High-Throughput Analysis of Clinical Flow Cytometry Data by Automated Gating.

Advancements in flow cytometers with capability to measure 15 or more parameters have enabled us to characterize cell populations at unprecedented levels of detail. Beyond discovery research, there is now a growing demand to dive deeper into evaluating the immune response in clinical trials for immune modulating compounds. However, for high-volume, complex flow cytometry data generated in clinical trials, conventional manual gating remains the standard of practice. Traditional manual gating is resource intense and becomes a bottleneck and an impractical method to complete high volumes of flow cytometry data analysis. Current efforts to automate "manual gating" have shown that computational algorithms can facilitate the analysis of daunting multi-parameter data; however, a greater degree of precision in comparison with traditional manual gating is needed for wide-scale adoption of automated gating methods. In an effort to more closely follow the manual gating process, our automated gating pipeline was created to include negative controls (Fluorescence Minus One [FMO]) to enhance the reliability of gate placement. We demonstrate that use of an automated pipeline, heavily relying on FMO controls for population discrimination, can analyze multi-parameter, large-scale clinical datasets with comparable precision and accuracy to traditional manual gating.

Template-based prediction of protein function.

We discuss recent approaches for structure-based protein function annotation. We focus on template-based methods where the function of a query protein is deduced from that of a template for which both the structure and function are known. We describe the different ways of identifying a template. These are typically based on sequence analysis but new methods based on purely structural similarity are also being developed that allow function annotation based on structural relationships that cannot be recognized by sequence. The growing number of available structures of known function, improved homology modeling techniques and new developments in the use of structure allow template-based methods to be applied on a proteome-wide scale and in many different biological contexts. This progress significantly expands the range of applicability of structural information in function annotation to a level that previously was only achievable by sequence comparison.

OnTheFly: a database of Drosophila melanogaster transcription factors and their binding sites.

We present OnTheFly (http://bhapp.c2b2.columbia.edu/OnTheFly/index.php), a database comprising a systematic collection of transcription factors (TFs) of Drosophila melanogaster and their DNA-binding sites. TFs predicted in the Drosophila melanogaster genome are annotated and classified and their structures, obtained via experiment or homology models, are provided. All known preferred TF DNA-binding sites obtained from the B1H, DNase I and SELEX methodologies are presented. DNA shape parameters predicted for these sites are obtained from a high throughput server or from crystal structures of protein-DNA complexes where available. An important feature of the database is that all DNA-binding domains and their binding sites are fully annotated in a eukaryote using structural criteria and evolutionary homology. OnTheFly thus provides a comprehensive view of TFs and their binding sites that will be a valuable resource for deciphering non-coding regulatory DNA.

Genome-wide structural analysis reveals novel membrane binding properties of AP180 N-terminal homology (ANTH) domains.

An increasing number of cytosolic proteins are shown to interact with membrane lipids during diverse cellular processes, but computational prediction of these proteins and their membrane binding behaviors remains challenging. Here, we introduce a new combinatorial computation protocol for systematic and robust functional prediction of membrane-binding proteins through high throughput homology modeling and in-depth calculation of biophysical properties. The approach was applied to the genomic scale identification of the AP180 N-terminal homology (ANTH) domain, one of the modular lipid binding domains, and prediction of their membrane binding properties. Our analysis yielded comprehensive coverage of the ANTH domain family and allowed classification and functional annotation of proteins based on the differences in local structural and biophysical features. Our analysis also identified a group of plant ANTH domains with unique structural features that may confer novel functionalities. Experimental characterization of a representative member of this subfamily confirmed its unique membrane binding mechanism and unprecedented membrane deforming activity. Collectively, these studies suggest that our new computational approach can be applied to genome-wide functional prediction of other lipid binding domains.

High-throughput computational structure-based characterization of protein families: START domains and implications for structural genomics.

SkyLine, a high-throughput homology modeling pipeline tool, detects and models true sequence homologs to a given protein structure. Structures and models are stored in SkyBase with links to computational function annotation, as calculated by MarkUs. The SkyLine/SkyBase/MarkUs technology represents a novel structure-based approach that is more objective and versatile than other protein classification resources. This structure-centric strategy provides a multi-dimensional organization and coverage of protein space at the levels of family, function, and genome. The concept of "modelability", the ability to model sequences on related structures, provides a reliable criterion for membership in a protein family ("leverage") and underlies the unique success of this approach. The overall procedure is illustrated by its application to START domains, which comprise a Biomedical Theme for the Northeast Structural Genomics Consortium as part of the Protein Structure Initiative. START domains are typically involved in the non-vesicular transport of lipids. While 19 experimentally determined structures are available, the family, whose evolutionary hierarchy is not well determined, is highly sequence diverse, and the ligand-binding potential of many family members is unknown. The SkyLine/SkyBase/MarkUs approach provides significant insights and predicts: (1) many more family members (approximately 4,000) than any other resource; (2) the function for a large number of unannotated proteins; (3) instances of START domains in genomes from which they were thought to be absent; and (4) the existence of two types of novel proteins, those containing dual START domain and those containing N-terminal START domains.

Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2'-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction.

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2'-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2'-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first beta sheet and second beta sheet of RNase HI of Escherichia coli might participate in substrate binding.

Contributions of the interdomain loop, amino terminus, and subunit interface to the ligand-facilitated dimerization of neurophysin: crystal structures and mutation studies of bovine neurophysin-I.

Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55-59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1-6, which interact with the loop and adjacent residues 53-54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1-6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1-6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary.

NMR investigation of main-chain dynamics of the H80E mutant of bovine neurophysin-I: demonstration of dimerization-induced changes at the hormone-binding site.

Neurophysins are hormone-binding proteins composed of two partially homologous domains. Ligand-binding (localized to the amino domain) and dimerization (involves both domains) are cooperatively linked by an as yet undefined allosteric mechanism. To help define this mechanism, we investigated the backbone dynamics of the unliganded monomeric state of the H80E mutant of bovine neurophysin-I by (15)N NMR. Model-free analysis of the NMR relaxation parameters indicated significantly greater flexibility in the carboxyl domain than in the amino domain, particularly at their dimerization interface segments. Amino domain residues critical to hormone binding were highly structured, constraining potential allosteric mechanisms. Model-free analysis additionally demonstrated chemical exchange effects, manifest as R(ex) terms, in 16 residues, 14 of which are located in the amino domain at, or immediately adjacent to, either the dimerization interface or the hormone-binding site. The chemical exchange process was further characterized using relaxation-compensated CPMG measurements, the results allowing assignment of the process to monomer-dimer exchange and calculation of the exchange kinetics, which were slow on the NMR time scale. An apparently different concentration-dependent process, distinguished from normal dimerization by its fast exchange behavior and pH-independence, also principally involved a subset of residues at and immediately adjacent to either the hormone-binding site or the amino domain dimerization interface. The data represent the first direct demonstration of an effect of dimerization in the unliganded state on neurophysin's hormone-binding site, the effect particularly involving residues that interact with hormone residue 2, and specifically identify Ser25 and Ile26 as likely intermediaries between the sites of dimerization and of hormone binding. Consistent with recent views of the role of anchor residues in protein interactions, we propose that dimerization proceeds by a fast pH-independent association of the well-structured amino domain interface that is rapidly communicated to the binding site for hormone residue 2, followed by a rate-determining pH-dependent interaction of the less structured carboxyl domain interface.

NMR structure and Mg2+ binding of an RNA segment that underlies the L7/L12 stalk in the E.coli 50S ribosomal subunit.

Helix 42 of Domain II of Escherichia coli 23S ribosomal RNA underlies the L7/L12 stalk in the ribosome and may be significant in positioning this feature relative to the rest of the 50S ribosomal subunit. Unlike the Haloarcula marismortui and Deinococcus radiodurans examples, the lower portion of helix 42 in E.coli contains two consecutive G*A oppositions with both adenines on the same side of the stem. Herein, the structure of an analog of positions 1037-1043 and 1112-1118 in the helix 42 region is reported. NMR spectra and structure calculations support a cis Watson-Crick/Watson-Crick (cis W.C.) G*A conformation for the tandem (G*A)2 in the analog and a minimally perturbed helical duplex stem. Mg2+ titration studies imply that the cis W.C. geometry of the tandem (G*A)2 probably allows O6 of G20 and N1 of A4 to coordinate with a Mg2+ ion as indicated by the largest chemical shift changes associated with the imino group of G20 and the H8 of G20 and A4. A cross-strand bridging Mg2+ coordination has also been found in a different sequence context in the crystal structure of H.marismortui 23S rRNA, and therefore it may be a rare but general motif in Mg2+ coordination.

IP-COSY, a totally in-phase and sensitive COSY experiment.

The IP-COSY experiment presented in this paper gives an in-phase spectral presentation in both the F(1) and F(2) dimensions by a combined use of a constant evolution time (CT) in t(1) and a symmetrical refocusing period before t(2). Compared with DQF-COSY and CT-COSY, IP-COSY further alleviates the effect of signal reduction due to a small ratio p (= J/linewidth), showing (1) improved lineshape and cross-peak definition and (2) especially enhancement in signals of the peaks of small active J coupling constant and the peaks of broader linewidth. A new strategy was adopted to eliminate or reduce effectively artifactual peaks by adding a 0.1-0.2 ms variation to the time delays of the CT period used for each scan of the FID in IP-COSY and CT-COSY. (3)J(H,H) coupling constants of larger than 4 Hz in the fingerprint region of peptides can be directly derived from the separation of doublets. IP-COSY cross peaks are stronger than those in DQF-COSY by 4-20-fold for tested peptides and oligonucleotides (MW < 8 kDa) with acquisition and processing parameters used in the work, and they are easier to identify than those in CT-COSY. The overall improvement in IP-COSY should make the detection/autodetection of the COSY cross peaks and the measurements of the various coupling constants more easily achieved, providing valuable information for the structure elucidation of peptides/small proteins and oligonucleotides.