mocyanine von E. californicum und P. imperator bei der Aktivierung zur Tyrosinase mittels SDS wurde experimentell best��tigt und die Stellung der Dodekamere der H��mocyanine als wesentlich bei der Aktivierung festgestellt. Im Zusammenhang mit anderen Arbeiten gilt damit die Relaxierung der Struktur unter SDS-Einfluss und der sterische Einfluss auf die verbindenden Untereinheiten b & c als wahrscheinliche Ursache f��r die Aktivierung zur Tyrosinase. Eigene Software zum sogenannten rigid body-Modellierung auf der Basis von R��ntgenkleinwinkelstreudaten wurde erstellt, um die Streudaten des hexameren H��mocyanins von Palinurus elephas und Palinurus argus unter Einfluss der Effektoren Urat und Koffein strukturell zu interpretieren. Die Software ist die erste Implementierung eines Monte Carlo-Algorithmus zum rigid body-Modelling. Sie beherrscht zwei Varianten des Algorithmus: In Verbindung mit simulated annealing k��nnen wahrscheinliche Konformationen ausgefiltert werden und in einer anschlie��enden systematischen Analyse kann eine Konformation geometrisch beschrieben werden. Andererseits ist ein weiterer, reiner Monte Carlo-Algorithmus in der Lage die Konformation als Dichteverteilung zu beschreiben.

Molecular organization of n-cyanobiphenyl liquid crystals on a molybdenite surface

The alignement and anchoring of liquid crystals on solid surfaces is a key problem for modern device technology that until now has been treated empirically, but that can now be tackled by atomistic computer simulations. Molecular dynamics (MD) simulations were used in this thesis work to study two films of 7 and 8 n-alkyl-4���cyanobiphenyl (7CB and 8CB) liquid crystals , with a thickness of 15 nm, confined between two (001) surfaces of MoS2 (molybdenite). The isotropic and nematic phases of both liquid crystals were simulated, and the resulting structures characterized structurally. A new force field was designed to model the interactions between the liquid crystal (LC) molecules and the surface of molybdenite, while an accurate force field developed previously was used to model the 7CB and 8CB molecules. The results show that the (001) molybdenite surface induces a planar orientation in both the liquid crystals. For the nematic phase of 8CB, one of the two solid/LC interfaces is composed of a first layer of molecules aligned parallel to the surface, followed by a second layer of molecules aligned perpendicular to the surface (also called, homeotropic). The effect of the surface appears to be local in nature as it is confined to the first 15 Angstr��m of the LC film. Conversely, for the nematic phase of 7CB, a planar ordering is established into the LC film. The LC molecules at the interface with the molybdenite appear to align preferentially their alkyl chains toward the solid substrate. The resulting tilt angle of molecules was found to be in good agreement with experimental measurements available in literature. Despite the fact that the MD simulations spanned a time range of more than 100 ns, the nematic phases of both 7CB and 8CB were found not to be completely formed. In order to confirm the findings presented in this thesis, we propose to extend the current study.

