How rotating ATP synthases can modulate membrane structure.

F1Fo-ATP synthase (ATP synthase) is a central membrane protein that synthetizes most of the ATP in the cell through a rotational movement driven by a proton gradient across the hosting membrane. In mitochondria, ATP synthases can form dimers through specific interactions between some subunits of the protein. The dimeric form of ATP synthase provides the protein with a spontaneous curvature that sustain their arrangement at the rim of the high-curvature edges of mitochondrial membrane (cristae). Also, a direct interaction with cardiolipin, a lipid present in the inner mitochondrial membrane, induces the dimerization of ATP synthase molecules along cristae. The deletion of those biochemical interactions abolishes the protein dimerization producing an altered mitochondrial function and morphology. Mechanically, membrane bending is one of the key deformation modes by which mitochondrial membranes can be shaped. In particular, bending rigidity and spontaneous curvature are important physical factors for membrane remodelling. Here, we discuss a complementary mechanism whereby the rotatory movement of the ATP synthase might modify the mechanical properties of lipid bilayers and contribute to the formation and regulation of the membrane invaginations.

Rhodamine-based sensor for real-time imaging of mitochondrial ATP in living fibroblasts.

Mitochondria are essential for the production and maintenance of ATP in the eukaryotic cell. To image and monitor intracellular ATP level without cell breakage, biological and chemical sensors were developed in the last years. Here, we have internalized a rhodamine-based sensor RSL+ into living cells and monitored the mitochondrial ATP levels in cultured mouse embryonic fibroblasts. To evaluate the robustness of the sensor we imaged the changes of the mitochondrial ATP levels under non-physiological conditions upon incubation with FCCP, oligomycin, azide, deoxyglucose or phosphoenolpyruvate; all compounds that interfere with ATP homeostasis of the cell. The ATP sensor allowed us to determine the mitochondrial ATP levels in human skin fibroblasts where we observe a similar amount of ATP compared to mouse embryonic fibroblasts. We propose the RSL+ to be a valuable tool for the assessment of mitochondrial dysfunction in human cells derived from mitochondrial OXPHOS patients and for basic studies on bioenergetics metabolism.

Structural basis of the interaction between integrin alpha6beta4 and plectin at the hemidesmosomes.

The interaction between the integrin alpha6beta4 and plectin is essential for the assembly and stability of hemidesmosomes, which are junctional adhesion complexes that anchor epithelial cells to the basement membrane. We describe the crystal structure at 2.75 A resolution of the primary alpha6beta4-plectin complex, formed by the first pair of fibronectin type III domains and the N-terminal region of the connecting segment of beta4 and the actin-binding domain of plectin. Two missense mutations in beta4 (R1225H and R1281W) linked to nonlethal forms of epidermolysis bullosa prevent essential intermolecular contacts. We also present two structures at 1.75 and 2.05 A resolution of the beta4 moiety in the absence of plectin, which reveal a major rearrangement of the connecting segment of beta4 on binding to plectin. This conformational switch is correlated with the way alpha6beta4 promotes stable adhesion or cell migration and suggests an allosteric control of the integrin.

Atomic crystal structure and sugar specificity of a ß-trefoil lectin domain from the ectomycorrhizal basidiomycete Laccaria bicolor.

Lectins from fruiting bodies are a diverse group of sugar-binding proteins from mushrooms that face the biologically relevant challenge of discriminating self- from non-self carbohydrate structures, therefore providing a basis for an innate defence system. Such a system entails both detection and destruction of invaders and/or feeders, and in contrast to more complex organisms with immense immune systems, these two functions normally rely on multitasking lectins, namely, lectins with different functional modules. Here, we present a novel fungal lectin, LBL, from the basidiomycete Laccaria bicolor. Using a diverse set of biophysical techniques, we unveil the fine details of the sugar-binding specificity of the N-terminal ß-trefoil of LBL (LBL152), whose structure has been determined at the highest resolution so far reported for such a fold. LBL152 binds complex poly-N-Acetyllactosamine polysaccharides and also robust LBL152 binding to Caenorhabditis elegans and Drosophila melanogaster cellular extracts was detected in microarray assays, with a seeming preference for the fruit fly adult and pupa stages over the larva stage. Prediction of the structure of the C-terminal part of LBL with AlphaFold reveals a tandem repeat of two structurally almost identical domains of around 110 amino acids each, despite sharing low sequence conservation.

