A Fluorescence Cross-Correlation-Spectroscopy-Based Immunoassay for Rapid, Selective, and Accurate Protein Sizing in Human Plasma, Applied to the von Willebrand Factor.

Multimeric abnormalities in plasma von Willebrand factor (VWF) cause bleeding or clotting disorders. Electrophoretic analysis of multimers is used to detect such abnormalities but is qualitative, slow, and difficult to standardize. Fluorescence correlation spectroscopy (FCS) is a good alternative but is affected by low selectivity and concentration bias. Here, we report the development of a homogeneous immunoassay based on dual-color fluorescence cross-correlation spectroscopy (FCCS) that overcomes these challenges. By performing a mild denaturation treatment followed by reacting with polyclonal antibodies, the concentration bias was drastically reduced. The use of a dual antibody assay improved selectivity. Diffusion times of immunolabeled VWF were measured with FCCS and standardized relative to calibrator measurements. The assay measures size changes in VWF using 1 µL of plasma and less than 10 ng of antibody per measurement and was validated over a 16-fold range of VWF antigen concentration (VWF:Ag), with a sensitivity of VWF:Ag 0.8%. Concentration bias and imprecision were less than 10%. Measurements were unaffected by hemolytic, icteric, or lipemic interference. Strong correlations were obtained with reference densitometric readouts (0.97 for calibrators, 0.85 for clinical samples), and significant differences were found between normal (n = 10), type 2A (n = 5), and type 2B (n = 5) von Willebrand's disease and acquired thrombotic thrombocytopenic purpura (n = 10) samples (p < 0.01). This FCCS based immunoassay accurately and selectively determines changes in the multimeric status of plasma VWF and may be used as a simpler, faster, and a standardizable alternative for multimer analysis, following further clinical validation in larger cohorts.

Structure of an Unfolding Intermediate of an RRM Domain of ETR-3 Reveals Its Native-like Fold.

Prevalence of one or more partially folded intermediates during protein unfolding with different secondary and ternary conformations has been identified as an integral character of protein unfolding. These transition-state species need to be characterized structurally for elucidation of their folding pathways. We have determined the three-dimensional structure of an intermediate state with increased conformational space sampling under urea-denaturing condition. The protein unfolds completely at 10 M urea but retains residual secondary structural propensities with restricted motion. Here, we describe the native state, observable intermediate state, and unfolded state for ETR-3 RRM-3, which has canonical RRM fold. These observations can shed more light on unfolding events for RRM-containing proteins.

Structural investigation on SPI-6-associated Salmonella typhimurium VirG-like stress protein that promotes pathogen survival in macrophages.

Enteric microbial pathogenesis, remarkably a complex process, is achieved by virulence factors encoded by genes located within regions of the bacterial genome termed pathogenicity islands. Salmonella pathogenicity islands (SPI) encodes proteins, that are essential virulence determinants for pathogen colonization and virulence. In addition to the well-characterized SPI-1 and SPI-2 proteins, which are required for bacterial invasion and intracellular replication, respectively, SPI-6 (formerly known as Salmonella enterica centisome 7 island [SCI]) encoding proteins are also known to play pivotal role in Salmonella pathogenesis. However, the underlying molecular mechanism of these proteins remained elusive. To gain molecular insights into SPI-6-associated proteins, in this study, a SPI-6 Salmonella typhimurium VirG-like protein (STV) is characterized using interdisciplinary experimental approaches including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and infection assays. The high-resolution crystal structure, determined by the single-wavelength anomalous dispersion (SAD) method, reveals that STV belongs to the LTxxQ motif family. Solution-state NMR spectroscopy studies reveal that STV form a dimer involving interconnected helices. Interestingly, functional studies show that STV influence pathogen persistence inside macrophages in vitro at later stages of infection. Altogether, our findings suggest that STV, a member of the LTxxQ stress protein family, modulates bacterial survival mechanism in macrophages through SPI-1 and SPI-2 genes, respectively.

The N-terminal RNA Recognition Motif of PfSR1 Confers Semi-specificity for Pyrimidines during RNA Recognition.