Dynamics, rheology and critical properties of colloidal fluid mixtures

Liquids under the influence of external fields exhibit a wide range of intriguing phenomena that can be markedly different from the behaviour of a quiescent system. This work considers two different systems ��� a glassforming Yukawa system and a colloid-polymer mixture ��� by Molecular Dynamics (MD) computer simulations coupled to dissipative particle dynamics. The former consists of a 50-50 binary mixture of differently-sized, like-charged colloids interacting via a screened Coulomb (Yukawa) potential. Near the glass transition the influence of an external shear field is studied. In particular, the transition from elastic response to plastic flow is of interest. At first, this model is characterised in equilibrium. Upon decreasing temperature it exhibits the typical dynamics of glassforming liquids, i.e. the structural relaxation time ���� grows strongly in a rather small temperature range. This is discussed with respect to the mode-coupling theory of the glass transition (MCT). For the simulation of bulk systems under shear, Lees-Edwards boundary conditions are applied. At constant shear rates ���� ��� 1/���� the relevant time scale is given by 1/���� and the system shows shear thinning behaviour. In order to understand the pronounced differences between a quiescent system and a system under shear, the response to a suddenly commencing or terminating shear flow is studied. After the switch-on of the shear field the shear stress shows an overshoot, marking the transition from elastic to plastic deformation, which is connected to a super-diffusive increase of the mean squared displacement. Since the average static structure only depends on the value of the shear stress, it does not discriminate between those two regimes. The distribution of local stresses, in contrast, becomes broader as soon as the system starts flowing. After a switch-off of the shear field, these additional fluctuations are responsible for the fast decay of stresses, which occurs on a time scale 1/���� . The stress decay after a switch-off in the elastic regime, on the other hand, happens on the much larger time scale of structural relaxation ����. While stresses decrease to zero after a switch-off for temperatures above the glass transition, they decay to a finite value for lower temperatures. The obtained results are important for advancing new theoretical approaches in the framework of mode-coupling theory. Furthermore, they suggest new experimental investigations on colloidal systems. The colloid-polymer mixture is studied in the context of the behaviour near the critical point of phase separation. For the MD simulations a new effective model with soft interaction potentials is introduced and its phase diagram is presented. Here, mainly the equilibrium properties of this model are characterised. While the self-diffusion constants of colloids and polymers do not change strongly when the critical point is approached, critical slowing down of interdiffusion is observed. The order parameter fluctuations can be determined through the long-wavelength limit of static structure factors. For this strongly asymmetric mixture it is shown how the relevant structure factor can be extracted by a diagonalisation of a matrix that contains the partial static structure factors. By presenting first results of this model under shear it is demonstrated that it is suitable for non-equilibrium simulations as well.

Crystal phases and glassy dynamics in monodisperse hard ellipsoids

We have performed Monte Carlo and molecular dynamics simulations of suspensions of monodisperse, hard ellipsoids of revolution. Hard-particle models play a key role in statistical mechanics. They are conceptually and computationally simple, and they offer insight into systems in which particle shape is important, including atomic, molecular, colloidal, and granular systems. In the high density phase diagram of prolate hard ellipsoids we have found a new crystal, which is more stable than the stretched FCC structure proposed previously . The new phase, SM2, has a simple monoclinic unit cell containing a basis of two ellipsoids with unequal orientations. The angle of inclination is very soft for length-to-width (aspect) ratio l/w=3, while the other angles are not. A symmetric state of the unit cell exists, related to the densest-known packings of ellipsoids; it is not always the stable one. Our results remove the stretched FCC structure for aspect ratio l/w=3 from the phase diagram of hard, uni-axial ellipsoids. We provide evidence that this holds between aspect ratios 3 and 6, and possibly beyond. Finally, ellipsoids in SM2 at l/w=1.55 exhibit end-over-end flipping, warranting studies of the cross-over to where this dynamics is not possible. Secondly, we studied the dynamics of nearly spherical ellipsoids. In equilibrium, they show a first-order transition from an isotropic phase to a rotator phase, where positions are crystalline but orientations are free. When over-compressing the isotropic phase into the rotator regime, we observed super-Arrhenius slowing down of diffusion and relaxation, and signatures of the cage effect. These features of glassy dynamics are sufficiently strong that asymptotic scaling laws of the Mode-Coupling Theory of the glass transition (MCT) could be tested, and were found to apply. We found strong coupling of positional and orientational degrees of freedom, leading to a common value for the MCT glass-transition volume fraction. Flipping modes were not slowed down significantly. We demonstrated that the results are independent of simulation method, as predicted by MCT. Further, we determined that even intra-cage motion is cooperative. We confirmed the presence of dynamical heterogeneities associated with the cage effect. The transit between cages was seen to occur on short time scales, compared to the time spent in cages; but the transit was shown not to involve displacements distinguishable in character from intra-cage motion. The presence of glassy dynamics was predicted by molecular MCT (MMCT). However, as MMCT disregards crystallization, a test by simulation was required. Glassy dynamics is unusual in monodisperse systems. Crystallization typically intervenes unless polydispersity, network-forming bonds or other asymmetries are introduced. We argue that particle anisometry acts as a sufficient source of disorder to prevent crystallization. This sheds new light on the question of which ingredients are required for glass formation.