Rotation of the c-Ring Promotes the Curvature Sorting of Monomeric ATP Synthases.

ATP synthases are proteins that catalyse the formation of ATP through the rotatory movement of their membrane-spanning subunit. In mitochondria, ATP synthases are found to arrange as dimers at the high-curved edges of cristae. Here, a direct link is explored between the rotatory movement of ATP synthases and their preference for curved membranes. An active curvature sorting of ATP synthases in lipid nanotubes pulled from giant vesicles is found. Coarse-grained simulations confirm the curvature-seeking behaviour of rotating ATP synthases, promoting reversible and frequent protein-protein contacts. The formation of transient protein dimers relies on the membrane-mediated attractive interaction of the order of 1.5 kB T produced by a hydrophobic mismatch upon protein rotation. Transient dimers are sustained by a conic-like arrangement characterized by a wedge angle of θ ≈ 50°, producing a dynamic coupling between protein shape and membrane curvature. The results suggest a new role of the rotational movement of ATP synthases for their dynamic self-assembly in biological membranes.

Directed, strong, and reversible immobilization of proteins tagged with a ß-trefoil lectin domain: a simple method to immobilize biomolecules on plain agarose matrixes.

A highly stable lipase from Geobacillus thermocatenolatus (BTL2) and the enhanced green fluorescent protein from Aquorea victoria (EGFP) were recombinantly produced N-terminally tagged to the lectin domain of the hemolytic pore-forming toxin LSLa from the mushroom Laetiporus sulphureus . Such a domain (LSL(150)), recently described as a novel fusion tag, is based on a ß-trefoil scaffold with two operative binding sites for galactose or galactose-containing derivatives. The fusion proteins herein analyzed have enabled us to characterize the binding mode of LSL(150) to polymeric and solid substrates such as agarose beads. The lectin-fusion proteins are able to be quantitatively bound to both cross-linked and non-cross-linked agarose matrixes in a very rapid manner, resulting in a surprisingly dynamic protein distribution inside the porous beads that evolves from heterogeneous to homogeneous along the postimmobilization time. Such dynamic distribution can be related to the reversible nature of the LSL(150)-agarose interaction. Furthermore, this latter interaction is temperature dependent since it is 4-fold stronger when the immobilization takes place at 25 °C than when it does at 4 °C. The strongest lectin-agarose interaction is also quite stable under a survey of different conditions such as high temperatures (up to 60 °C) or high organic solvent concentrations (up to 60% of acetonitrile). Notably, the use of cross-linked agarose would endow the system with more robustness due to its better mechanical properties compared to the noncross-linked one. The stability of the LSL(150)-agarose interaction would prevent protein leaching during the operation process unless high pH media are used. In summary, we believe that the LSL(150) lectin domain exhibits interesting structural features as an immobilization domain that makes it suitable to reversibly immobilize industrially relevant enzymes in very simple carriers as agarose.

Overexpression of the White Opaque Switching Master Regulator Wor1 Alters Lipid Metabolism and Mitochondrial Function in Candida albicans.

Candida albicans is a commensal yeast that inhabits the gastrointestinal tract of humans; increased colonization of this yeast in this niche has implicated the master regulator of the white-opaque transition, Wor1, by mechanisms not completely understood. We have addressed the role that this transcription factor has on commensalism by the characterization of strains overexpressing this gene. We show that WOR1 overexpression causes an alteration of the total lipid content of the fungal cell and significantly alters the composition of structural and reserve molecular species lipids as determined by lipidomic analysis. These cells are hypersensitive to membrane-disturbing agents such as SDS, have increased tolerance to azoles, an augmented number of peroxisomes, and increased phospholipase activity. WOR1 overexpression also decreases mitochondrial activity and results in altered susceptibility to certain oxidants. All together, these changes reflect drastic alterations in the cellular physiology that facilitate adaptation to the gastrointestinal tract environment.

