Kevin Truong

Cadherins in embryonic and neural morphogenesis

Cadherins not only maintain the structural integrity of cells and tissues but also control a wide array of cellular behaviours. They are instrumental for cell and tissue polarization, and they regulate cell movements such as cell sorting, cell migration and cell rearrangements. Cadherins may also contribute to neurite outgrowth and pathfinding, and to synaptic specificity and modulation in the central nervous system.

Calmodulin target database.

The intracellular calcium sensor protein calmodulin (CaM) interacts with a large number of proteins to regulate their biological functions in response to calcium stimulus. This molecular recognition process is diverse in its mechanism, but can be grouped into several classes based on structural and sequence information. We have developed a web-based database (http://calcium.uhnres.utoronto.ca/ctdb) for this family of proteins containing CaM binding sites or, as we propose to call it herein, CaM recruitment signaling (CRS) motifs. At present the CRS motif found in approximately 180 protein sequences in the databases can be divided into four subclasses, each subclass representing a distinct structural mode of molecular recognition involving CaM. The database can predict a putative CRS location within a given protein sequence, identify the subclass to which it may belong, and structural and biophysical parameters such as hydrophobicity, hydrophobic moment, and propensity for a -helix formation.

The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo.

Intermolecular and intramolecular FRET between two spectrally overlapping green fluorescent protein variants fused to two different host proteins or at two different sites within the same protein offers a unique opportunity to monitor real-time protein-protein interactions or protein conformational changes. By using fluorescence digital imaging microscopy, one can visualize the location of green fluorescent proteins within a living cell and follow the time course of the changes in FRET corresponding to cellular events at a millisecond time resolution. The observation of such dynamic molecular events in vivo provides vital insight into the action of biological molecules.

FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule.

Intracellular Ca2+ acts as a second messenger that regulates numerous physiological cellular phenomena including development, differentiation and apoptosis. Cameleons, a class of fluorescent indicators for Ca2+ based on green fluorescent proteins (GFPs) and calmodulin (CaM), have proven to be a useful tool in measuring free Ca2+ concentrations in living cells. Traditional cameleons, however, have a small dynamic range of fluorescence resonance energy transfer (FRET), making subtle changes in Ca2+ concentrations difficult to detect and study in some cells and organelles. Using the NMR structure of CaM bound to the CaM binding peptide derived from CaM-dependent kinase kinase (CKKp), we have rationally designed a new cameleon that displays a two-fold increase in the FRET dynamic range within the physiologically significant range of cytoplasmic Ca2+ concentration of 0.05-1 μM.

Identification and characterization of subfamily-specific signatures in a large protein superfamily by a hidden Markov model approach

BackgroundMost profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. The next step of such database studies should include the development of classification systems capable of distinguishing between subfamilies within a structurally and functionally diverse superfamily. This would be helpful in elucidating sequence-structure-function relationships of proteins.ResultsHere, we present a method to diagnose sequences into subfamilies by employing hidden Markov models (HMMs) to find windows of residues that are distinct among subfamilies (called signatures). The method starts with a multiple sequence alignment (MSA) of the subfamily. Then, we build a HMM database representing all sliding windows of the MSA of a fixed size. Finally, we construct a HMM histogram of the matches of each sliding window in the entire superfamily. To illustrate the efficacy of the method, we have applied the analysis to find subfamily signatures in two well-studied superfamilies: the cadherin and the EF-hand protein superfamilies. As a corollary, the HMM histograms of the analyzed subfamilies revealed information about their Ca2+ binding sites and loops.ConclusionsThe method is used to create HMM databases to diagnose subfamilies of protein superfamilies that complement broad profile and motif databases such as BLOCKS, PROSITE, Pfam, SMART, PRINTS and InterPro.