Computational strategies to include protein flexibility in Ligand Docking and Virtual Screening

The dynamic character of proteins strongly influences biomolecular recognition mechanisms. With the development of the main models of ligand recognition (lock-and-key, induced fit, conformational selection theories), the role of protein plasticity has become increasingly relevant. In particular, major structural changes concerning large deviations of protein backbones, and slight movements such as side chain rotations are now carefully considered in drug discovery and development. It is of great interest to identify multiple protein conformations as preliminary step in a screening campaign. Protein flexibility has been widely investigated, in terms of both local and global motions, in two diverse biological systems. On one side, Replica Exchange Molecular Dynamics has been exploited as enhanced sampling method to collect multiple conformations of Lactate Dehydrogenase A (LDHA), an emerging anticancer target. The aim of this project was the development of an Ensemble-based Virtual Screening protocol, in order to find novel potent inhibitors. On the other side, a preliminary study concerning the local flexibility of Opioid Receptors has been carried out through ALiBERO approach, an iterative method based on Elastic Network-Normal Mode Analysis and Monte Carlo sampling. Comparison of the Virtual Screening performances by using single or multiple conformations confirmed that the inclusion of protein flexibility in screening protocols has a positive effect on the probability to early recognize novel or known active compounds.

Computersimulationen zum Einfluss topologischer Beschr��nkungen auf Polymere

Topologische Beschr��nkungen beeinflussen die Eigenschaften von Polymeren. Im Rahmen dieser Arbeit wird mit Hilfe von Computersimulationen im Detail untersucht, inwieweit sich die statischen Eigenschaften von kollabierten Polymerringen, Polymerringen in konzentrierten L��sungen und aus Polymerringen aufgebauten B��rsten mit topologischen Beschr��nkungen von solchen ohne topologische Beschr��nkungen unterscheiden. Des Weiteren wird analysiert, welchen Einfluss geometrische Beschr��nkungen auf die topologischen Eigenschaften von einzelnen Polymerketten besitzen. Im ersten Teil der Arbeit geht es um den Einfluss der Topologie auf die Eigenschaften einzelner Polymerketten in verschiedenen Situationen. Da allerdings gerade die effiziente Durchf��hrung von Monte-Carlo-Simulationen von kollabierten Polymerketten eine gro��e Herausforderung darstellt, werden zun��chst drei Bridging-Monte-Carlo-Schritte f��r Gitter- auf Kontinuumsmodelle ��bertragen. Eine Messung der Effizienz dieser Schritte ergibt einen Beschleunigungsfaktor von bis zu 100 im Vergleich zum herk��mmlichen Slithering-Snake-Algorithmus. Darauf folgt die Analyse einer einzelnen, vergr��berten Polystyrolkette in sph��rischer Geometrie hinsichtlich Verschlaufungen und Knoten. Es wird gezeigt, dass eine signifikante Verknotung der Polystrolkette erst eintritt, wenn der Radius des umgebenden Kapsids kleiner als der Gyrationsradius der Kette ist. Des Weiteren werden sowohl Monte-Carlo- als auch Molekulardynamiksimulationen sehr gro��er Ringe mit bis zu einer Million Monomeren im kollabierten Zustand durchgef��hrt. W��hrend die Konfigurationen aus den Monte-Carlo-Simulationen aufgrund der Verwendung der Bridging-Schritte sehr stark verknotet sind, bleiben die Konfigurationen aus den Molekulardynamiksimulationen unverknotet. Hierbei zeigen sich signifikante Unterschiede sowohl in der lokalen als auch in der globalen Struktur der Ringpolymere. Im zweiten Teil der Arbeit wird das Skalierungsverhalten des Gyrationsradius der einzelnen Polymerringe in einer konzentrierten L��sung aus v��llig flexiblen Polymerringen im Kontinuum untersucht. Dabei wird der Anfang des asymptotischen Skalierungsverhaltens, welches mit dem Modell des ���fractal globules��� konsistent ist, erreicht. Im abschlie��enden, dritten Teil dieser Arbeit wird das Verhalten von B��rsten aus linearen Polymeren mit dem von Ringpolymerb��rsten verglichen. Dabei zeigt sich, dass die Struktur und das Skalierungsverhalten beider Systeme mit identischem Dichteprofil parallel zum Substrat deutlich voneinander abweichen, obwohl die Eigenschaften beider Systeme in Richtung senkrecht zum Substrat ��bereinstimmen. Der Vergleich des Relaxationsverhaltens einzelner Ketten in herk��mmlichen Polymerb��rsten und Ringb��rsten liefert keine gravierenden Unterschiede. Es stellt sich aber auch heraus, dass die bisher verwendeten Erkl��rungen zur Relaxationsverhalten von herk��mmlichen B��rsten nicht ausreichen, da diese lediglich den anf��nglichen Zerfall der Korrelationsfunktion ber��cksichtigen. Bei der Untersuchung der Dynamik einzelner Monomere in einer herk��mmlichen B��rste aus offenen Ketten vom Substrat hin zum offenen Ende zeigt sich, dass die Monomere in der Mitte der Kette die langsamste Relaxation besitzen, obwohl ihre mittlere Verr��ckung deutlich kleiner als die der freien Endmonomere ist.

