Interactive Sonification for Structural Biology and Structure-based Drug Design

The visualisation of structural biology data can be quite challenging as the datasets are complex, in particular the intrinsic dynamics/flexibility. Therefore some researchers have looked into the use of sonification for the display of proteins. Combining sonification and visualisation appears to be well fitted to this problem, but at the time of writing there are no plugins available for any of the major molecular visualisation applications. Therefore we set out to develop a sonification plugin for one of those applications, released as open-source software, in order to facilitate scrutiny and evaluation from as many parties as possible. This paper presents our open source sonification plugin for UCSF Chimera, which we have developed in collaboration with medicinal chemists and structural biologists. We determined two tasks that we deemed were not well represented visually and developed sonifications for them. Furthermore, we extended a general-purpose Chimera tool to map attributes of protein residues to pitch. We evaluated one of the tasks with eight participants and present the results of this evaluation

Structural Analysis of Transient Receptor Potential Vanilloid Type 1 (TRPV1) Channel Protein and Proline Mimics using Computational Techniques

Chapter I The Transient Receptor Potential (TRP) family of ion channels encompasses more than 30 members, which are expressed in many different tissues and cell types.1 Transient Receptor Potential Vanilloid Type 1 (TRPV1) is part of the TRP family gated by vanilloids, heat and protons.2 Molecular modeling will be used in order obtain structural and functional data on TRPV1 in its membrane bound environment. In particular, the transmembrane and C-terminal domain regions of TRPV1 are of particular interest. The S1-S4 region of the channel is the putative ligand-binding segment, while the C-terminal domain is suggested to respond to temperature and is regulated by phosphotidylinosides (PIP2). Despite the crucial roles in mediating signal transductions at both peripheral and central nervous systems, TRP channels are poorly understood in the context of structures and mechanisms.4 A molecular model of the published transmembrane section of TRPV1 along with the putative, unstructured C-terminal domain was created using their respective homology models and inserted into their membranes.5 Simulations were performed using both a lipid membrane containing PIP2 and one without PIP2 in order to determine its role in TRPV1 activation/deactivation. Molecular dynamics simulations could provide pivotal information about ligand binding, voltage sensing, interaction with heat/cold and proton binding for TRPV1. MD simulations alluded to the fact that when both temperature and PIP2 are present a greater degree of conformational change is observed. A greater understanding of the structure of TRPV1 could provide important details on how to alleviate certain diseases such as pain, asthma and diabetes. Chapter II Proline is unlike any other natural amino acid; it is the only amino acid that contains a pyrrolidine ring structure and is a secondary amine.56 Pseudoproline was derived in order to address the solubility and aggregation difficulties that can arise when performing FMOC solid phase synthesis of peptides.60 The presence of pseudoproline in a peptide overcomes aggregation by disrupting helices and β-sheets; leading causes in peptide aggregation.60 Derived from serine, cysteine or threonine via cyclo-condensation reaction with aldehydes or ketones, pseudoproline is commercially available, however, it undergo peptide synthesis through SPPS. A new proline mimic be utilized by SPPS; additionally, it is hypothesized to also decrease aggregation and increase solubility. The proposed mechanism for the proline mimic increased stability is due to a hypothesized formation of stable β hairpin turn during peptide synthesis. Density functional theory (DFT) calculations were performed in order to determine the equilibrium constant (K) and total energy of peptides containing proline, pseudoproline or the proline mimic. Molecular dynamic simulations were used in order to generate theoretical Ramachandran plots, which provided essential insight into the secondary structure of all three peptides

Region based gene expression via reanalysis of publicly available microarray data sets.

A DNA microarray is a high-throughput technology used to identify relative gene expression. One of the most widely used platforms is the Affymetrix® GeneChip® technology which detects gene expression levels based on probe sets composed of a set of twenty-five nucleotide probes designed to hybridize with specific gene targets. Given a particular Affymetrix® GeneChip® platform, the design of the probes is fixed. However, the method of analysis is dynamic in nature due to the ability to annotate and group probes into uniquely defined groupings. This is particularly important since publicly available repositories of microarray datasets, such as ArrayExpress and NCBI’s Gene Expression Omnibus (GEO) have made millions of samples readily available to be reanalyzed computationally without the need for new biological experiments. One way in which the analysis can dynamically change is by correcting the mapping between probe sets and targets by creating custom Chip Description Files (CDFs) to arrange which probes belong to which probe set based on the latest genomic information or specific annotations of interest. Since default probe sets in Affymetrix® GeneChip® platforms are specific for a gene, transcript or exon, the analyses are then limited to profile differential expression at the gene, transcript or individual exon level. However, it has been revealed that untranslated regions (UTRs) of mRNA have important impacts on the regulation of proteins. We therefore developed a new probe mapping protocol that addresses three issues of Affymetrix® GeneChip® data analyses: removing nonspecific probes, updating probe target mapping based on the latest genome information and grouping the probes into region (UTR, individual exon), gene and transcript level targets of interest to support a better understanding of the effect of UTRs and individual exons on gene expression levels. Furthermore, we developed an R package, affyCustomCdf, for users to dynamically create custom CDFs. The affyCustomCdf tool takes annotations in a General/Gene Transfer Format File (GTF), aligns probes to gene annotations via Nested Containment List (NCList) indexing and generates a custom Chip Description File (CDF) to regroup probes into probe sets based on a region (UTR and individual exon), transcript or gene level. Our results indicate that removing probes that no longer align to the genome without mismatches or align to multiple locations can help to reduce false-positive differential expression, as can removal of probes in regions overlapping multiple genes. Moreover, our method based on regions can detect changes that would have been missed by analysis based on gene and transcript. It also allows for a better understanding of 3’ UTR dynamics through the reanalysis of publicly available data

