Center Structure

Core 1, Project 1: Isolation of the Native Macromolecular Environment

Specific Aims

This project seeks to establish efficient and rapid macromolecular complex isolation conditions. We seek to develop new approaches to the isolation of macromolecular complexes, based on innovative extensions of our current, highly successful methodologies. These complexes will be isolated in a form such that we can determine their state in the living cell the moment before their isolation. This goal can be divided into three specific aims:

Aim 1: Ultrafast Affinity Isolation. Armed with new innovations, it should be possible to isolate macromolecular complexes in a matter of seconds, instead of hours. Upon cell lysis, many macromolecular assemblies degrade with time from their pristine in vivo state. Our goal is simply to isolate these complexes quickly, before significant decay occurs, allowing us to trap even the most transient of interactions in the cell. In order to achieve this, we will improve on our current cryolytic methods, apply new developments in magnetic nanoparticles, produce new superfast-associating tags, and design a practical automated affinity isolation device.

Aim 2: Affinity Nanodomain Isolation. We will take advantage of our fast, mild isolation conditions to isolate relatively large subcellular assemblies (termed nanodomains) associated with our tagged macromolecules, trapping and preserving the specific larger environment surrounding the tag. We will rely on sophisticated new mass spectrometric and microarray methods to analyze the resulting complex macromolecular mixtures.

Aim 3: Near-Neighbor Analysis. We will use well-characterized, well-behaved standard crosslinkers and optimize their crosslinking conditions to facilitate further analysis. The complex readout we anticipate will be filtered, catalogued and analyzed by microarray and new mass spectrometric methods.

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Core 1, Project 2: Analysis of the Composition and Dynamics of Macromolecular Complexes

We will comprehensively characterize the nature of macromolecular complexes and how they change in response to time and circumstance.

SPECIFIC AIMS

Aim 1: Composition: Identification and elucidation of the macromolecular composition of isolated interactome nanodomains.

Aim 2: Interactions: : Definition of the interactions between components of the macromolecular complexes.

Aim 3: Quantitation: : Quantitation of components in macromolecular complexes relative to each other as well to the whole cell.

Aim 4: Regulated Modifications: : Quantitative elucidation of posttranslational modifications that act as switches in the information pathway.

Aim 5: Dynamics: : Determination of changes in time of the composition, interactions, quantities, and modifications of macromolecular complexes.

Aim 6: Interactome Refinement: : Refinement of the data generated above, taking it to the next level of resolution.

Aim 7: Morphology: : Definition of the architecture of the macromolecular complexes.

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Core 1, Project 3: Integration of Data Into Spatial and Temporal Models of Subcellular Processes

SPECIFIC AIMS

A comprehensive structural description of proteins, nucleic acids, and their assemblies will help us discover the principles that underlie cellular processes and bridge the gaps between genome sequencing, functional genomics, proteomics, and systems biology. Our objective is to contribute to this effort by developing and applying a computational system for enumerating structures and trajectories of macromolecular assemblies that are consistent with all available information from experimental methods, physical theories, and statistical preferences extracted from biological databases. Thus, we will maximize efficiency, accuracy, resolution, and completeness of the structural coverage of macromolecular assemblies.

Aim 1: Develop an approach to enumerating static structures of a given assembly that are consistent with all available structural information. This aim will include development of methods, implementation in the MODELLER program (http://salilab.org/modeller), and testing with the aid of model systems. The representation will allow for hierarchy in the system and the corresponding restraints. The scoring function will express the input information in the common reference frame, be it from experiment, physics, or statistics, as probabilistic restraints on spatial aspects of the model (eg, distances, angles, volume, and symmetry).

Aim 2: Develop bioinformatics and physics-based methods for derivation of spatial restraints on two subunits. We will examine each given assembly from four primary aspects: infer pairs of interacting subunits and their relative orientation from sufficient sequence and potentially structure similarity to a known pair of interacting partners; predict potential binding sites on a given protein structure from the occurrence of known binding sites in related structures; derive preferences for different relative spatial orientations of two protein structures by binary docking calculations; assess the utility of these binary spatial restraints for the modeling of higher order assemblies in the context of Aim 1.

Aim 3: Develop an approach to assembling snapshots of a dynamic macromolecular system into an ensemble of trajectories. We will provide a framework for analyzing the scope of possible trajectories and defining future experiments to narrow down the number of possibilities. We will also introduce means for imposing temporal restraints derived from experiment, such as “proteins A and B interact first before protein A can join to form a ternary complex”.

