Studying the folding of peptide dendrimers using molecular simulation methods

Tese de mestrado, Bioquímica, Universidade de Lisboa, Faculdade de Ciências, 2009Dendrimers are a family of branched compounds that share a common layout where wedges emerge radially from a core by means of a regular branching pattern. Peptide dendrimers are a specific kind of dendrimers formed by alternating functional amino acids with branching diamino acids. There has been increasing interest in the synthesis of peptide-based dendritic architectures modelling specific aspects of biological function. Some results are already available, demonstrating these molecules ability to act as enzyme models and to mimic natural ligands. Unfortunately, most studies concerning peptide dendrimers lack structural information at the molecular level. The theoretical study published so far, reported peptide dendrimers presenting shapes close to spheres, though experimental studies on the same systems suggest the existence of more disordered states. Herein, we characterize five third-generation peptide dendrimers (B1, B1H, B1HH, B1HHH and C1) through multiple long molecular dynamics simulations (MD), and analyse their conformational details and folding preferences in solution. Special emphasis is placed on the analysis of conformational trends representative of the examined models. The conformational sampling results, obtained through MM/MD simulations, were scrutinized using several approaches. Namely, histogram analysis, phi-psi dihedral distributions, inter-residue distance matrices, shape analysis and principal coordinate analysis. The adequacy of each approach to discern the conformation space of peptide dendrimers is discussed. Using these analysis procedures we were able to observe two distinct types of behaviour (sphere-like and bowl-like structures), both asserting the enormous structural flexibility characterizing these molecules; and the myriad of conformational states available to them. Our conclusions can be interpreted together with the available experimental results, contributing to a synergistic understanding of the structure-function relation in peptide dendrimers, and casting the bases for novel knowledge-based applications.Os dendrímeros são uma família de compostos ramificados que partilham uma arquitectura comum, onde diferentes cadeias emergem radialmente de um mesmo núcleo (ou centro) através de um padrão de ramificação regular. Os dendrímeros peptídicos, são uma classe particular de dendrímeros, constituída por estruturas que incorporam de forma alternada e iterativa resíduos de amino-ácidos funcionais (resíduos de espaçamento) com resíduos de diamino-ácidos ramificados (resíduos de ramificação). Os resíduos de diamino-ácidos ramificados promovem a bifurcação das cadeias peptídicas e a aquisição da estrutura dendrítica. A possibilidade de sintetizar dendrímeros com composições que mimetizem as funções de moléculas biológicas, constitui o aliciante para a investigação neste campo cientifico. Em particular, a síntese planeada e controlada de estruturas dendríticas baseadas nos componentes apresentados pelas moléculas biológicas, como peptidos ou glícidos, constitui um desafio atractivo pelas potenciais aplicações que dai podem emergir. De facto, já foram reportados dendrímeros peptídicos que modelam aspectos específicos de funções biológicas, tais como: modelos enzimáticos para catálise dirigida (“enzimas artificiais”); mimetização de co-factores naturais (de que e exemplo a vitamina B12); transportadores de fármacos, pois quando acopladas aos ligandos adequados estas moléculas tem a capacidade de aderir à membrana celular. É também importante referir que várias destas moléculas têm sido estudadas enquanto modelos de folding das proteínas naturais, pois investigações experimentais indiciam que alguns dendrímeros peptídicos podem apresentar, em solução, uma estrutura compacta semelhante à das proteínas globulares. Contudo, a maioria dos estudos experimentais realizados até à data são omissos no que concerne a informação estrutural, e carecem do enquadramento adequado a nível molecular e atómico. O único estudo teórico publicado sobre dendrímeros peptídicos parece confirmar a ideia de que, em solução estas moléculas apresentam, de facto, formas semelhantes a esferas, isto apesar de existirem evidências experimentais que sugerem a existência de estados conformacionais mais desordenados, nesses mesmos sistemas. Considerando o grande interesse que estas moléculas tem vindo a despertar, a verdade é que pouco se sabe sobre o seu arranjo estrutural tridimensional, e sobre os processos que a ele conduzem (folding). Nesta tese tentamos preencher algumas destas lacunas. Para tal, procedemos à caracterização de cinco dendrímeros peptídicos de terceira geração (que designamos por B1, B1H, B1HH, B1HHH e C1) com diferentes constituintes peptídicos. Os sistemas que escolhemos como objecto de estudo, estão directamente relacionados com a coordenação da aquocobalamina (análogo da vitamina B12) a dendrímeros peptídicos, ainda que apenas três deles tenham sido sintetizados e caracterizados experimentalmente (B1, B1H e C1). Deste modo, pretendemos não só investigar as suas preferências conformacionais, mas também inferir possíveis relações entre a sua estrutura e a capacidade para desempenhar uma função análoga à das moléculas biológicas (transcobalamina). É importante salientar que de entre os dendrímeros que foram sintetizados experimentalmente, e que são também aqui estudados, os que apresentam maior capacidade de coordenação com a aquacobalamina, são os que possuem um menor número de resíduos com potencial de