Most organisms rely on innate immune receptors to recognize conserved molecular structures from invading microbes. Two essential innate immune receptors, RIG-I and MDA5, detect viral double-stranded RNA in the cytoplasm. The inflammatory response triggered by these RIG-I-like receptors (RLRs) is one of the first and most important lines of defense against infection. RIG-I recognizes short RNA ligands with 5′-triphosphate caps. MDA5 recognizes long kilobase-scale genomic RNA and replication intermediates. Ligand binding induces conformational changes and oligomerization of RLRs that activate the signaling partner MAVS on the mitochondrial and peroxisomal membranes. This signaling process is under tight regulation, dependent on post-translational modifications of RIG-I and MDA5, and on regulatory proteins including unanchored ubiquitin chains and a third RLR, LGP2. Here, we review recent advances that have shifted the paradigm of RLR signaling away from the conventional linear signaling cascade. In the emerging RLR signaling model, large multimeric signaling platforms generate a highly cooperative, self-propagating, and context-dependent signal, which varies with the subcellular localization of the signaling platform.

Background: Antifreeze proteins bind to ice crystals and inhibit their growth. Results: The crystal structure of a potent beetle antifreeze protein was determined by direct methods. Conclusion: Ordered crystallographic waters on the protein surface match several planes of hexagonal ice. Significance: The structure is the largest determined ab initio without heavy atoms, and its ordered waters suggest a molecular basis for ice binding.

I. General methods. Fmoc-protected α-amino acids, Wang resin, and NovaPEG Wang resin were purchased from Novabiochem (San Diego, CA). Fmoc-protected β 3 -amino acids were purchased from PepTech Corp. (Burlington, MA) or prepared from the analogous Fmocprotected α-amino acids, following previously established synthetic procedures. 1 N,N-Dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), N-methylmorpholine (NMM), trifluoroacetic acid (TFA) and piperidine were purchased from American Bioanalytical (Natick, MA). 1-Hydroxy-7-azabenzotriazole (HOAt) was purchased from Chempep, Inc. (Miami, FL).(7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) was purchased from Oakwood Products, Inc. (West Columbia, SC). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Microwave-assisted peptide syntheses were performed in a CEM MARS 5 system (Matthews, NC). LC-MS spectra were acquired with a Waters XEVO Q-TOF mass spectrometer (Milford, MA). 1 H NMR spectra were acquired on a Bruker 400 MHz instrument (Billerica, MA). Reversed-phase HPLC was performed using a Varian Prostar HPLC module (Palo Alto, CA) and YMC semi-preparative columns (Kyoto, Japan). Circular dichroism (CD) spectra were acquired with a Jasco J-810 Spectropolarimeter (Tokyo, Japan) equipped with a Peltier temperature control. Kinetic measurements were performed on a Molecular Devices Spectramax M5 Microplate Reader (Sunnyvale, CA).

Rhodopsin is a vertebrate dim-light photoreceptor consisting of a seven-helical transmembrane opsin protein and a chromophore, 11-cis retinal, linked to the protein via a protonated Schiff base (PSB). 1 Following photon absorption, 11-cis retinal isomerizes to the all-trans configuration to initiate the visual transduction cascade. Since optimal transduction depends on the thermal stability of rhodopsin, the thermal decay process has long been a subject of interest, yet the chemistry behind it has remained unclear. [2][3][4] Baylor et al. demonstrated that the thermal isomerization of rhodopsin, which enhances dark noise and reduces photosensitivity, generates the same physiological response as photoisomerization. 5,6 Furthermore, rhodopsin was reported to undergo thermal decay characterized by a decrease in visible absorption at 500 nm and an increase in UV absorption at 380 nm upon incubation at 37-55°C. 7,8 The observed thermal decay was attributed to hydrolysis of the PSB. Further analysis by HPLC showed that the concentration of all-trans retinal extracted from rhodopsin increased after incubation in the dark at 55°C. In this case, the thermally-induced isomerization from 11-cis to all-trans was proposed to be initiated by a different process -thermal denaturation. 9 We recently proposed that a hydrogen-bonding network at the retinal binding site 10 is involved in the counterion switch between dark state rhodopsin and metarhodopsin I, an intermediate precursor to transducin photoactivation. 11,12 This theory has prompted our current investigation of the thermal properties of the putative hydrogen-bonding network through solvent deuterium isotope effects. In this report, we attempt to clarify and characterize the chemical reactions responsible for the thermal decay process, with particular emphasis on (1) thermal decay marked by a decrease in optical density at 500 nm (OD 500 ), (2) thermal isomerization of 11-cis to all-trans retinal, and (3) hydrolysis of the PSB. Our results suggest that although PSB hydrolysis and retinal isomerization affect the thermal decay of rhodopsin, denaturation of the secondary structure is not significant. Moreover, we discover that the rates of thermal decay, isomerization, and PSB hydrolysis are two-to three-fold slower in D 2 O than H 2 O. Based on these results, we conclude that the rate-determining step of these thermal processes involves the breaking of hydrogen bonds, which probably stabilize the tertiary structure of rhodopsin, thereby contributing to the high thermal stability.To examine the kinetics of thermal decay, we measured differences in the UV-vis absorption of rhodopsin at appropriate time intervals at 59°C to determine the rates of thermal decay in * elsa.yan@yale.edu.Supporting Information Available: Experimental details (PDF) are available free of charge via the Internet at http://pubs.acs.org. Figure 1A and B). Absorption at 280 nm corresponds to the aromatic amino acids of opsin, while the signal at 500 nm corresponds to rhodopsin with the 11-cis retinal...

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