Aim 4: Apply the methods developed in Aims 1-3 to systems studied in Core 2. We will collaborate with experimentalists to convert the experimental measurements into formal spatial and temporal restraints and interpret the results of our calculations. We also expect to improve the computational platform in response to the realities of experimental data.

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Core 2, Project 1: Chromosomal Interactomes and Dynamics

SPECIFIC AIMS

We will utilize and build on the tools developed in Core 1, which will allow us to probe for both stable and transient protein-protein and protein-DNA interactions. We will first define the interactome involved in the replication of DNA in S. cerevisiae. As our techniques continue to develop we will expand our studies to other chromosomal sites, including a description of the interactome on the right arm of chromosome III.

Aim 1: Comprehensive definition of chromatin interactomes. We plan to elucidate the sets of proteins that associate with any given region of a chromosome, and how they are arranged upon the DNA sequence. This will allow us to “walk across” a chromosome and determine the identity, organization, and dynamics of specifically bound proteins. We will investigate the timing of association and positioning of these chromatin complexes and, from this data, we will describe how dynamic activities such as replication, chromatin maintenance and transcription are driven.

Aim 2: Dynamic picture of DNA replication. We plan to perform a comprehensive analysis of DNA replication complexes in order to identify and describe the macromolecular assemblies that form at the origins of replication and observe their transition to progressing replication forks. We will follow the dynamics of these complexes in a cell cycle dependent manner and determine their detailed choreography with chromatin during replication.

Core 2, Project 2: Processing and Export of RNAs

SPECIFIC AIMS

The main aim of the proposed research will be to study the late stages of the nuclear information pathway; in particular, to look in detail at the composition of late messenger and ribosomal RNPs and to understand how the molecular interactions therein contribute to the regulation of processing and export of RNPs.

Aim 1: Isolation of intact rRNPs and mRNPs. We will use rapid affinity isolation techniques on tagged proteins and RNAs to analyze different macromolecular RNP assemblies of the later nuclear information pathway.

Aim 2: Characterization of the late processing stages. We will dissect the composition, relative amounts and timing of association of the RNP assemblies.

Aim 3: Comparision between late rRNP and mRNP processing. We will use comparisons of the similarities and differences between mRNP and rRNP processing to shed light on the processes of RNP maturation as a whole.

Aim 4: Building a 4D picture of late processing of RNPs. We will refine our interactome maps to get higher detail pictures of the organization and dynamics of each macromolecular complex. We will then integrate our data using computational methods to generate 4D, “time and space” models of the late RNP processing machineries in action.

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Core 2, Project 3 Research Proposal: Mapping the HIV-1 Virus-Host Interactome

SPECIFIC AIMS

We will use the new approaches outlined in Core 1 to identify the factors that interact directly with the HIV-1 machinery during viral replication. We will use a system in which viruses have been molecularly engineered to incorporate a potent immunological or biochemical tag; this system demonstrably circumvents the problems others have had in generating viable tagged strains. We also seek to recover host proteins that interact specifically with the virus as it progresses through its natural life cycle. As these engineered viruses were generated through a process that is based on replication competency in culture, the tagged viral proteins likely undergo the same interactions encountered by the wild type virus. We believe that this system will afford us a more authentic view of both the transient and stable molecular interactions that form during the normal course of HIV infection. Success of these integrated approaches will open up new studies on HIV-host cell interactions.

Aim 1: Tagging the Virus. We will perform a comprehensive mutagenesis of the HIV-1 genome and selectively recover infectious, replication-competent tagged viruses.

Aim 2: Isolating Hybrid Virus-Host Macromolecular Complexes. We will use the tagged viruses for the quantitative recovery of the hybrid complexes that the tagged viral proteins form with host proteins during virus replication.

Aim 3: Characterization of the Hybrid Complexes. We will identify the interacting proteins using mass spectrometry, and assess their role in the HIV-1 infectious cycle.

Core 2, Project 4: Human Cytomegalovirus

SPECIFIC AIMS

The long-term objective of this research program is to elucidate the function of human cytomegalovirus (HCMV) genes that regulate the interaction of the virus with its host cell and thereby control the processes of viral replication and pathogenesis. This proposal is designed to identify the dynamically interacting cellular protein partners of viral immediate-early proteins, viral products that function at the very start of infection.

Aim 1: Construct recombinant derivatives of the HCMV AD169 laboratory strain and the FIX clinical isolate that express Protein A-tagged and GFP-tagged immediate-early proteins, and identify the viral and cellular proteins that are present in complexes with these proteins within infected human cells by single and two-stage mass spectrometry.

Aim 2: Confirm putative interactions by independent methods and test the biochemical consequences of the interactions.

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