160-fold acceleration of the Smith-Waterman algorithm using a field programmable gate array (FPGA)

BackgroundTo infer homology and subsequently gene function, the Smith-Waterman (SW) algorithm is used to find the optimal local alignment between two sequences. When searching sequence databases that may contain hundreds of millions of sequences, this algorithm becomes computationally expensive.ResultsIn this paper, we focused on accelerating the Smith-Waterman algorithm by using FPGA-based hardware that implemented a module for computing the score of a single cell of the SW matrix. Then using a grid of this module, the entire SW matrix was computed at the speed of field propagation through the FPGA circuit. These modifications dramatically accelerated the algorithms computation time by up to 160 folds compared to a pure software implementation running on the same FPGA with an Altera Nios II softprocessor.ConclusionThis design of FPGA accelerated hardware offers a new promising direction to seeking computation improvement of genomic database searching.

Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics.

Genetically-coded, fluorescence resonance energy transfer (FRET) biosensors are widely used to study molecular events from single cells to whole organisms. They are unique among biosensors because of their spontaneous fluorescence and targeting specificity to both organelles and tissues. In this review, we discuss the theoretical basis of FRET with a focus on key parameters responsible for designing FRET biosensors that have the highest sensitivity. Next, we discuss recent applications that are grouped into four common biosensor design patterns—intermolecular FRET, intramolecular FRET, FRET from substrate cleavage and FRET using multiple colour fluorescent proteins. Lastly, we discuss recent progress in creating fluorescent proteins suitable for FRET purposes. Together these advances in the development of FRET biosensors are beginning to unravel the interconnected and intricate signalling processes as they are occurring in living cells and organisms.

Engineering a Photoactivated Caspase-7 for Rapid Induction of Apoptosis

Apoptosis is a cell death program involved in the development of multicellular organisms, immunity, and pathologies ranging from cancer to HIV/AIDS. We present an engineered protein that causes rapid apoptosis of targeted cells in monolayer culture after stimulation with blue light. Cells transfected with the protein switch L57V, a tandem fusion of the light-sensing LOV2 domain and the apoptosis-executing domain from caspase-7, rapidly undergo apoptosis within 60 min after light stimulation. Constant illumination of under 5 min or oscillating with 1 min exposure had no effect, suggesting that cells have natural tolerance to a short duration of caspase-7 activity. Furthermore, the overexpression of Bcl-2 prevented L57V-mediated apoptosis, suggesting that although caspase-7 activation is sufficient to start apoptosis, it requires mitochondrial contribution to fully commit.

A fluorescent cassette-based strategy for engineering multiple domain fusion proteins.

BackgroundThe engineering of fusion proteins has become increasingly important and most recently has formed the basis of many biosensors, protein purification systems, and classes of new drugs. Currently, most fusion proteins consist of three or fewer domains, however, more sophisticated designs could easily involve three or more domains. Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all.ResultsTherefore, to aid in the efficient construction of fusion proteins involving multiple domains, we have created a new expression vector that allows us to rapidly generate a library of cassettes. Cassettes have a standard vector structure based on four specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesive end restriction enzymes, they can be fused in any order and number of times. Furthermore, the insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of fluorescence.ConclusionsFinally, the utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags.

TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector

Delivery and expression of multiple genes is an important requirement in a range of applications such as the engineering of synthetic signaling pathways and the induction of pluripotent stem cells. However, conventional approaches are often inefficient, nonstoichiometric and may limit the maximum number of genes that can be simultaneously expressed. We here describe a versatile approach for multiple gene delivery using a single expression vector by mimicking the protein expression strategy of RNA viruses. This was accomplished by first expressing the genes together with TEV protease as a single fusion protein, then proteolytically self‐cleaving the fusion protein into functional components. To demonstrate this method in E. coli cells, we analyzed the translation products using SDS‐PAGE and showed that the fusion protein was efficiently cleaved into its components, which can then be purified individually or as a binding complex. To demonstrate this method in mammalian cells, we designed a differential localization scheme and used live cell imaging to observe the distinctive subcellular targeting of the processed products. We also showed that the stoichiometry of the processed products was consistent and corresponded with the frequency of appearance of their genes on the expression vector. In summary, the efficient expression and separation of up to three genes was achieved in both E. coli and mammalian cells using a single TEV protease self‐processing vector.