Functional map of arrestin binding to phosphorylated opsin, with and without agonist

Arrestins desensitize G protein-coupled receptors (GPCRs) and act as mediators of signalling. Here we investigated the interactions of arrestin-1 with two functionally distinct forms of the dim-light photoreceptor rhodopsin. Using unbiased scanning mutagenesis we probed the individual contribution of each arrestin residue to the interaction with the phosphorylated apo-receptor (Ops-P) and the agonist-bound form (Meta II-P). Disruption of the polar core or displacement of the C-tail strengthened binding to both receptor forms. In contrast, mutations of phosphate-binding residues (phosphosensors) suggest the phosphorylated receptor C-terminus binds arrestin differently for Meta II-P and Ops-P. Likewise, mutations within the inter-domain interface, variations in the receptor-binding loops and the C-edge of arrestin reveal different binding modes. In summary, our results indicate that arrestin-1 binding to Meta II-P and Ops-P is similarly dependent on arrestin activation, although the complexes formed with these two receptor forms are structurally distinct

Insights into congenital stationary night blindness based on the structure of G90D rhodopsin

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102109/1/embr201344.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102109/2/embr201344.reviewer_comments.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102109/3/embr201344-sup-0001.pd

AAscan, PCRdesign and MutantChecker: A Suite of Programs for Primer Design and Sequence Analysis for High-Throughput Scanning Mutagenesis

Scanning mutagenesis is a powerful protein engineering technique used to study protein structure-function relationship, map binding sites and design more stable proteins or proteins with altered properties. One of the time-consuming tasks encountered in application of this technique is the design of primers for site-directed mutagenesis. Here we present an open-source multi-platform software AAscan developed to design primers for this task according to a set of empirical rules such as melting temperature, overall length, length of overlap regions, and presence of GC clamps at the 3’ end, for any desired substitution. We also describe additional software tools which are used to analyse a large number of sequencing results for the presence of desired mutations, as well as related software to design primers for ligation independent cloning. We have used AAscan software to design primers to make over 700 mutants, with a success rate of over 80%. We hope that the open-source nature of our software and ready availability of freeware tools used for its development will facilitate its adaptation and further development. The software is distributed under GPLv3 licence and is available at http://www.psi.ch/lbr/aascan.ISSN:1932-620

AAscan software interface.

<p>(1) Input text box for reference sequence. (2) Options for primer design. (3) Output window containg primer forward and (4) reverse primers.</p

Two-fragment PCR mutagenesis strategy for difficult mutants.

<p>Each mutagenesis primer is used together with another primer annealing approximately opposite the mutation site, e.g. origin region of the plasmid. The resulting two PCR fragments are re-combined by CloneEZ or Gibson reaction to form the original circular plasmid.</p

Arrestin-1 engineering facilitates complex stabilization with native rhodopsin

Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin−arrestin-1 complexes that eventually decay to opsin, retinal and arrestin-1. Via a multi-dimensional screening setup, we identified and combined arrestin-1 mutants that form lasting complexes with light-activated and phosphorylated rhodopsin in harsh conditions, such as high ionic salt concentration. Two quadruple mutants, D303A + T304A + E341A + F375A and R171A + T304A + E341A + F375A share similar heterologous expression and thermo-stability levels with wild type (WT) arrestin-1, but are able to stabilize complexes with rhodopsin with more than seven times higher half-maximal inhibitory concentration (IC50) values for NaCl compared to the WT arrestin-1 protein. These quadruple mutants are also characterized by higher binding affinities to phosphorylated rhodopsin, light-activated rhodopsin and phosphorylated opsin, as compared with WT arrestin-1. Furthermore, the assessed arrestin-1 mutants are still specifically associating with phosphorylated or light-activated receptor states only, while binding to the inactive ground state of the receptor is not significantly altered. Additionally, we propose a novel functionality for R171 in stabilizing the inactive arrestin-1 conformation as well as the rhodopsin–arrestin-1 complex. The achieved stabilization of the active rhodopsin–arrestin-1 complex might be of great interest for future structure determination, antibody development studies as well as drug-screening efforts targeting G protein-coupled receptors (GPCRs).ISSN:2045-232

Immobilization of arrestin-3 on different biosensor platforms for evaluating GPCR binding

G protein-coupled receptors (GPCRs) are a large and ubiquitous family of membrane receptors of great pharmacological interest. Cell-based assays are the primary tool for assessing GPCR interactions and activation but their design and intrinsic complexity limit their application. Biosensor-based assays that directly and specifically report GPCR-protein binding (e.g. arrestin or G protein) could provide a good alternative. We present an approach based on the stable immobilization of different arrestin-3 proteins (wild type, and two mutants, mutant X (arrestin-3 I386A) and mutant Y (arrestin-3 R393E)) via histidine tags on NTA(Ni2+)-coated sensors in a defined orientation. Using biolayer interferometry (BLI), surface plasmon resonance (SPR), and quartz crystal microbalance with dissipation (QCM-D), we were able to follow the interaction between the different arrestin-3 proteins and a representative GPCR, jumping spider rhodopsin-1 (JSR1), in a label-free manner in real-time. The interactions were quantified as binding affinity, association and dissociation rate constants. The combination of surface-based biosensing methods indicated that JSR1 showed the strongest binding to arrestin mutant Y. Taken together, this work introduces direct label-free, biosensor-based screening approaches that can be easily adapted for testing interactions of proteins and other compounds with different GPCRs.ISSN:1463-9084ISSN:1463-907

Influence of secondary structure and melting temperatures of primers on the success of mutagenesis.

<p>Features of primer pairs (ΔTm) are counted once, features of single primers (ΔG) were counted separately for the reverse and forward primer of a pair and are represented in one graph. Solid bars represent the number of primers with a particular value of a feature (left axis), striped bar represent the fraction failed (right axis). The standard deviation of the fraction failed was calculated as (<i>f</i>*(1-<i>f</i>)/<i>N</i>)^1/2, where <i>f</i> is fraction failed and <i>N</i> is the total number of primers in a particular category. Success (black) or failure (grey) of mutagenesis, as well as fraction failed (striped) in dependence of <b>(A)</b> the ΔG of hairpins formed by the primers, <b>(B)</b> the ΔG of homodimers formed by the primers, <b>(C)</b> primer melting temperatures calculated for mutation of the native DNA (early PCR cycles), <b>(D)</b> primer melting temperatures calculated for mutation of the DNA containing the mutation (later cycles of PCR), <b>(F)</b> quality score of the GC clamp and <b>(G)</b> GC content of the primer.</p