Calmodulin-dependent KCNE4 dimerization controls membrane targeting.

The voltage-dependent potassium channel Kv1.3 participates in the immune response. Kv1.3 is essential in different cellular functions, such as proliferation, activation and apoptosis. Because aberrant expression of Kv1.3 is linked to autoimmune diseases, fine-tuning its function is crucial for leukocyte physiology. Regulatory KCNE subunits are expressed in the immune system, and KCNE4 specifically tightly regulates Kv1.3. KCNE4 modulates Kv1.3 currents slowing activation, accelerating inactivation and retaining the channel at the endoplasmic reticulum (ER), thereby altering its membrane localization. In addition, KCNE4 genomic variants are associated with immune pathologies. Therefore, an in-depth knowledge of KCNE4 function is extremely relevant for understanding immune system physiology. We demonstrate that KCNE4 dimerizes, which is unique among KCNE regulatory peptide family members. Furthermore, the juxtamembrane tetraleucine carboxyl-terminal domain of KCNE4 is a structural platform in which Kv1.3, Ca2+/calmodulin (CaM) and dimerizing KCNE4 compete for multiple interaction partners. CaM-dependent KCNE4 dimerization controls KCNE4 membrane targeting and modulates its interaction with Kv1.3. KCNE4, which is highly retained at the ER, contains an important ER retention motif near the tetraleucine motif. Upon escaping the ER in a CaM-dependent pattern, KCNE4 follows a COP-II-dependent forward trafficking mechanism. Therefore, CaM, an essential signaling molecule that controls the dimerization and membrane targeting of KCNE4, modulates the KCNE4-dependent regulation of Kv1.3, which in turn fine-tunes leukocyte physiology.

Endocannabinoids and cannabinoid analogues block human cardiac Kv4.3 channels in a receptor-independent manner.

Endocannabinoids are amides and esters of long chain fatty acids that can modulate ion channels through both receptor-dependent and receptor-independent effects. Nowadays, their effects on cardiac K(+) channels are unknown even when they can be synthesized within the heart. We have analyzed the direct effects of endocannabinoids, such as anandamide (AEA), 2-arachidonoylglycerol (2-AG), the endogenous lipid lysophosphatidylinositol, and cannabinoid analogues such as palmitoylethanolamide (PEA), and oleoylethanolamide, as well as the fatty acids from which they are endogenously synthesized, on human cardiac Kv4.3 channels, which generate the transient outward K(+) current (I(to1)). Currents were recorded in Chinese hamster ovary cells, which do not express cannabinoid receptors, by using the whole-cell patch-clamp. All these compounds inhibited I(Kv4.3) in a concentration-dependent manner, AEA and 2-AG being the most potent (IC(50) approximately 0.3-0.4 microM), while PEA was the least potent. The potency of block increased as the complexity and the number of C atoms in the fatty acyl chain increased. The effects were not mediated by modifications in the lipid order and microviscosity of the membrane and were independent of the presence of MiRP2 or DPP6 subunits in the channel complex. Indeed, effects produced by AEA were reproduced in human atrial I(to1) recorded in isolated myocytes. Moreover, AEA effects were exclusively apparent when it was applied to the external surface of the cell membrane. These results indicate that at low micromolar concentrations the endocannabinoids AEA and 2-AG directly block human cardiac Kv4.3 channels, which represent a novel molecular target for these compounds.

Characterization of the control catabolite protein of gluconeogenic genes repressor by fluorescence cross-correlation spectroscopy and other biophysical approaches.

