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

A tool for visualizing protein motions in time-resolved crystallography

Time-resolved serial femtosecond crystallography (TR-SFX) at an x-ray free electron laser enables protein structural changes to be imaged on time-scales from femtoseconds to seconds. It can, however, be difficult to grasp the nature and timescale of global protein motions when structural changes are not isolated near a single active site. New tools are, therefore, needed to represent the global nature of electron density changes and their correlation with modeled protein structural changes. Here, we use TR-SFX data from bacteriorhodopsin to develop and validate a method for quantifying time-dependent electron density changes and correlating them throughout the protein. We define a spherical volume of difference electron density about selected atoms, average separately the positive and negative electron difference densities within each volume, and walk this spherical volume through all atoms within the protein. By correlating the resulting difference electron density amplitudes with time, our approach facilitates an initial assessment of the number and timescale of structural intermediates and highlights quake-like motions on the sub-picosecond timescale. This tool also allows structural models to be compared with experimental data using theoretical difference electron density changes calculated from refined resting and photo-activated structures.ISSN:2329-777

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

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

Various strategies for primer design.

<p>Green – insert, black – vector, red – additional optional sequences to be incorporated between the insert and the vector. Overhangs can be added to the vector-replicating primers (A), to the insert-replicating primers (B) or they can be split and added to both primers (C).</p

Mutagenesis by overlapping PCR reaction.

<p>Mutagenesis by overlapping PCR reaction.</p