The 3′ Splice Site of Influenza A Segment 7 mRNA Can Exist in Two Conformations: A Pseudoknot and a Hairpin

The 3′ splice site of influenza A segment 7 is used to produce mRNA for the M2 ion-channel protein, which is critical to the formation of viable influenza virions. Native gel analysis, enzymatic/chemical structure probing, and oligonucleotide binding studies of a 63 nt fragment, containing the 3′ splice site, key residues of an SF2/ASF splicing factor binding site, and a polypyrimidine tract, provide evidence for an equilibrium between pseudoknot and hairpin structures. This equilibrium is sensitive to multivalent cations, and can be forced towards the pseudoknot by addition of 5 mM cobalt hexammine. In the two conformations, the splice site and other functional elements exist in very different structural environments. In particular, the splice site is sequestered in the middle of a double helix in the pseudoknot conformation, while in the hairpin it resides in a two-by-two nucleotide internal loop. The results suggest that segment 7 mRNA splicing can be controlled by a conformational switch that exposes or hides the splice site

Unveiling the Polymerase Complex of Negative Stranded RNA Viruses

Negative stranded RNA viruses (NSVs) are among the most common human pathogens which cause pandemics and epidemics. This group includes many notable members such as influenza, mumps and Ebola viruses. These viruses are identifiable by their negative polarity genome which is associated with the nucleocapsid (NP) protein and assembled into higher order structures. The RNA-nucleocapsid complex or ribonucleoprotein (RNP) serves as the template for transcription and replication by the viral RNA-dependent RNA polymerase (vRdRp). Though progress has been made in the study of these viruses, knowledge is lacking with regards to the polymerase complex. Here, we utilize structural biology and mutational analysis to identify components of the polymerase complex that will be targets for drug design. NSVs typically cause high mortality outbreaks by transmission from animal reservoirs. In fact, in 2013 H7N9 avian influenza A virus emerged as human infections and in 2017 the number of infections raised to 688. This reaffirms that influenza virus is a global health threat and requires antiviral drugs in the effort to control influenza virus. Frequently used anti-influenza drugs target neuraminidase; however, there have been strains that show resistance to these neuraminidase inhibitors. The PB2cap binding domain of the influenza RNA polymerase is an innovative target for development of anti-influenza drugs. In this study, we have solved the crystal structure of the PB2cap binding domain of influenza A H1N1 virus alone and in complex with its binding partner. Utilizing this structure, we have identified critical interactions that will aid in the design of antivirals. The emergence of mumps virus outbreaks throughout the United States in the past five years indicates that the MMR vaccine is not the most efficient source of protection and reaffirms the need for inhibitors that target the virus.Here, we have utilized cryogenic electron microscopy (cryoEM) to analyze the RNA encapsidation of mumps virus nucleocapsid and mutational analysis of the phosphoprotein to probe the interactions involved in uncoiling the nucleocapsid. This data adds to the available knowledge about mumps virus infection and could potentially aid in the design of inhibitors

Nucleic Acids Conjugates for Biotechnological Applications

Life in biological systems is maintained by the cooperative actions of various biomolecules. With the development of chemical and biological technologies related to nucleic acids, the details of the mechanisms of such cooperative actions between nucleic acids and other biomolecules have been elucidated and further applied in various applications. In the papers published in this Special Issue, advanced research works involved in nucleic acid conjugates are reported in wide application fields, such as artificial gene regulation, biomolecular sensing, and therapeutics from leading scientists in nucleic acids chemistry and engineering

Molecular Simulation Approaches to Proteins Structure and Dynamics and to Ligand Design

