BackgroundNanoparticles in contact with biological fluids interact with proteins and other biomolecules, thus forming a dynamic corona whose composition varies over time due to continuous protein association and dissociation events. Eventually equilibrium is reached, at which point the continued exchange will not affect the composition of the corona.ResultsWe developed a simple and effective dynamic model of the nanoparticle protein corona in a body fluid, namely human plasma. The model predicts the time evolution and equilibrium composition of the corona based on affinities, stoichiometries and rate constants. An application to the interaction of human serum albumin, high density lipoprotein (HDL) and fibrinogen with 70 nm N-iso-propylacrylamide/N-tert-butylacrylamide copolymer nanoparticles is presented, including novel experimental data for HDL.ConclusionsThe simple model presented here can easily be modified to mimic the interaction of the nanoparticle protein corona with a novel biological fluid or compartment once new data will be available, thus opening novel applications in nanotoxicity and nanomedicine.
Guanylate cyclase activating protein 1 (GCAP1) is a neuronal Ca(2+) sensor (NCS) that regulates the activation of rod outer segment guanylate cyclases (ROS-GCs) in photoreceptors. In this study, we investigated the Ca(2+)-induced effects on the conformation and the thermal stability of four GCAP1 variants associated with hereditary human cone dystrophies. Ca(2+) binding stabilized the conformation of all the GCAP1 variants independent of myristoylation. The myristoylated wild-type GCAP1 was found to have the highest Ca(2+) affinity and thermal stability, whereas all the mutants showed decreased Ca(2+) affinity and significantly lower thermal stability in both apo and Ca(2+)-loaded forms. No apparent cooperativity of Ca(2+) binding was detected for any variant. Finally, the non-myristoylated mutants were still capable of activating ROS-GC1, but the measured cyclase activity was shifted toward high, nonphysiological Ca(2+) concentrations. Thus, we conclude that distorted Ca(2+)-sensor properties could lead to cone dysfunction.
Photoreceptor cells finely adjust their sensitivity and electrical response according to changes in light stimuli as a direct consequence of the feedback and regulation mechanisms in the phototransduction cascade. In this study, we employed a systems biology approach to develop a dynamic model of vertebrate rod phototransduction that accounts for the details of the underlying biochemistry. Following a bottom-up strategy, we first reproduced the results of a robust model developed by Hamer et al. (Vis. Neurosci., 2005, 22(4), 417), and then added a number of additional cascade reactions including: (a) explicit reactions to simulate the interaction between the activated effector and the regulator of G-protein signalling (RGS); (b) a reaction for the reformation of the G-protein from separate subunits; (c) a reaction for rhodopsin (R) reconstitution from the association of the opsin apoprotein with the 11-cis-retinal chromophore; (d) reactions for the slow activation of the cascade by opsin. The extended network structure successfully reproduced a number of experimental conditions that were inaccessible to prior models. With a single set of parameters the model was able to predict qualitative and quantitative features of rod photoresponses to light stimuli ranging over five orders of magnitude, in normal and altered conditions, including genetic manipulations of the cascade components. In particular, the model reproduced the salient dynamic features of the rod from Rpe65(-/-) animals, a well established model for Leber congenital amaurosis and vitamin A deficiency. The results of this study suggest that a systems-level approach can help to unravel the adaptation mechanisms in normal and in disease-associated conditions on a molecular basis.
Ca(2+)-sensor proteins regulate a variety of intracellular processes by adopting specific conformations in response to finely tuned changes in Ca(2+)-concentration. Here we present a surface plasmon resonance (SPR)-based approach, which allows for simultaneous detection of conformational dynamics of four Ca(2+)-sensor proteins (calmodulin, recoverin, GCAP1, and GCAP2) operating in the vertebrate phototransduction cascade, over variations in Ca(2+) concentration in the 0.1-0.6 μM range. By working at conditions that quantitatively mimic those found in the cell, we show that the method is able to detect subtle differences in the dynamics of each Ca(2+)-sensor, which appear to be influenced by the presence of free Mg(2+) at physiological concentration and by posttranslational modifications such as myristoylation. Comparison between the macroscopic Ca(2+)-binding constants, directly measured by competition with a chromophoric chelator, and the concerted binding-conformational switch detected by SPR at equilibrium reveals the relative contribution of the conformational change process to the SPR signal. This process appears to be influenced by the presence of other cations that perturb Ca(2+)-binding and the conformational transition by competing with Ca(2+), or by pure electrostatic screening. In conclusion, the approach described here allows a comparative analysis of protein conformational changes occurring under physiologically relevant molecular crowding conditions in ultrathin biosensor layers.