Simulations of amphiphilic peptides at the air-water interface

Amphiphile Peptide, Pro-Glu-(Phe-Glu)n-Pro, Pro-Asp-(Phe-Asp)n-Pro, und Phe-Glu-(Phe-Glu)n-Phe, k��nnen so aus n alternierenden Sequenzen von hydrophoben und hydrophilen Aminos��uren konstruiert werden, dass sie sich in Monolagen an der Luft-Wasser Grenzfl��che anordnen. In biologischen Systemen k��nnen Strukturen an der organisch-w��ssrigen Grenzfl��che als Matrix f��r die Kristallisation von Hydroxyapatit dienen, ein Vorgang der f��r die Behandlung von Osteoporose verwendet werden kann. In der vorliegenden Arbeit wurden Computersimulationenrneingesetzt, um die Strukturen und die zugrunde liegenden Wechselwirkungen welche die Aggregation der Peptide auf mikroskopischer Ebene steuern, zu untersuchen. Atomistische Molekulardynamik-Simulationen von einzelnen Peptidstr��ngen zeigen, dass sie sich leicht an der Luft-Wasser Grenzfl��che anordnen und die F��higkeit haben, sich in ��-Schleifen zu falten, selbst f��r relativ kurze Peptidl��ngen (n = 2). Seltene Ereignisse wie diese (i.e. Konformations��nderungen) erfordern den Einsatz fortgeschrittener Sampling-Techniken. Hier wurde ���Replica Exchange��� Molekulardynamik verwendet um den Einfluss der Peptidsequenzen zu untersuchen. Die Simulationsergebnisse zeigten, dass Peptide mit k��rzeren azidischen Seitenketten (Asp vs. Glu) gestrecktere Konformationen aufwiesen als die mit l��ngeren Seitenketten, die in der Lage waren die Prolin-Termini zu erreichen. Dar��ber hinaus zeigte sich, dass die Prolin-Termini (Pro vs. Phe) notwendig sind, um eine 2D-Ordnung innerhalb derrnAggregate zu erhalten. Das Peptid Pro-Asp-(Phe-Asp)n-Pro, das beide dieser Eigenschaften enth��lt, zeigt das geordnetste Verhalten, eine geringe Verdrehung der Hauptkette, und ist in der Lage die gebildeten Aggregate durch Wasserstoffbr��cken zwischen den sauren Seitenketten zu stabilisieren. Somit ist dieses Peptid am besten zur Aggregation geeignet. Dies wurde auch durch die Beurteilung der Stabilit��t von experimentnah-aufgesetzten Peptidaggregaten, sowie der Neigung einzelner Peptide zur Selbstorganisation von anf��nglich ungeordneten Konfigurationen unterst��tzt. Da atomistische Simulationen nur auf kleine Systemgr����en und relativ kurze Zeitskalen begrenzt sind, wird ein vergr��bertes Modell entwickelt damit die Selbstorganisation auf einem gr����eren Ma��stab studiert werden kann. Da die Selbstorganisation an der Grenzfl��che vonrnInteresse ist, wurden existierenden Vergr��berungsmethoden erweitert, um nicht-gebundene Potentiale f��r inhomogene Systeme zu bestimmen. Die entwickelte Methode ist analog zur iterativen Boltzmann Inversion, bildet aber das Update f��r das Interaktionspotential basierend auf der radialen Verteilungsfunktion in einer Slab-Geometrie und den Breiten des Slabs und der Grenzfl��che. Somit kann ein Kompromiss zwischen der lokalen Fl��ssigketsstruktur und den thermodynamischen Eigenschaften der Grenzfl��che erreicht werden. Die neue Methode wurde f��r einen Wasser- und einen Methanol-Slab