Alternative splicing confers a complexity to the mRNA landscape of apicomplexans, resulting in a high proteomic diversity. The Plasmodium falciparum Ser/Arg-rich protein 1 (PfSR1) is the first protein to be confirmed as an alternative splicing factor in this class of parasitic protists [1]. A recent study [2] showed a purine bias in RNA binding among cognate RNA substrates of PfSR1. Here, we have investigated the role played by the amino-terminal RNA recognition motif (RRM1) of PfSR1 from the solution structure of its complex with ACAUCA RNA hexamer to understand how its mechanism of RNA recognition compares to human orthologs and to the C-terminal RRM. RNA binding by RRM1 is mediated through specific recognition of a cytosine base situated 5' of one or more pyrimidine bases by a conserved tyrosine residue on ß1 and a glutamate residue on the ß4 strand. Affinity is conferred through insertion of a 3' pyrimidine into a positively charged pocket. Retention of fast dynamics and ITC binding constants indicate the complex to be of moderate affinity. Using calorimetry and mapping of NMR chemical shift perturbations, we have also ascertained the purine preference of PfSR1 to be a property of the carboxy terminal pseudo-RRM (RRM2), which binds RNA non-canonically and with greater affinity compared to RRM1. Our findings show conclusive evidence of complementary RNA sequence recognition by the two RRMs, which may potentially aid PfSR1 in binding RNA with a high sequence specificity.

Structural delineation of stem-loop RNA binding by human TAF15 protein.

Human TATA binding protein associated factor 2 N (TAF15) and Fused in sarcoma (FUS) are nucleic acid binding proteins belonging to the conserved FET family of proteins. They are involved in diverse processes such as pre-mRNA splicing, mRNA transport, and DNA binding. The absence of information regarding the structural mechanism employed by the FET family in recognizing and discriminating their cognate and non-cognate RNA targets has hampered the attainment of consensus on modes of protein-RNA binding for this family. Our study provides a molecular basis of this RNA recognition using a combination of solution-state NMR spectroscopy, calorimetry, docking and molecular dynamics simulation. Analysis of TAF15-RRM solution structure and its binding with stem-loop RNA has yielded conclusive evidence of a non-canonical mode of RNA recognition. Rather than classical stacking interactions that occur across nitrogen bases and aromatic amino acids on ribonucleoprotein sites, moderate-affinity hydrogen bonding network between the nitrogen bases in the stem-loop RNA and a concave face on the RRM surface primarily mediate TAF15-RRM RNA interaction. We have compared the binding affinities across a set of single-stranded RNA oligonucleotides to conclusively establish that RNA binding is dependent upon structural elements in the RNA rather than sequence.

Sequence-specific resonance assignments of human TAF15-RRM and TAF15-RRM-RanBP2.

Human TATA binding protein associated factor 2 N (TAF15) is a RNA/DNA binding protein involved in many aspects of RNA and DNA metabolism. TAF15 contains an N-terminal transcriptional activation domain and C-terminal region comprising the RNA recognition motif (RRM) and RanBP2 type zinc finger domains with interspersed RGG motifs. In this study we report the complete backbone and side chain resonance assignments of human TAF15-RRM and backbone assignments of TAF15-RRM-RanBP2.

Dynamic association of PfEMP1 and KAHRP in knobs mediates cytoadherence during Plasmodium invasion.

Plasmodium falciparum infected erythrocytes display membrane knobs that are essential for their adherence to vascular endothelia and for prevention of clearance by the spleen. The knob associated histidine rich protein (KAHRP) is indispensable to knob formation and has been implicated in the recruitment and tethering of P. falciparum erythrocyte membrane protein-1 (PfEMP1) by binding to its cytoplasmic domain termed VARC. However, the precise mechanism of interaction between KAHRP and VARC is not very well understood. Here we report that both the proteins co-localize to membrane knobs of P. falciparum infected erythrocytes and have identified four positively charged linear sequence motifs of high intrinsic mobility on KAHRP that interact electrostatically with VARC in solution to form a fuzzy complex. The current study provides molecular insight into interaction between KAHRP and VARC in solution that takes place at membrane knobs.