Developing methods to construct ring pucker free energy hypersurfaces applied to the analysis of glycosidase enzyme catalytic mechanisms

Includes bibliographical references.Carbohydrates consist of one or more sub-units usually various 5- and 6-membered cycles (furanoses and pyranoses) which can twist, bend or flip into a variety of conformers that differ in strain - this is ring puckering. These puckers notably the strained puckering conformers are observed during enzymatically assisted bond formation or cleavage of the glycosidic bonds of carbohydrate substrates. In this thesis, the free energy of ring puckering is calculated by implementing the Hill-Reilly reduced coordinate pucker description into the sampling enhancing Free Energies from Adaptive Reaction Coordinate Forces (FEARCF) method. FEARCF non-Boltzmann simulations of prototypical sugars β-Dribose and β-D-glucose converged to yield free energy pucker surfaces and volumes when using several semi-empirical QM methods - AM1, PM3, PM3CARB-1 and SCC-DFTB. From this, the accessible puckering conformations and minimum free energy paths of puckering were reasoned An analysis of the furanose and pyranose free energy pucker surfaces and volumes compared with both Density Functional Theory RB3LYP/6-311++G** optimised structures and a Hartree-Fock free energy surface revealed that SCC-DFTB provides the best semi-empirical description of 5- and 6- membered carbohydrate ring deformation. This illustrates that necessary high energy ring conformations observed in enzymatic binding sites requires the enzyme to induce and preserve high energy conformations required for successful hydrolyses and synthesis of the glycosidic bond. To further test this hypothesis, a 5- and 6-membered cycle were studied within enzymatic environments. The polysaccharide cellulose contains β 1-4 linked glucose subunit and is degraded by cellulase, a glycosidase. Specifically, the retaining cellobiohydrolase I (CBHI) of Trichoderma Reesei which cleaves cellobiose units from crystalline cellulose.The free energy volumes of puckering for the glucose sub-unit (in the catalytic position of an 8 unit cellulosic fragment - cellooctaose) were calculated and explored in vacuum, water and in the active site of CBHI. It was observed that the binding pocket of enzymes limits the ring pucker and that the active site amino acids preferentially stabilise certain puckering conformations. For CBHI, the first part of the glycosidase reaction is the glycosylation step. This was driven to completion during SCC-DFTB QM/MD FEARCF calculations where GLU212, ASP214 and GLU217 and part of the substrate were treated quantum mechanically. The general hybrid orbital method was used to connect the QM and MM regions. The free energy barriers of glycosylation were computed and the puckering statistics during the conversion of cellooctaose to products were correlated with this. Guanosine, a 5-membered ribose derivative is phosphorylated by Purine Nucleoside Phosphorylase (PNP) in order to salvage the guanine base. The effect of the PNP protein environment on ring pucker was studied by using FEARCF SCC-DFTB QM/MD non Boltzmann free energy calculations to quantify the pucker change induced in guanosine when changing environment from vacuum, to water and to the protein. In vacuo, the E4 and E1 pucker conformers were observed as minima. Upon solvation, the puckering phase space became less restricted with the 3T4 and 2T3 pucker conformers as minima. In the PNP active site pucker became restricted with only the 4E conformer observed

Re-evaluation of analytical chemistry techniques in studying DNA structures

This work describes the use of analytical chemistry techniques to examine the structural changes that DNA adopts when subjected to a number of external/internal factors. A self-complementary sequence, d(CG)9, and a non-self-complementary sequence (mixed sequence) were used to study the conformational effects displayed by each type of oligonucleotide sequence. The structural changes adopted by DNA was examined using a variety of analytical techniques, such as: nuclear magnetic resonance imaging (NMR), differential scanning calorimetry (DSC), ultra violet visible (UV-Vis) spectroscopy, circular dichroism (CD) spectroscopy, and high-performance liquid chromatography (HPLC). 1) d(CG)9 and a mixed sequence in the B- and Z-DNA conformation was examined by CD and UV-Vis at a concentration of 1mM using a home-made cuvette called a Flexicell with a minimum pathlength of 0.129± 0.015 mm. The CD and UV-Vis spectra’s produced were found to be reliable when compared to commercial cuvettes with a pathlength of 1 cm and sample concentration of 10 µM. 2) d(CG)9 was lyophilized and reconstituted using either water or buffer to determine if d(CG)9 adopts a different structure when reconstituted using different conditions. It was determined that lyophilized d(CG)9 adopts a hairpin conformation when reconstituted with water, and a B-DNA duplex when reconstituted with a buffer containing NaCl. 3) d(CG)9 was thermally denatured using DSC to determine if DSC can be a viable method to study oligonucleotides. It was determined that d(CG)9 undergoes a two-state unfolding pathway. 4) Nuclear Overhauser Effect spectroscopy (NOESY) and correlation spectroscopy (COSY) were used to examine the conformational differences of 2’-deoxyadenosine when incubated in water. From the distance and torsion angle constraints obtained from NOESY and COSY respectively, and from existing crystal structures, it was found the structures that were determined by NMR spectroscopy were misleading because of spectral artifacts. 5) A mixed sequence was treated with organic modifying agents to determine the minimal condition required for DNA denaturation when different modifiers were used. It was determined that urea at a concentration of 8 M and at a pH of 12.5 is sufficient to denature the mixed sequence duplex