im Vakuum demonstriert, sowie f��r ein einzelnes Benzolmolek��l an der Vakuum-Wasser Grenzfl��che, eine Anwendung die von besonderer Bedeutung in der Biologie ist, in der oft das thermodynamische/Grenzfl��chenpolymerisations-Verhalten zus��tzlich der strukturellen Eigenschaften des Systems erhalten werden m��ssen. Daraufrnbasierend wurde ein vergr��bertes Modell ��ber einen Fragment-Ansatz parametrisiert und die Affinit��t des Peptids zur Vakuum-Wasser Grenzfl��che getestet. Obwohl die einzelnen Fragmente sowohl die Struktur als auch die Wahrscheinlichkeitsverteilungen an der Grenzfl��che reproduzierten, diffundierte das Peptid als Ganzes von der Grenzfl��che weg. Jedoch f��hrte eine Reparametrisierung der nicht-gebundenen Wechselwirkungen f��r eines der Fragmente der Hauptkette in einem Trimer dazu, dass das Peptid an der Grenzfl��che blieb. Dies deutet darauf hin, dass die Kettenkonnektivit��t eine wichtige Rolle im Verhalten des Petpids an der Grenzfl��che spielt.

Synthesis of constrained peptidomimetics for therapeutic, diagnostic and theranostic applications

This thesis work deals, principally, with the development of different chemical protocols ranging from environmental sustainability peptide synthesis to asymmetric synthesis of modified tryptophans to a series of straightforward procedures for constraining peptide backbones without the need for a pre-formed scaffold. Much efforts have been dedicated to the structural analysis in a biomimetic environment, fundamental for predicting the in vivo conformation of compounds, as well as for giving a rationale to the experimentally determined bioactivity. The conformational analyses in solution has been done mostly by NMR (2D gCosy, Roesy, VT, titration experiments, molecular dynamics, etc.), FT-IR and ECD spectroscopy. As a practical application, 3D rigid scaffolds have been employed for the synthesis of biological active compounds based on peptidomimetic and retro-mimetic structures. These mimics have been investigated for their potential as antiflammatory agents and actually the results obtained are very promising. Moreover, the synthesis of Amo ring permitted the development of an alternative high effective synthetic pathway for obtaining Linezolid antibiotic. The final section is, instead, dedicated to the construction of a new biosensor based on zeolite L SAMs functionalized with the integrin ligand c[RGDfK], that has showed high efficiency for the selective detection of tumor cells. Such kind of sensor could, in fact, enable the convenient, non-invasive detection and diagnosis of cancer in early stages, from a few drops of a patient's blood or other biological fluids. In conclusion, the researches described herein demonstrate that the peptidomimetic approach to 3D definite structures, allows unambiguous investigation of the structure-activity relationships, giving an access to a wide range bioactive compounds of pharmaceutical interest to use not only as potential drugs but also for diagnostic and theranostic applications.