Determination of the physical parameters underlying protein-DNA interactions is crucial for understanding the regulation of gene expression. In particular, knowledge of the stoichiometry of the complexes is a prerequisite to determining their energetics and functional molecular mechanisms. However, the experimental determination of protein-DNA complex stoichiometries remains challenging. We used fluorescence cross-correlation spectroscopy (FCCS) to investigate the interactions of the control catabolite protein of gluconeogenic genes, a key metabolic regulator in Gram-positive bacteria, with two oligonucleotides derived from its target operator sequences, gapB and pckA. According to our FCCS experiments, the stoichiometry of binding is twofold larger for the pckA target than for gapB. Correcting the FCCS data for protein self-association indicated that control catabolite protein of gluconeogenic genes forms dimeric complexes on the gapB target and tetrameric complexes on the pckA target. Analytical ultracentrifugation coupled with fluorescence anisotropy and hydrodynamic modeling allowed unambiguous confirmation of this result. The use of multiple complementary techniques to characterize these complexes should be employed wherever possible. However, there are cases in which analytical ultracentrifugation is precluded, due to protein stability, solubility, or availability, or, more obviously, when the studies are carried out in live cells. If information concerning the self-association of the protein is available, FCCS can be used for the direct and simultaneous determination of the affinity, cooperativity, and stoichiometry of protein-DNA complexes in a concentration range and conditions relevant to the regulation of these interactions.

Modulation of the atrial specific Kv1.5 channel by the n-3 polyunsaturated fatty acid, alpha-linolenic acid.

Epidemiological, clinical and experimental studies suggest that the cardioprotective effect of fish intake is mainly due to the antiarrhythmic properties of marine n-3 polyunsaturated fatty acids (PUFA), which modulate ion currents. Emerging evidences point to similar effects of alpha-linolenic acid (ALA), a vegetable n-3 PUFA, but much less is known about its effects on the specific cardiac ion channels. Using electrophysiology, protein biochemistry and fluorescence anisotropy measurements, we tested the effects of ALA on the atrial specific Kv1.5 channel. In stably transfected Ltk(-) cells, ALA blocked Kv1.5 channels in a time- and voltage-dependent manner with an IC(50) value of 3.7+/-0.3 microM. ALA at 2.5 microM inhibited the Kv1.5 current, shifted the midpoint of the activation curve by -8.8+/-4.3 mV (p<0.05), accelerated the activation kinetics of Kv1.5 due to a negative shift in its voltage dependency and slowed its deactivation process. Marine n-3 PUFA eicosapentaenoic and docosahexaenoic (EPA and DHA) acids, but not ALA, reduced the steady-state levels of Kv1.5 protein. DHA, but not ALA, increased the cell membrane order parameter. These results demonstrate that ALA directly blocks atria-specific Kv1.5 channels without modifying their expression or the bilayer order. Together, these effects suggest that the antiarrhythmic potential of diets enriched with plant-derived n-3 PUFA result, in part, from direct effects on cardiac ion channels.

Investigating transcriptional regulation by fluorescence spectroscopy, from traditional methods to state-of-the-art single-molecule approaches.

Gene expression regulation, in particular at the level of transcription, has been demonstrated to play a key role in the development of human diseases, including cancer, and in bacteria it is crucial for proliferation as well as for pathogenicity. Transcriptional regulation is based on complex networks of interactions, including those of the regulatory proteins with the operator DNAs, which are further modulated by ligands. Thus, understanding transcriptional regulation mechanisms requires a thorough analysis of the physical parameters underlying the interactions involved. Among the panoply of methods available, fluorescence spectroscopy-based approaches have been widely used for the assessment of the thermodynamics and structural dynamics of biomolecular interactions. Here we will discuss the application of three fluorescence spectroscopy methods--fluorescence anisotropy and fluorescence correlation and cross-correlation spectroscopy--for the investigation of protein-DNA, protein-protein, and protein-ligand interactions. The weaknesses and the strengths of each method will be highlighted on the basis of our experience in the analysis of the interactions of bacterial repressors implicated in transcriptional regulation in bacilli.

Study of rabbit erythrocytes membrane solubilization by sucrose monomyristate using laurdan and phasor analysis.