coordenação. Este aparente paradoxo é por si só interessante e pode estar interligada com aspectos mais estruturais. Como temos por objectivo compreender as alterações e a variabilidade subjacentes às estruturas tridimensionais dos diferentes dendrímeros, empregamos metodologias adequadas ao detalhe da escala que pretendemos investigar. Nomeadamente, métodos computacionais de simulação molecular (MM/MD). Optámos portanto por simular cada um destes cinco sistemas através de múltiplas e longas simulações de dinâmica molecular, utilizando a água enquanto solvente explícito. Com efeito, no trabalho que conduziu a esta tese, realizamos simulações que contabilizam aproximadamente 1 μs-1 para cada um dos dendrímeros em estudo. No que respeita a estes sistemas, isto é muito superior ao tempo simulado em estudos anteriores. Nas últimas décadas a investigacao científica tem beneficiado imenso do avanço das técnicas de simulação computacional, que providenciam resultados e formas de escrutinar sistemas, que são de outra forma normalmente inacessíveis. A dinâmica molecular, especificamente, permite “seguir” a evolução temporal dos átomos que constituem um sistema, através da integração das equações de Newton para o movimento de corpos. É inclusive um dos métodos computacionais de eleição para estudar fenómenos biomoleculares. Os resultados obtidos com esta técnica de amostragem conformacional permitiram-nos analisar e identificar de forma adequada, os detalhes estruturais de cada um dos dendrímeros peptídicos. Colocamos especial ênfase nos arranjos estruturais mais estáveis. As conformações tridimensionais obtidas a partir das trajectórias resultantes das simulações, foram agrupadas de forma a obtermos os ensembles conformacionais característicos de cada dendrímero. Sobre estes conjuntos de conformações realizamos várias análises. Começamos por investigar algumas das propriedades que caracterizam estes sistemas, como o raio de giração, o número total de ligações de hidrogénio, a distância máxima entre os dois átomos mais afastados de cada estrutura, a superfície acessível ao solvente, entre outros. O raio de giração revelou ser a propriedade que individualmente, melhor espelha as variações intrínsecas a estes sistemas. Adicionalmente, procedemos também a caracterização da distribuição dos valores de phi-psi característicos dos diedros de cada um dos dendrímeros. Complementamos esta análise com o estudo das matrizes que reflectem as distãncias mínimas entre os resíduos de todas as conformações. Posteriormente aplicamos metodologias de análise conformacional que envolvem a determinação da energia livre, associada a diferentes coordenadas reaccionais (ou de folding) para cada estrutura nos diferentes ensembles, obtendo assim as correspondentes superfícies energéticas (folding landscapes). Utilizamos esta abordagem por forma a obter folding landscapes bi- e tridimensionais. Em especifíco, utilizamos como coordenadas de folding os valores do raio de giração, do root mean square deviation (RMSD), dos componentes principais do tensor do raio de giração diagonalizado, e os valores para a posição relativa das diferentes conformações, num espaço concordante com a matriz de RMSD, utilizando para tal o método de análise das coordenadas principais (PCoorA). Utilizando o tensor do raio de giração, foi possível investigar a forma dos arranjos estruturais de cada dendrímero peptídico, tendo inclusive sido definido um espaço tridimensional baseado nos componentes principais do tensor diagonalizado (espaço de giração). A capacidade de cada uma destas abordagens para discriminar de forma adequada o espaço das conformações dos dendrímeros peptídicos é discutida ao longo da tese. Dos diversos procedimentos de análise conformacional empregues, resulta uma clara indicação de que, em solução, os dendrímeros peptídicos podem apresentar dois comportamentos preferenciais distintos: estruturas compactas que privilegiam as interacções entre os diferentes resíduos, semelhantes a esferas (sphere-like); e estruturas “abertas” com as diferentes ramificações espaçadas, em que as interacções entre resíduos não adjacentes são minimizadas, semelhantes a taças (bowl-like). Ambas estas configurações atómicas consubstanciam a enorme flexibilidade estrutural que parece caracterizar estas moléculas, dando provas da miríade de estados conformacionais que lhes estão acessíveis. Foi ainda possível verificar a existência de evidências que suportam a ideia de que estas moléculas possuem uma grande robustez estrutural. Isto é, pequenas alterações na composição dos resíduos de amino-ácidos que as constituem não parecem desencadear alterações conformacionais significativas nos arranjos estruturais preferenciais. Através da comparação entre o coeficiente de difusao experimental disponível para um dos dendrímeros, e o coeficiente de difusão calculado com base nas trajectórias obtidas por simulação, foi possível verificar que os modelos utilizados, reflectem de forma adequada os sistemas experimentais. Concluiu-se também que o campo de força (force field) GROMOS 53A6 possui a capacidade de transferabilidade apropriada para lidar com estas moléculas. Os pontos fortes e fracos dos nossos modelos são discutidos ao longo da tese. Durante este trabalho foi ainda desenvolvida e implementada uma metodologia que permite o cálculo eficiente do RMSD entre estruturas dendríticas. As conclusões apresentadas nesta tese podem ser interpretadas juntamente com os resultados experimentais disponíveis, de forma a contribuir para uma compreensão sinérgica da relação entre a estrutura e a função dos dendrímeros peptídicos, lançando as fundações para aplicações inovadoras