Structure and dynamics of ionic liquid,electrode interfaces

In the early 20th century, Gouy, Chapman, and Stern developed a theory to describe the capacitance and the spatial ion distribution of diluted electrolytes near an electrode. After a century of research, considerable progress has been made in the understanding of the electrolyte/electrode interface. However, its molecular-scale structure and its variation with an applied potential is still under debate. In particular for room-temperature ionic liquids, a new class of solventless electrolytes, the classical theories for the electrical double layer are not applicable. Recently, molecular dynamics simulations and phenomenological theories have attempted to explain the capacitance of the ionic liquid/electrode interface with the molecular-scale structure and dynamics of the ionic liquid near the electrode. rnHowever, experimental evidence is very limited. rnrnIn the presented study, the ion distribution of an ionic liquid near an electrode and its response to applied potentials was examined with sub-molecular resolution. For this purpose, a new sample chamber was constructed, allowing in situ high energy X-ray reflectivity experiments under potential control, as well as impedance spectroscopy measurements. The combination of structural information and electrochmical data provided a comprehensive picture of the electric double layer in ionic liquids. Oscillatory charge density profiles were found, consisting of alternating anion- and cation-enriched layers at both, cathodic and anodic, potentials. This structure was shown to arise from the same ion-ion correlations dominating the liquid bulk structure that were observed as a distinct X-ray diffraction peak. Therefore, existing physically motivated models were refined and verified by comparison with independent measurements. rnrnThe relaxation dynamics of the interfacial structure upon potential variation were studied by time resolved X-ray reflectivity experiments with sub-millisecond resolution. The observed relaxation times during charging/discharging are consistent with the impedance spectroscopy data revealing three processes of vastly different characteristic time-scales. Initially, the ion transport normal to the interface happens on a millisecond-scale. Another 100-millisecond-scale process is associated with molecular reorientation of electrode-adsorbed cations. Further, a minute-scale relaxation was observed, which is tentatively assigned to lateral ordering within the first layer.

Development of models and methods to simulate peptide-assisted nucleation and growth of calcium-minerals