Application of three-dimensional interactive graphics in X-ray crystallographic analysis

A program called XpY was written for the PDP-10/LDS-1 at the Princeton University Computer Graphics Laboratory, for generating and displaying models of dinucleoside phosphates. The molecule GpC, a member of this class and a fragment of the nucleic acid RNA, was subjected to X-ray diffraction analysis. The paper describes the importance of model building in X-ray analysis, and shows step by step how XpY was used to deduce the atomic coordinates of GpC from the experimental data. The program documentation is also included as an Appendix. A subjective critique of graphics is made in the Conclusions section

The development of hybrid quantum classical computational methods for carbohydrate and hypervalent phosphoric systems

Includes bibliographical references.Ab initio, density functional theory, and semi-empirical methods serve as major computational tools for quantum mechanical calculations of medium to large molecular systems. Semi-empirical methods are most effectively used in a hybrid quantum mechanics/molecular mechanics (QM/MM) dynamics framework. However, semi-empirical methods have been designed to provide accurate results for organic molecules, but often fail to treat hypervalent species accurately due to their use of an sp basis. Recently, significant breakthroughs have been made with the incorporation of d-orbitals into the semi-empirical framework, thereby allowing for accurate modeling of both hypervalent and transition metal systems. Here I consider two methods that adopt this new methodology, namely AM1/d-PhoT and AM1*. Our major focus is the simulation of chemical biological and more specifically chemical glycobiological problems of biochemical interest. When I tested the ability of both AM1/d-PhoT and AM1* to reproduce key metrics in chemical glycobiology (i.e., sugar ring pucker, phosphate participation in transferase reactions) these methods, in combination with the published parameters, performed very poorly. Using the AM1/d-PhoT and AM1* Hamiltonians I set out to re-parameterize these methods aiming to produce holistic biochemical QM/MM toolsets able to simulate fundamental problems of binding and enzyme reactivity in chemical glycobiology. We called these methods AM1/d-CB1 and AM1*-CB1. In the development of these parameter sets I focused specifically on proton transfer, carbohydrate ring puckering, bond polarization, amino acid interactions, and phosphate interactions (facets important to chemical glycobiology). Both AM1/d-CB1 and AM1*-CB1 make use of a variable property optimization parameter approach for the glycan molecular class and its chemical environment. The accuracy of these methods is evaluated for carbohydrates, amino acids and phosphates present in catalytic domains of glycoenzymes, and the are shown to be more accurate for key performance indices (puckering, etc.) and on average across all simulation derived properties (QM/MM polarization, protein performance, etc.) than all other NDDO semiempirical methods currently being used. A major objective of the newly developed AM1/d-CB1 and AM1*-CB1 is to provide a platform to accurately model reactions central to chemical glycobiology using hybrid QM/MM molecular dynamics (MD) simulations. AM1/d-CB1 is applied to a well-known reaction involving purine nucleoside phosphorylase (PNP) and results lead me to conclude that the method shows promise for modelling glycobiological QM/MM systems

Characterizing RNA ensembles from NMR data with kinematic models

International audienceFunctional mechanisms of biomolecules often manifest themselves precisely in transient conformational substates. Researchers have long sought to structurally characterize dynamic processes in non-coding RNA, combining experimental data with computer algorithms. However, adequate exploration of conformational space for these highly dynamic molecules, starting from static crystal structures, remains challenging. Here, we report a new conformational sampling procedure, KGSrna, which can efficiently probe the native ensemble of RNA molecules in solution. We found that KGSrna ensembles accurately represent the conformational landscapes of 3D RNA encoded by NMR proton chemical shifts. KGSrna resolves motionally averaged NMR data into structural contributions; when coupled with residual dipolar coupling data, a KGSrna ensemble revealed a previously uncharacterized transient excited state of the HIV-1 trans-activation response element stem-loop. Ensemble-based interpretations of averaged data can aid in formulating and testing dynamic, motion-based hypotheses of functional mechanisms in RNAs with broad implications for RNA engineering and therapeutic intervention

