Direct visualization of electric-field-stimulated ion conduction in a potassium channel.

Understanding protein function would be facilitated by direct, real-time observation of chemical kinetics in the atomic structure. The selectivity filter (SF) of the K channel provides an ideal model, catalyzing the dehydration and transport of K ions across the cell membrane through a narrow pore. We used a "pump-probe" method called electric-field-stimulated time-resolved X-ray crystallography (EFX) to initiate and observe K conduction in the NaK2K channel in both directions on the timescale of the transport process.

An evolution-based model for designing chorismate mutase enzymes.

The rational design of enzymes is an important goal for both fundamental and practical reasons. Here, we describe a process to learn the constraints for specifying proteins purely from evolutionary sequence data, design and build libraries of synthetic genes, and test them for activity in vivo using a quantitative complementation assay. For chorismate mutase, a key enzyme in the biosynthesis of aromatic amino acids, we demonstrate the design of natural-like catalytic function with substantial sequence diversity.

Learning the pattern of epistasis linking genotype and phenotype in a protein.

Understanding the pattern of epistasis-the non-independence of mutations-is critical for relating genotype and phenotype. However, the combinatorial complexity of potential epistatic interactions has severely limited the analysis of this problem. Using new mutational approaches, we report a comprehensive experimental study of all 2 mutants that link two phenotypically distinct variants of the Entacmaea quadricolor fluorescent protein-an opportunity to examine epistasis up to the 13 order.

Electric-field-stimulated protein mechanics.

The internal mechanics of proteins-the coordinated motions of amino acids and the pattern of forces constraining these motions-connects protein structure to function. Here we describe a new method combining the application of strong electric field pulses to protein crystals with time-resolved X-ray crystallography to observe conformational changes in spatial and temporal detail. Using a human PDZ domain (LNX2) as a model system, we show that protein crystals tolerate electric field pulses strong enough to drive concerted motions on the sub-microsecond timescale.

Evolution-based design of proteins.

Statistical analysis of protein sequences indicates an architecture for natural proteins in which amino acids are engaged in a sparse, hierarchical pattern of interactions in the tertiary structure. This architecture might be a key and distinguishing feature of evolved proteins-a design principle providing not only for foldability and high-performance function but also for robustness to perturbation and the capacity for rapid adaptation to new selection pressures. Here, we describe an approach for systematically testing this design principle for natural-like proteins by (1) computational design of synthetic sequences that gradually add or remove constraints along the hierarchy of interacting residues and (2) experimental testing of the designed sequences for folding and biochemical function.

Surface sites for engineering allosteric control in proteins.

Statistical analyses of protein families reveal networks of coevolving amino acids that functionally link distantly positioned functional surfaces. Such linkages suggest a concept for engineering allosteric control into proteins: The intramolecular networks of two proteins could be joined across their surface sites such that the activity of one protein might control the activity of the other. We tested this idea by creating PAS-DHFR, a designed chimeric protein that connects a light-sensing signaling domain from a plant member of the Per/Arnt/Sim (PAS) family of proteins with Escherichia coli dihydrofolate reductase (DHFR).

Dynamic scaffolding in a G protein-coupled signaling system.

The INAD scaffold organizes a multiprotein complex that is essential for proper visual signaling in Drosophila photoreceptor cells. Here we show that one of the INAD PDZ domains (PDZ5) exists in a redox-dependent equilibrium between two conformations--a reduced form that is similar to the structure of other PDZ domains, and an oxidized form in which the ligand-binding site is distorted through formation of a strong intramolecular disulfide bond. We demonstrate transient light-dependent formation of this disulfide bond in vivo and find that transgenic flies expressing a mutant INAD in which PDZ5 is locked in the reduced state display severe defects in termination of visual responses and visually mediated reflex behavior.

Evolutionary information for specifying a protein fold.

Classical studies show that for many proteins, the information required for specifying the tertiary structure is contained in the amino acid sequence. Here, we attempt to define the sequence rules for specifying a protein fold by computationally creating artificial protein sequences using only statistical information encoded in a multiple sequence alignment and no tertiary structure information. Experimental testing of libraries of artificial WW domain sequences shows that a simple statistical energy function capturing coevolution between amino acid residues is necessary and sufficient to specify sequences that fold into native structures.

The structural basis for red fluorescence in the tetrameric GFP homolog DsRed.

Green fluorescent protein (GFP) has rapidly become a standard tool for investigating a variety of cellular activities, and has served as a model system for understanding spectral tuning in chromophoric proteins. Distant homologs of GFP in reef coral and anemone display two new properties of the fluorescent protein family: dramatically red-shifted spectra, and oligomerization to form tetramers. We now report the 1.

A molecular pathway for light-dependent photoreceptor apoptosis in Drosophila.

Light-induced photoreceptor apoptosis occurs in many forms of inherited retinal degeneration resulting in blindness in both vertebrates and invertebrates. Though mutations in several photoreceptor signaling proteins have been implicated in triggering this process, the molecular events relating light activation of rhodopsin to photoreceptor death are yet unclear. Here, we uncover a pathway by which activation of rhodopsin in Drosophila mediates apoptosis through a G protein-independent mechanism.

A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade.

How are signalling molecules organized into different pathways within the same cell? In Drosophila, the inaD gene encodes a protein consisting of five PDZ domains which serves as a scaffold to assemble different components of the phototransduction cascade, including the principal light-activated ion channels, the effector phospholipase C-beta and protein kinase C. Null inaD mutants have a dramatically reorganized subcellular distribution of signalling molecules, and a total loss of transduction complexes. Also, mutants defective in a single PDZ domain produce signalling complexes that lack the target protein and display corresponding defects in their physiology.

Regulation of PLC-mediated signalling in vivo by CDP-diacylglycerol synthase.

CDP-diacylglycerol synthase (CDS) is an enzyme required for the regeneration of the signalling molecule phosphatidylinositol-4,5-bisphosphate (PtdlnsP2) from phosphatidic acid. A photo-receptor cell-specific isoform of CDS from Drosophila is a key regulator of phototransduction, a G-protein-coupled signalling cascade mediated by phospholipase C. cds mutants cannot sustain a light-activated current as a result of depletion of PtdlnsP2.

Arrestin function in inactivation of G protein-coupled receptor rhodopsin in vivo.

Arrestins have been implicated in the regulation of many G protein-coupled receptor signaling cascades. Mutations in two Drosophila photoreceptor-specific arrestin genes, arrestin 1 and arrestin 2, were generated. Analysis of the light response in these mutants shows that the Arr1 and Arr2 proteins are mediators of rhodopsin inactivation and are essential for the termination of the phototransduction cascade in vivo.

Ectopic expression of ultraviolet-rhodopsins in the blue photoreceptor cells of Drosophila: visual physiology and photochemistry of transgenic animals.

We have generated transgenic flies expressing R7 cell-specific opsins in the major class of photoreceptor cells of the Drosophila retina and characterized their spectral properties using high-resolution microspectrophotometry and sensitivity recordings. We show that the Rh3 and Rh4 opsin genes encode UV-sensitive opsins with similar spectral properties (lambda max = 345 nm and 375 nm), and that Rh3 corresponds to the R7p and R7marg class of visual pigments. We have also generated Rh3 and Rh4 isoform-specific antibodies and present an R7 cell map of the Drosophila retina.