In den vergangenen Jahren wurden einige bislang unbekannte Ph��nomene experimentell beobachtet, wie etwa die Existenz unterschiedlicher Pr��-Nukleations-Strukturen. Diese haben zu einem neuen Verst��ndnis von Prozessen, die auf molekularer Ebene w��hrend der Nukleation und dem Wachstum von Kristallen auftreten, beigetragen. Die Auswirkungen solcher Pr��-Nukleations-Strukturen auf den Prozess der Biomineralisation sind noch nicht hinreichend verstanden. Die Mechanismen, mittels derer biomolekulare Modifikatoren, wie Peptide, mit Pr��-Nukleations-Strukturen interagieren und somit den Nukleationsprozess von Mineralen beeinflussen k��nnten, sind vielf��ltig. Molekulare Simulationen sind zur Analyse der Formation von Pr��-Nukleations-Strukturen in Anwesenheit von Modifikatoren gut geeignet. Die vorliegende Arbeit beschreibt einen Ansatz zur Analyse der Interaktion von Peptiden mit den in L��sung befindlichen Bestandteilen der entstehenden Kristalle mit Hilfe von Molekular-Dynamik Simulationen.rnUm informative Simulationen zu erm��glichen, wurde in einem ersten Schritt die Qualit��t bestehender Kraftfelder im Hinblick auf die Beschreibung von mit Calciumionen interagierenden Oligoglutamaten in w��ssrigen L��sungen untersucht. Es zeigte sich, dass gro��e Unstimmigkeiten zwischen etablierten Kraftfeldern bestehen, und dass keines der untersuchten Kraftfelder eine realistische Beschreibung der Ionen-Paarung dieser komplexen Ionen widerspiegelte. Daher wurde eine Strategie zur Optimierung bestehender biomolekularer Kraftfelder in dieser Hinsicht entwickelt. Relativ geringe Ver��nderungen der auf die Ionen���Peptid van-der-Waals-Wechselwirkungen bezogenen Parameter reichten aus, um ein verl��ssliches Modell f��r das untersuchte System zu erzielen. rnDas umfassende Sampling des Phasenraumes der Systeme stellt aufgrund der zahlreichen Freiheitsgrade und der starken Interaktionen zwischen Calciumionen und Glutamat in L��sung eine besondere Herausforderung dar. Daher wurde die Methode der Biasing Potential Replica Exchange Molekular-Dynamik Simulationen im Hinblick auf das Sampling von Oligoglutamaten justiert und es erfolgte die Simulation von Peptiden verschiedener Kettenl��ngen in Anwesenheit von Calciumionen. Mit Hilfe der Sketch-Map Analyse konnten im Rahmen der Simulationen zahlreiche stabile Ionen-Peptid-Komplexe identifiziert werden, welche die Formation von Pr��-Nukleations-Strukturen beeinflussen k��nnten. Abh��ngig von der Kettenl��nge des Peptids weisen diese Komplexe charakteristische Abst��nde zwischen den Calciumionen auf. Diese ��hneln einigen Abst��nden zwischen den Calciumionen in jenen Phasen von Calcium-Oxalat Kristallen, die in Anwesenheit von Oligoglutamaten gewachsen sind. Die Analogie der Abst��nde zwischen Calciumionen in gel��sten Ionen-Peptid-Komplexen und in Calcium-Oxalat Kristallen k��nnte auf die Bedeutung von Ionen-Peptid-Komplexen im Prozess der Nukleation und des Wachstums von Biomineralen hindeuten und stellt einen m��glichen Erkl��rungsansatz f��r die F��higkeit von Oligoglutamaten zur Beeinflussung der Phase des sich formierenden Kristalls dar, die experimentell beobachtet wurde.

Accurate Predictions of Water Cluster Formation, (H2O)n=2-10

An efficient mixed molecular dynamics/quantum mechanics model has been applied to the water cluster system. The use of the MP2 method and correlation consistent basis sets, with appropriate correction for BSSE, allows for the accurate calculation of electronic and free energies for the formation of clusters of 2���10 water molecules. This approach reveals new low energy conformers for (H2O)n=7,9,10. The water heptamer conformers comprise five different structural motifs ranging from a three-dimensional prism to a quasi-planar book structure. A prism-like structure is favored energetically at low temperatures, but a chair-like structure is the global Gibbs free energy minimum past 200 K. The water nonamers exhibit less complexity with all the low energy structures shaped like a prism. The decamer has 30 conformers that are within 2 kcal/mol of the Gibbs free energy minimum structure at 298 K. These structures are categorized into four conformer classes, and a pentagonal prism is the most stable structure from 0 to 320 K. Results can be used as benchmark values for empirical water models and density functionals, and the method can be applied to larger water clusters.

The Search for Low Energy Conformational Families of Small Peptides: Searching for Active Conformations of Small Peptides in the Absence of a Known Receptor

Breast cancer is the most common cancer among women. Tamoxifen is the preferred drug for estrogen receptor-positive breast cancer treatment, yet many of these cancers are intrinsically resistant to tamoxifen or acquire resistance during treatment. Therefore, scientists are searching for breast cancer drugs that have different molecular targets. Previous work revealed that 8-mer and cyclic 9-mer peptides inhibit breast cancer in mouse and rat model systems, interacting with an unknown receptor, while peptides smaller than eight amino acids did not inhibit breast cancer. We have shown that the use of replica exchange molecular dynamics predicts structure and dynamics of active peptides, leading to the discovery of smaller peptides with full biological activity. These simulations identified smaller peptide analogs with a conserved turn, a ��-turn formed in the larger peptides. These analogs inhibit estrogen-dependent cell growth in a mouse uterine growth assay, a test showing reliable correlation with human breast cancer inhibition. We outline the computational methods that were tried and used with the experimental information that led to the successful completion of this research.