Computational Studies of Glycan Conformations in Glycoproteins

N-glycans refer to oligosaccharide chains covalently attached to the side chain of asparagine (Asn) residues, and the majority of proteins synthesized in the endoplasmic reticulum (ER) are N-glycosylated. N-glycans can modulate the structural properties of proteins due to their close proximity to their parent proteins and their interactions between the glycan and the protein surface residues. In addition, N-glycans provide specific regions of recognition for cellular and molecular recognition. Despite their biological importance, the structural understanding of glycans and the impact of glycosylation to glycan or protein structure are lacking. I have explored the conformational freedom of glycans and their conformational preferences in different environments using structural databases and computer simulations. First, I have developed an algorithm to reliably annotate a given atomic structure of glycans. This algorithm is important because many glycan molecules in the crystal structure database are misannotated or contain errors. Using the algorithm, a database of glycans found in the PDB is constructed and available to the public. Second, the impact of glycosylation on the glycan conformation has been examined. Contrary to the common belief that the glycan conformations are independent to the protein structure, it appears that the protein structure can significantly affect the glycan structure upon glycosylation. This observation is significant because it may provide insight into protein-glycan interaction and opens up the possibility of a template-based glycan modeling approach. Third, the differences in conformational preference between glycans in solution and in glycoproteins has been examined. Using molecular dynamics (MD) simulations, the conformational preference of N-glycan pentassacharide in solution is exhaustively studied. Surprisingly, the conformational distribution is dominated by a single major conformational state and several minor conformational states. The dominant conformational state adopts a more extended conformation, thus it appears that entropy plays an important role in determining the conformational state. On the other hand, in glycoproteins, glycans can interact with surrounding protein side chains and, as a result, several conformational states are more equally populated. Based on these observations, a protocol is proposed for modeling the glycan portion of a known protein structure. It is typically more managable to acquire an atomic resolution structure or aglycoprotein (glycoprotein without glycan). In addition, the glycoform and the glycosylation site can be identified independently by mass spectrometry or NMR. The proposed modeling protocol assumes the glycosylation site, glycoform, and aglycoprotein structure are already known, and builds glycan structure models on top of the known aglycoprotein structure. The performance of the modeling protocol is greatly improved by using appropriate template structures. This protocol can be used to generate the initial model for MD simulations or refinement of low resolution models from experiments (small angle X-ray scattering and electron microscopy)

Molecular Dynamics Study of Single Stranded Peptide Nucleic Acids

A PNA molecule is a DNA strand where the sugar-phosphate backbone has been replaced by a structurally homomorphous pseudopeptide chain consisting of N(2-aminoethyl)-glycine units. PNA binds strongly to both DNA and RNA. However, an analysis of the X-ray and NMR data show that the dihedral angles of PNA/DNA or PNA/DNA complexes are very different from those of DNA:DNA or RNA:RNA complexes. In addition, the PNA strand is very flexible. One way to improve the binding affinity of PNA for DNA/RNA is to design a more pre-organized PNA structure. An effective way to rigidify the PNA strand is to introduce ring structures into the backbone. In several experimental studies, the ethylenediamine portion of aminoethylglycine peptide nucleic acids (aegPNA) has been replaced with one or more (S,S)- trans cyclopentyl (cpPNA) units. This substitution has met with varied success in terms of DNA/RNA recognition. In the present work, molecular modeling studies were performed to investigate PNA and modified PNA analogs. Molecular dynamics (MD) simulations is a principal tool in the theoretical study of biological molecules. This computational method calculates the time dependent behavior of a molecular system and provides detailed information on the fluctuations and conformational changes. The MD simulation uses an empirical parameterized energy functions. These parameters play an important role in the quality of the simulations. Therefore, novel empirical force field parameters were developed for cyclopentane modified PNA analogs. We demonstrate that our parameterization can accurately reproduce high level quantum mechanical calculations. Detailed investigations on the conformational and dynamical properties of single stranded aegPNA and cpPNA were undertaken to determine how the cyclopentane ring will improve binding and to determine the contributions of both entropy and dihedral angle preference to the observed stronger binding. The effects of single and multiple modifications of the PNA backbone were also analyzed to understand changes in conformational and dynamical properties

Studies on DNA polymorphism

Possible variations in the secondary structure and the A - B transition in DNA's of varying primary base sequence and composition have been studied by the techniques of X ray diffraction and molecular model building. The DNA's studied are from both bacteria and from eukaryotic cells. In addition a DNA of viral origin, from the bacteriophage OW-14, has been investigated. A computerised model building study of the changes induced in DNA secondary structure by the binding of intercalating drugs has also been carried out. A linked atom least squares routine has been extended and used to refine the models presented. The routine enables standard values for the parameters defining the covalent stereochemistry of the structure to be retained. Methods of calculating the Fourier transforms of the models produced are discussed, and this enables some comparisons to be made between the observed diffraction data and those predicted by the models. Structures studied include the intercalation complexes involving ethidium or daunomycin, general intercalation models for DNA and models for the conformation of the putrescene groups in OW-14 DNA