The study of surfactant and bio membranes interaction is particularly complex due to the diversity in lipid composition and the presence of proteins in natural membranes. Even more difficult is the study of this interaction in vivo since cellular damage may complicate the interpretation of the results, therefore for most of the studies in this field either artificial or model systems are used. One of the model system most used to study biomembranes are erythrocytes due to their relatively simple structure (they lack nuclei and organelles having only the plasma membrane), their convenient experimental manipulation and availability. In this context, we used rabbit erythrocytes as a model membrane and Laurdan (6-lauroyl-2-dimethylaminonaphthalene) as the fluorescent probe to study changes promoted in the membrane by the interaction with the sucrose monoester of myristic acid, ß-d-fructofuranosyl-6-O-myristoyl-α-d-glucopyranoside (MMS). Surfactant and erythrocytes interaction was studied by measuring hemoglobin release and the changes in water content in the membrane sensed by Laurdan. Using two-photon excitation, three types of measurements were performed: Generalized Polarization (analyzed as average GP values), Fluorescence Lifetime Imaging, FLIM (analyzed using phasor plots) and Spectral imaging (analyzed using spectral phasor). Our data indicate that at sublytical concentration of surfactant (20µM MMS), there is a decrease of about 35% in erythrocytes size, without changes in Laurdan lifetime or emission spectra. We also demonstrate that as hemolysis progress, Laurdan lifetime increased due to the decrease in hemoglobin (strong quencher of Laurdan emission) content inside the erythrocytes. Under these conditions, Laurdan spectral phasor analyses can extract the information on the water content in the membrane in the presence of hemoglobin. Our results indicate an increase in membrane fluidity in presence of MMS.

Dynamic cellular maps of molecular species: Application to drug-target interactions.

The design of living cell studies aimed at deciphering the mechanism of action of drugs targeting proteins with multiple functions, expressed in a wide range of concentrations and cellular locations, is a real challenge. We recently showed that the antitumor drug plitidepsin (APL) localizes sufficiently close to the elongation factor eEF1A2 so as to suggest the formation of drug-protein complexes in living cells. Here we present an extension of our previous micro-spectroscopy study, that combines Generalized Polarization (GP) images, with the phasor approach and fluorescence lifetime imaging microscopy (FLIM), using a 7-aminocoumarin drug analog (APL*) as fluorescence tracer. Using the proposed methodology, we were able to follow in real time the formation and relative distribution of two sets of APL-target complexes in live cells, revealing two distinct patterns of behavior for HeLa-wt and APL resistant HeLa-APL-R cells. The information obtained may complement and facilitate the design of new experiments and the global interpretation of the results obtained with other biochemical and cell biology methods, as well as possibly opening new avenues of study to decipher the mechanism of action of new drugs.

Enhanced Cytotoxic Activity of Mitochondrial Mechanical Effectors in Human Lung Carcinoma H520 Cells: Pharmaceutical Implications for Cancer Therapy.

Cancer cell mitochondria represent an attractive target for oncological treatment as they have unique hallmarks that differ from their healthy counterparts, as the presence of a stronger membrane potential that can be exploited to specifically accumulate cytotoxic cationic molecules. Here, we explore the selective cytotoxic effect of 10-N-nonyl acridine orange (NAO) on human lung carcinoma H520 cells and compare them with healthy human lung primary fibroblasts. NAO is a lipophilic and positively charged molecule that promotes mitochondrial membrane adhesion that eventually leads to apoptosis when incubated at high micromolar concentration. We found an enhanced cytotoxicity of NAO in H520 cancer cells. By means Fluorescence lifetime imaging microscopy (FLIM) we also confirmed the formation of H-dimeric aggregates originating from opposing adjacent membranes that interfere with the mitochondrial membrane structure. Based on our results, we suggest the mitochondrial membrane as a potential target in cancer therapy to mechanically control the cell proliferation of cancer cells.

Early events in the binding of the pPS10 replication protein RepA to single iteron and operator DNA sequences.