Computational Design and Experimental Discovery of an Anti-estrogenic Peptide Derived from Alpha-Fetoprotein

Breast cancer is the most common cancer among women, and tamoxifen is the preferred drug for estrogen receptor-positive breast cancer treatment. Many of these cancers are intrinsically resistant to tamoxifen or acquire resistance during treatment. Consequently, there is an ongoing need for breast cancer drugs that have different molecular targets. Previous work has shown that 8-mer and cyclic 9-mer peptides inhibit breast cancer in mouse and rat models, interacting with an unsolved receptor, while peptides smaller than eight amino acids did not. We show that the use of replica exchange molecular dynamics predicts the structure and dynamics of active peptides, leading to the discovery of smaller peptides with full biological activity. Simulations identified smaller peptide analogues with the same conserved reverse turn demonstrated in the larger peptides. These analogues were synthesized and shown to inhibit estrogen-dependent cell growth in a mouse uterine growth assay, a test showing reliable correlation with human breast cancer inhibition.

In search of CS2(H2O)n = 1���4 clusters

Gaussian-3 and MP2/aug-cc-pVnZ methods have been used to calculate geometries and thermochemistry of CS2(H2O)n, where n = 1���4. An extensive molecular dynamics search followed by optimization using these two methods located two dimers, six trimers, six tetramers, and two pentamers. The MP2/aug-cc-pVDZ structure matched best with the experimental result for the CS2(H2O) dimer, showing that diffuse functions are necessary to model the interactions found in this complex. For larger CS2(H2O)n clusters, the MP2/aug-cc-pVDZ minima are significantly different from the MP2(full)/6-31G* structures, revealing that the G3 model chemistry is not suitable for investigation of sulfur containing van der Waals complexes. Based on the MP2/aug-cc-pVTZ free energies, the concentration of saturated water in the atmosphere and the average amount of CS2 in the atmosphere, the concentrations of these clusters are predicted to be on the order of 105CS2(H2O) clusters���cm���3 and 102 CS2(H2O)2 clusters���cm���3 at 298.15���K. The MP2/aug-cc-pVDZ scaled harmonic and anharmonic frequencies of the most abundant dimer cluster at 298���K are presented, along with the MP2/aug-cc-pVDZ scaled harmonic frequencies for the CS2(H2O)n structures predicted to be present in a low-temperature molecular beam experiment.

Antiestrogenic and anticancer activities of peptides derived from the active site of alpha-fetoprotein

Cyclo[EKTOVNOGN] (AFPep), a cyclic 9-amino acid peptide derived from the active site of alpha-fetoprotein, has been shown to prevent carcinogen-induced mammary cancer in rats and inhibit the growth of ER+ human breast cancer xenografts in mice. Recently, studies using replica exchange molecular dynamics predicted that the TOVN region of AFPep might form a dynamically stable putative Type I beta-turn, and thus be biologically active without additional amino acids. The studies presented in this paper were performed to determine whether TOVN and other small analogs of AFPep would inhibit estrogen-stimulated cancer growth and exhibit a broad effective-dose range. These peptides contained nine or fewer amino acids, and were designed to bracket or include the putative pharmacophoric region (TOVN) of AFPep. Biological activities of these peptides were evaluated using an immature mouse uterine growth inhibition assay, a T47D breast cancer cell proliferation assay, and an MCF-7 breast cancer xenograft assay. TOVN had very weak antiestrogenic activity in comparison to AFPep's activity, whereas TOVNO had antiestrogenic and anticancer activities similar to AFPep. OVNO, which does not form a putative Type I beta-turn, had virtually no antiestrogenic and anticancer activities. A putative proteolytic cleavage product of AFPep, TOVNOGNEK, significantly inhibited E2-stimulated growth in vivo and in vitro over a wider dose range than AFPep or TOVNO. We conclude that TOVNO has anticancer potential, that TOVNOGNEK is as effective as AFPep in suppressing growth of human breast cancer cells, and that it does so over a broader effective-dose range.