Electronic Spectroscopy of Jet-Cooled Benzylidenecyclobutane, a Sterically Hindered Styrene

The electronic spectrum of the styrene derivative, benzylidenecyclobutane, seeded in a supersonic jet expansion has been recorded using resonantly enhanced two-photon ionization spectroscopy. The main vibronic features in the spectrum are associated with a low frequency progression assigned to the torsional motion of the phenyl ring. Analysis of the observed torsional levels reveals an excited state potential energy surface characteristic of a planar equilibrium geometry which undergoes large amplitude motion and a ground state surface having a minimum at a torsional angle of 25° between the phenyl and vinyl groups. Ab initio calculations of the ground state torsional potential surface predict a minimum in the range of 28°-26°, depending on the size of the basis set. In these structures the cyclobutane ring adopts a puckering angle between 17° and 19°. Deuterated isotopomers have also been synthesized and their corresponding photoionization spectra analyzed to reveal the mixing between the torsion and other low frequency modes such as cyclobutane ring puckering. The extent of this mixing is found to be sensitive to the sites of deuteration on the molecule. © 1996 American Institute of Physics

Electronic Spectroscopy of Jet-Cooled Benzylidenecyclobutane, a Sterically Hindered Styrene

The electronic spectrum of the styrene derivative, benzylidenecyclobutane, seeded in a supersonic jet expansion has been recorded using resonantly enhanced two-photon ionization spectroscopy. The main vibronic features in the spectrum are associated with a low frequency progression assigned to the torsional motion of the phenyl ring. Analysis of the observed torsional levels reveals an excited state potential energy surface characteristic of a planar equilibrium geometry which undergoes large amplitude motion and a ground state surface having a minimum at a torsional angle of 25° between the phenyl and vinyl groups. Ab initio calculations of the ground state torsional potential surface predict a minimum in the range of 28°-26°, depending on the size of the basis set. In these structures the cyclobutane ring adopts a puckering angle between 17° and 19°. Deuterated isotopomers have also been synthesized and their corresponding photoionization spectra analyzed to reveal the mixing between the torsion and other low frequency modes such as cyclobutane ring puckering. The extent of this mixing is found to be sensitive to the sites of deuteration on the molecule. © 1996 American Institute of Physics