The early steps in vertebrate vision require fast interactions between Rh (rhodopsin) and Gt (transducin), which are classically described by a collisional coupling mechanism driven by the free diffusion of monomeric proteins on the disc membranes of rod and cone cells. Recent findings, however, point to a very low mobility for Rh and support a substantially different supramolecular organization. Moreover, Rh-G(t) interactions seem to possibly occur even prior to light stimuli, which is also difficult to reconcile with the classical scenario. We investigated the kinetics of interaction between native Rh and G(t) in different conditions by surface plasmon resonance and analysed the results in the general physiological context by employing a holistic systems modelling approach. The results from the present study point to a mechanism that is intermediate between pure collisional coupling and physical scaffolding. Such a 'dynamic scaffolding', in which prevalently dimeric Rh and G(t) interact in the dark by forming transient complexes (~25% of G(t) is precoupled to Rh), does not slow down the phototransduction cascade, but is compatible with the observed photoresponses on a broad scale of light stimuli. We conclude that Rh molecules and Rh-G(t) complexes can both absorb photons and trigger the visual cascade.
Calcium-signaling in cells requires a fine-tuned system of calcium-transport proteins involving ion channels, exchangers, and ion-pumps but also calcium-sensor proteins and their targets. Thus, control of physiological responses very often depends on incremental changes of the cytoplasmic calcium concentration, which are sensed by calcium-binding proteins and are further transmitted to specific target proteins. This Review will focus on calcium-signaling in vertebrate photoreceptor cells, where recent physiological and biochemical data indicate that a subset of neuronal calcium sensor proteins named guanylate cyclase-activating proteins (GCAPs) operate in a calcium-relay system, namely, to make gradual responses to small changes in calcium. We will further integrate this mechanism in an existing computational model of phototransduction showing that it is consistent and compatible with the dynamics that are characteristic for the precise operation of the phototransduction pathways.
We determined the conditions under which surface plasmon resonance can be used to monitor at real-time the Ca(2+)-induced conformational transitions of the sensor protein recoverin immobilized over a sensor chip. The equilibrium and the kinetics of conformational transitions were detected and quantified over a physiological range of Ca(2+) and protein concentrations similar to those found within cells. Structural analysis suggests that the detection principle reflects changes in the hydrodynamic properties of the protein and is not due to a mass effect. The phenomenon appears to be related to changes in the refractive index at the metal/dielectric interface.
Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination. The photoexcitation and adaptation machinery in photoreceptor cells consists of protein complexes that can form highly ordered supramolecular structures and control the homeostasis and mutual dependence of the secondary messengers cyclic guanosine monophosphate (cGMP) and Ca2+. The visual pigment in rod photoreceptors, the G protein-coupled receptor rhodopsin is organized in tracks of dimers thereby providing a signaling platform for the dynamic scaffolding of the G protein transducin. Illuminated rhodopsin is turned off by phosphorylation catalyzed by rhodopsin kinase (GRK1) under control of Ca2+-recoverin. The GRK1 protein complex partly assembles in lipid raft structures, where shutting off rhodopsin seems to be more effective. Re-synthesis of cGMP is another crucial step in the recovery of the photoresponse after illumination. It is catalyzed by membrane bound sensory guanylate cyclases (GCs) and is regulated by specific neuronal Ca2+-sensor proteins called guanylate cyclase-activating proteins (GCAPs). At least one GC (ROS-GC1) was shown to be part of a multiprotein complex having strong interactions with the cytoskeleton and being controlled in a multimodal Ca2+-dependent fashion. The final target of the cGMP signaling cascade is a cyclic nucleotide-gated (CNG) channel that is a hetero-oligomeric protein located in the plasma membrane and interacting with accessory proteins in highly organized microdomains. We summarize results and interpretations of findings related to the inhomogeneous organization of signaling units in photoreceptor outer segments.
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