A phage-displayed single-chain Fab library optimized for rapid production of single-chain IgGs.

Phage-displayed synthetic antibody (Ab) repertoires have become a major source of affinity reagents for basic and clinical research. Specific Abs identified from such libraries are often screened as fragments antigen binding (Fabs) produced in bacteria, and those with desired biochemical characteristics are reformatted for production as full-length immunoglobulin G (IgG) in mammalian cells. The conversion of Fabs to IgGs is a cumbersome and often rate-limiting step in the development of Abs. Moreover, biochemical properties required for lead IgG development are not always shared by the Fabs, and these issues are not uncovered until a significant effort has been spent on Abs that ultimately will not be useful. Thus, there is a need for simple and rapid techniques to convert phage-displayed Fabs to IgGs at an early stage of the Ab screening process. We report the generation of a highly diverse phage-displayed synthetic single-chain Fab (scFab) library, in which the light and heavy chains were tethered with an optimized linker. Following selection, pools of scFabs were converted to single-chain IgGs (scIgGs) en masse, enabling facile screening of hundreds of phage-derived scIgGs. We show that this approach can be used to rapidly screen for and select scIgGs that target cell-surface receptors, and scIgGs behave the same as conventional IgGs.

Subdiffraction resolution microscopy methods for analyzing centrosomes organization.

In this chapter, we describe the current methods of examining the structure of centrosomes by fluorescence subdiffraction microscopy. By using recently developed microscopy techniques, centrosomal proteins can now be examined in cells with a resolution of only a few nanometers, a level of molecular detail beyond the reach of traditional cell biology methods as confocal and widefield microscopy. We emphasize imaging by three-dimensional structured illumination microscopy, stochastic optical reconstruction microscopy, and quantitative approaches to image data analysis.

Scalable high throughput selection from phage-displayed synthetic antibody libraries.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

Visualization of arrestin recruitment by a G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) are critically regulated by ß-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ß2 adrenergic receptor (ß2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ß-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-ß-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ß2AR-ß-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ß2AR and ß-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of ß-arrestin 1 to the ß2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of ß-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of ß-arrestin 1 when coupled to the ß2AR. A molecular model of the ß2AR-ß-arrestin signalling complex was made by docking activated ß-arrestin 1 and ß2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

Phospholipase D controls Dictyostelium development by regulating G protein signaling.

Dictyostelium discoideum cells normally exist as individual amoebae, but will enter a period of multicellular development upon starvation. The initial stages of development involve the aggregation of individual cells, using cAMP as a chemoattractant. Chemotaxis is initiated when cAMP binds to its receptor, cAR1, and activates the associated G protein, Gα2ßγ. However, chemotaxis will not occur unless there is a high density of starving cells present, as measured by high levels of the secreted quorum sensing molecule, CMF. We previously demonstrated that cells lacking PldB bypass the need for CMF and can aggregate at low cell density, whereas cells overexpressing pldB do not aggregate even at high cell density. Here, we found that PldB controlled both cAMP chemotaxis and cell sorting. PldB was also required by CMF to regulate G protein signaling. Specifically, CMF used PldB, to regulate the dissociation of Gα2 from Gßγ. Using fluorescence resonance energy transfer (FRET), we found that along with cAMP, CMF increased the dissociation of the G protein. In fact, CMF augmented the dissociation induced by cAMP. This augmentation was lost in cells lacking PldB. PldB appears to mediate the CMF signal through the production of phosphatidic acid, as exogenously added phosphatidic acid phenocopies overexpression of pldB. These results suggest that phospholipase D activity is required for CMF to alter the kinetics of cAMP-induced G protein signaling.

Limb-girdle muscular dystrophy type 2A can result from accelerated autoproteolytic inactivation of calpain 3.

Loss-of-function mutations in calpain 3 have been shown to cause limb-girdle muscular dystrophy type 2A (LGMD2A), an autosomal recessive disorder that results in gradual wasting of the muscles of the hip and shoulder areas. Due to the inherent instability of calpain 3, recombinant expression of the full-length enzyme has not been possible, making in vitro analysis of specific LGMD2A-causing mutations difficult. However, because calpain 3 is highly similar in amino acid sequence to calpain 2, the recently solved crystal structure of full-length, Ca2+-bound, calpastatin-inhibited rat calpain 2 has allowed us to model calpain 3 as a Ca2+-bound homodimer. The model revealed three distinct areas of the enzyme that undergo a large conformational change upon Ca2+ binding. Located in these areas are several residues that undergo mutation to cause LGMD2A. We investigated the in vitro effects of six of these mutations by making the corresponding mutations in rat calpain 2. All six mutations examined in this study resulted in a decrease in enzyme activity. All but one of the mutations caused an increased rate of autoproteolytic degradation of the enzyme as witnessed by SDS-PAGE, indicating the decrease in enzyme activity is caused, at least in part, by an increase in the rate of autoproteolytic degradation. The putative in vivo effects of these mutations on calpain 3 activity are discussed with respect to their ability to cause LGMD2A.

Calcium-bound structure of calpain and its mechanism of inhibition by calpastatin.

Calpains are non-lysosomal calcium-dependent cysteine proteinases that selectively cleave proteins in response to calcium signals and thereby control cellular functions such as cytoskeletal remodelling, cell cycle progression, gene expression and apoptotic cell death. In mammals, the two best-characterized members of the calpain family, calpain 1 and calpain 2 (micro-calpain and m-calpain, respectively), are ubiquitously expressed. The activity of calpains is tightly controlled by the endogenous inhibitor calpastatin, which is an intrinsically unstructured protein capable of reversibly binding and inhibiting four molecules of calpain, but only in the presence of calcium. To date, the mechanism of inhibition by calpastatin and the basis for its absolute specificity have remained speculative. It was not clear how this unstructured protein inhibits calpains without being cleaved itself, nor was it known how calcium induced changes that facilitated the binding of calpastatin to calpain. Here we report the 2.4-A-resolution crystal structure of the calcium-bound calpain 2 heterodimer bound by one of the four inhibitory domains of calpastatin. Calpastatin is seen to inhibit calpain by occupying both sides of the active site cleft. Although the inhibitor passes through the active site cleft it escapes cleavage in a novel manner by looping out and around the active site cysteine. The inhibitory domain of calpastatin recognizes multiple lower affinity sites present only in the calcium-bound form of the enzyme, resulting in an interaction that is tight, specific and calcium dependent. This crystal structure, and that of a related complex, also reveal the conformational changes that calpain undergoes on binding calcium, which include opening of the active site cleft and movement of the domains relative to each other to produce a more compact enzyme.

Calpastatin simultaneously binds four calpains with different kinetic constants.

Calpastatin is the endogenous, specific protein inhibitor of the calcium-dependent protease, calpain. Using an active site knock-out m-calpain mutant we have studied the enzyme's calcium-dependent binding to calpastatin by surface plasmon resonance without the complication of proteolysis. Calpastatin was capable of simultaneously binding four molecules of calpain. Its four inhibitory domains (CAST1, 2, 3, and 4) were individually expressed in Escherichia coli and the kinetics of their interaction with calpain was separately compared. Their K(d) values ranged from picomolar to nanomolar in the order CAST1>4>3>2. They have similar k(on) values but the k(off) values ranged over three orders of magnitude and can account for the differences in affinity.