Clustering of Epigenetic Methylation and Oxidative Damage: Effects on Duplex DNA Solution Structure, Thermodynamic Stability and Local Dynamics

All known living organisms use DNA to store genetic templates used for development, proper function and reproduction. The structural integrity of DNA is therefore of extreme importance and cellular machinery continuously regulates our DNA either through addition of covalent molecules to regulate the transcription of genes or the removal of DNA lesions propagating from the exposure to reactive molecules. One of the most common DNA lesions, 8-oxoguanine (8OG), is a prominent, pro-mutagenic DNA adduct present at a baseline level from consistent generation of reactive oxygen species through oxidative metabolism or at greater concentrations through exposure to ionizing radiation and other toxins. Its mutagenic potential is attributed to its ability in the syn- conformation, to mimic thymine during DNA replication, resulting in a mispair with adenine. In contrast, 5-methylcytosine (5MC), occurs from the covalent addition of a methyl group to a cytosine base by a DNA methyltransferase. 5MC acts as an epigenetic gene regulator, often found densely packed within CpG islands upstream of transcriptionally inactive genes. It can be estimated that each human diploid cell contains hundreds of CpG dinucleotides undergoing active methylation while also harboring 8OG. Previous results obtained by Kasymov et al, showed reduced endonuclease activity by hOGG1 for substrates containing 5MC adjacent and cross strand from 8OG. In addition, the work presented by Maltseva et al, conveyed that the enzymatic methylation rates by maintenance DNA methyltransferases were severely impacted when 8OG is adjacent to the methylation target. These results prompted us to investigate the clustering of these two modifications in greater detail. We present the results of solution NMR structure determination, thermodynamic stability analysis and molecular dynamics simulations on the DNA sequence 5’-d(CGCGAATTCGCG)-3’ with clustered 5MC and 8OG in CpG dinucleotides. NMR spectroscopy and restrained molecular dynamics were used to refine the structure of 11 DNA duplexes containing different methylation and oxidation patterns. The results reveal that 8OG induces local unwinding 5’ to itself and 31P chemical shifts indicate an increase in the BII phosphate backbone conformation 3’ relative to 8OG. Melting temperatures of the duplexes was shown to decrease with the addition of 8OG in all contexts. Surprisingly, the addition of 5MC in two separate instances led to lower Tm values of already oxidized DNA samples. 1D-1H NMR linewidths indicate 8OG increases the base dynamics while incorporation of 5MC leads to a stabilizing effect. Our results indicate that addition of 8OG to a fully-methylated CpG induces a sequence dependent stabilizing effect. Molecular dynamics trajectories were analyzed for BI/BII phosphate conformation populations, conformational flexibility and local dynamics. Comparison of helical geometries and backbone angles indicated that our MD simulations accurately and reliably reproduced our NMR structures within one standard deviation. Principal component analysis was carried out to highlight the most dominant modes of motion for CpG sites with clustered 5MC and 8OG. Particularly, we report significant differences in concerted atomic displacements, with the 8OG:5MC base pair displaying the greatest dynamic effects

Euclidean distance geometry and applications

Euclidean distance geometry is the study of Euclidean geometry based on the concept of distance. This is useful in several applications where the input data consists of an incomplete set of distances, and the output is a set of points in Euclidean space that realizes the given distances. We survey some of the theory of Euclidean distance geometry and some of the most important applications: molecular conformation, localization of sensor networks and statics.Comment: 64 pages, 21 figure

Automated and assisted RNA resonance assignment using NMR chemical shift statistics

The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and 1H and 13C chemical shifts. Statistics of resonances from regularly Watson-Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1′/C1′ chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92-100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding 13C resonances, which provides an important step toward automated structure determination of RNA