Molecular simulations approaches are powerful tools for structural biology and drug discovery. They provide additional and complementary information on structure, dynamics and energetics of biomolecules whose structures have been determined experimentally [1,2,3,4,5,6,7]. In particular, Molecular Dynamics (MD) simulations [8], along with elastic network analysis [9], offer insights into molecular fluctuations, conformational changes and allosteric mechanisms. In addition, molecular simulation can be used to design novel and potent ligands to a specific target (either a protein or DNA) as well as to estimate ligands potency [10,11]. Attempts at predicting protein structures using bioinformatics and MD are also increasingly successful [12,13,14,15], as well as approaches that use solely simulation tools [16]. The development of new algorithms and the continuously growing computer power currently allow for the simulation of more and more complex biological systems, such as protein aggregates [7,17,18,19,20] and protein/DNA complexes [21]. In this context, a number of theoretical techniques (namely molecular dynamics simulations, elastic network analysis, electrostatic modeling and binding energy predictions) have here been applied to the study of specific proteins. On the basis of X-ray protein structures, molecular simulations have provided a detailed description of internal motions and interactions, which are not evident from the experimental data and have functional implications. First, we have used MD to investigate structural features, focusing on the differences between the solid state and the aqueous solution structures. Over 80% of data in the PDB [22] are X-ray structures, making protein crystallography the major resource in structural biology. Nevertheless, in a few cases, the structural details might be affected by environmental features, such as the presence of small compounds in the buffering solution and/or crystal packing contacts due to the periodic lattice. Here, a comparative MD study has been performed on the Catabolite Activator Protein (CAP), in both the crystal phase and in the aqueous solution. CAP is a bacterial DNA-binding transcription regulator whose activity is controlled by the binding of the intracellular mediator cyclic Adenosine MonoPhosphate (cAMP). CAP is a homodimeric protein and each subunit is formed by a cyclic nucleotide- and a DNA-binding domain. Inspection of the available CAP X-ray structure within the crystal environment [23] suggests that packing contacts do affect the native conformation of the ligand activated protein. Anticipating our results, we have found that indeed the conformation of the protein in solution is different, and that these differences may play a role for CAP biological function. Next, we have used molecular simulations to target structural flexibility. Conformational fluctuations often play a key role for the protein function and MD simulations can provide information on large-scale concerted motions of proteins [24,25,26]. We have addressed this point in the context of the Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) cation channel. The tetrameric HCN channels are opened by membrane hyperpolarization, while their activation is allosterically modulated by the binding of cAMP in the cytoplasm. The cytoplasmic part of the HCN2 channel, which is responsible for the channel modulation, has been here investigated by MD simulations and elastic network analysis, on the basis of the available X-ray structure [27], to earn new insights into the molecular mechanism triggered by cAMP. We have found that, in the presence of cAMP, the protein undergoes a quaternary structure oscillation, in which each subunit moves as a rigid body. This fluctuation, which is not observed in the absence of cAMP, could facilitate the channel opening transition. Finally, we have moved our attention to an issue relevant for structure-based drug design. Within a long-standing collaboration with Prof. Cattaneo\u2019s lab (SISSA and Motivations and Summary 7 Layline Genomics), our group has been interested in the design of mimics of proteins involved in the biochemical pathways that lead to the Alzheimer\u2019s disease. Here, on the basis of structural information [28], we have designed a peptide that could specifically target trkA, the high affinity receptor of the Nerve Growth Factor (NGF), which is a protein that plays a critical role for the development, survival and maintenance of neurons in the vertebrate nervous system and activates signaling pathways related to neuroprotection. The results of this research will be tested at the Prof. Cattaneo\u2019s Lab in order to validate the theoretical findings and assess the potency and the effects of such a ligand

Modeling and simulations of single stranded rna viruses

The presented work is the application of recent methodologies on modeling and simulation of single stranded RNA viruses. We first present the methods of modeling RNA molecules using the coarse-grained modeling package, YUP. Coarse-grained models simplify complex structures such as viruses and let us study general behavior of the complex biological systems that otherwise cannot be studied with all-atom details. Second, we modeled the first all-atom T=3, icosahedral, single stranded RNA virus, Pariacoto virus (PaV). The x-ray structure of PaV shows only 35% of the total RNA genome and 88% of the capsid. We modeled both missing portions of RNA and protein. The final model of the PaV demonstrated that the positively charged protein N- terminus was located deep inside the RNA. We propose that the positively charged N- terminal tails make contact with the RNA genome and neutralize the negative charges in RNA and subsequently collapse the RNA/protein complex into an icosahedral virus. Third, we simulated T=1 empty capsids using a coarse-grained model of three capsid proteins as a wedge-shaped triangular capsid unit. We varied the edge angle and the potentials of the capsid units to perform empty capsid assembly simulations. The final model and the potential are further improved for the whole virus assembly simulations. Finally, we performed stability and assembly simulations of the whole virus using coarse-grained models. We tested various strengths of RNA-protein tail and capsid protein-capsid protein attractions in our stability simulations and narrowed our search for optimal potentials for assembly. The assembly simulations were carried out with two different protocols: co-transcriptional and post-transcriptional. The co-transcriptional assembly protocol mimics the assembly occurring during the replication of the new RNA. Proteins bind the partly transcribed RNA in this protocol. The post-transcriptional assembly protocol assumes that the RNA is completely transcribed in the absence of proteins. Proteins later bind to the fully transcribed RNA. We found that both protocols can assemble viruses, when the RNA structure is compact enough to yield a successful virus particle. The post-transcriptional protocol depends more on the compactness of the RNA structure compared to the co-transcriptional assembly protocol. Viruses can exploit both assembly protocols based on the location of RNA replication and the compactness of the final structure of the RNA.PhDCommittee Chair: Stephen C. Harvey; Committee Member: Adegboyega Oyelere; Committee Member: Loren Williams; Committee Member: Rigoberto Hernandez; Committee Member: Roger Wartel