Interaction of DNA with groove binding ligands

Synthetic molecules that target the major groove in a sequence-selective way are a major goal in molecular medicine. Recently a major step has been taken toward achieving this goal: a novel cylinder has been developed that binds strongly into the major groove of DNA. Experimental techniques have provided some information regarding the binding strength and preferred binding sites of the cylinder on DNA. From all the experimental data it is clear that the parent cylinder binds in the major groove and is able to induce dramatic conformational changes in the DNA; these are unprecedented effects with synthetic DNA binders. However, gaining molecular level information in such a macromolecular system is challenging. Molecular dynamics (MD) simulations can provide information at the molecular level that is complementary to experiment and therefore are an ideal way to get a better understanding of this system. In this work we present the results of various MD simulations designed to probe the DNA-cylinder system. We have studied the effect of using CHARMM22 and CHARMM27 as the force-field for the simulations. Results showed that uncomplexed DNA simulated with CHARMM22 was less stable in the B-form than the comparable strand of DNA simulated with CHARMM27. Investigations into the effects of the cylinders charge and shape are also reported. Multi-nanosecond simulations were performed using two related synthetic cylinders, one with two Fe(II) metal centers and the other with two Cu(I) centers, and DNA. Finally the role of DNA within the system was investigated by performing a series of simulations of the cylinders with d(ATATATATATAT)2, d(CGCGCGCGCGCG)2 and d(CGCGCATATACG). Simulations with these DNA strands has only produced one system (CCu2+ with d(ATATATATATAT)) where the cylinder causes a conformational change in the DNA

Insights into the N-Sulfation Mechanism: Molecular Dynamics Simulations of the N-Sulfotransferase Domain of Ndst1 and Mutants

Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. the N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS), yielding sulfated sugar chains and 3'-phosphoadenosine-5'-phosphate (PAP). for the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. in this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. in addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional do Desenvolvimento Cientifico e TecnologicoCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilUniv Fed Rio Grande do Sul, Ctr Biotecnol, Porto Alegre, RS, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, BrazilFAPESP: 2010/52426-3Web of Scienc

Experimental and Theoretical Study on the OH-Reaction Kinetics and Photochemistry of Acetyl Fluoride (CH3C(O)F), an Atmospheric Degradation Intermediate of HFC-161 (C2H5F)

The direct reaction kinetic method of low pressure fast discharge flow (DF) with resonance fluorescence monitoring of OH (RF) has been applied to determine rate coefficients for the overall reactions OH + C2H5F (EtF) (1) and OH + CH3C(O)F (AcF) (2). Acetyl fluoride reacts slowly with the hydroxyl radical, the rate coefficient at laboratory temperature is k(2)(300 K) = (0.74 +/- 0.05) x 10(-14) cm(3) molecule(-1) s(-1) (given with 2 sigma statistical uncertainty). The temperature dependence of the reaction does not obey the Arrhenius law and it is described well by the two-exponential rate expression of k(2)(300-410 K) = 3.60 x 10(-3) exp(-10500/T) + 1.56 x 10(-13) exp(-910/T) cm(3) molecule(-1) s(-1). The rate coefficient of k(1) = (1.90 +/- 0.19) x 10(-13) cm(3) molecule(-1) s(-1) has been determined for the EtF-reaction at room temperature (T = 298 K). Microscopic mechanisms for the OH + CH3C(O)F reaction have also been studied theoretically using the ab initio CBS-QB3 and G4 methods. Variational transition state theory was employed to obtain rate coefficients for the OH + CH3C(O)F reaction as a function of temperature on the basis of the ab initio data. The calculated rate coefficients are in good agreement with the experimental data. It is revealed that the reaction takes place predominantly via the indirect H-abstraction mechanism involving H-bonded prereactive complexes and forming the nascent products of H2O and the CH2CFO radical. The non-Arrhenius behavior of the rate coefficient at temperatures below 500 K is ascribed to the significant tunneling effect of the in-the-plane H-abstraction dynamic bottleneck. The production of FC(O)OH + CH3 via the addition/elimination mechanism is hardly competitive due to the significant barriers along the reaction routes. Photochemical experiments of AcF were performed at 248 nm by using exciplex lasers. The total photodissociation quantum yield for CH3C(O)F has been found significantly less than unity; among the primary photochemical processes, C-C bond cleavage is by far dominating compared with CO-elimination. The absorption spectrum of AcF has also been determined by displaying a strong blue shift compared with the spectra of aliphatic carbonyls. Consequences of the results on atmospheric chemistry have been discussed