RepA protein, encoded in the Pseudomonas pPS10 replicon, is a stable dimer in solution (dRepA), acting as a self-repressor of repA transcription through binding to an inverted repeat operator. However, RepA monomers (mRepA) are required to initiate plasmid replication upon binding to four directly repeated DNA sequences (iterons). RepA is composed of two winged-helix (WH) domains: C-terminal WH2 is the main DNA-binding domain (DBD) for both target sequences, whereas N-terminal WH1 acts as dimerization interface in dRepA, but becomes a second DBD in mRepA. On the basis of CD spectroscopy, hydrodynamics, X-ray crystallography and model building studies, we proposed previously that the activation of RepA initiator implies a large structural change in WH1, coupled to protein monomerization and interdomain compaction. Here, we report novel features in the process. Binding curves of RepA to an iteron, followed by fluorescence anisotropy in solution and by surface plasmon resonance on immobilized DNA, exhibit the profiles characteristic of transitions between three states. In contrast, RepA-R93C, a monomeric activated mutant, exhibits a single binding transition. This suggests the presence of an intermediate species in the iteron-induced dissociation and structural transformation of RepA. High concentrations of bovine serum albumin or ovalbumin (macromolecular crowding) enhance RepA affinity for an iteron in solution and, in gel mobility-shift assays, result in the visualization of novel protein-DNA complexes. RepA-induced DNA bending requires the binding of two WH domains: either both WH2 in dimers (operator) or WH1 plus WH2 in monomers (iteron).

Structure and dynamics of lysozyme encapsulated in a silica sol-gel matrix.

Proteins entrapped in sol-gel matrices have been extensively studied during the last 15 years, showing that most of them can be encapsulated with retention of their native structure and functionality and with enhanced stability. However, relatively little is known about the structural and dynamical details of the biomolecule-matrix interactions. To achieve this goal, the model protein hen egg white lysozyme (HEWL) has been entrapped in sol-gel matrices prepared from tetraethyl orthosilicate through an alcohol-free sol-gel route, and the photophysical properties of its fluorescent tryptophans have been determined using both steady-state and time-resolved fluorescence techniques. By combining fluorescence spectra, quenching experiments, lifetimes, and time-resolved fluorescence anisotropy measurements, we have obtained information on the structure, dynamics, and solvation properties of the entrapped protein. Our results show that the environment of HEWL within the silica pore as well as its internal dynamics is similar to that in aqueous solution, except that the protein showed no or, depending on conditions, very much slower global motion but retained its internal angularly restricted (hindered) segmental rotation upon entrapment. The experiments carried out at different experimental conditions indicate that, below the isoelectric point of the protein, a strong electrostatic interaction is established between the protein molecule and the negatively charged sol-gel walls, which is ultimately responsible for the total arrest of the overall rotation of the protein, but without significant effect upon its segmental rotational relaxation. The electrostatic nature of the interaction is clearly established since either reducing the positive charge of the protein (by increasing the pH toward its isoelectric point) or increasing the ionic strength of the solution (shielding against the attractive interaction) leads to a situation in which the protein freely rotates within the matrix pore, albeit an order of magnitude more slowly than that in free solution under similar macroscopic solution conditions, and still retains its segmental rotational properties.

Translational and rotational motions of proteins in a protein crowded environment.

Fluorescence correlation spectroscopy (FCS) was used to measure the translational diffusion of labeled apomyoglobin (tracer) in concentrated solutions of ribonuclease A and human serum albumin (crowders), as a quantitative model system of protein diffusive motions in crowded physiological environments. The ratio of the diffusion coefficient of the tracer protein in the protein crowded solutions and its diffusion coefficient in aqueous solution has been interpreted in terms of local apparent viscosities, a molecular parameter characteristic for each tracer-crowder system. In all protein solutions studied in this work, local translational viscosity values were larger than the solution bulk viscosity, and larger than rotational viscosities estimated for apomyoglobin in the same crowding solutions. Here we propose a method to estimate local apparent viscosities for the tracer translational and rotational diffusion directly from the bulk viscosity of the concentrated protein solutions. As a result of this study, the identification of protein species and the study of hydrodynamic changes and interactions in model crowded protein solutions by means of FCS and time-resolved fluorescence depolarization techniques may be expected to be greatly simplified.

Conformational changes in human integrin alphaIIbbeta3 after platelet activation, monitored by FRET.

Integrin alpha(IIb)beta(3), an abundant heterodimeric receptor at the surface of blood platelets, binds adhesive proteins after platelet activation and plays a primary role in haemostasis. In solution, it has been observed mainly in two conformations: the bent and the extended forms. Based on X-ray crystallography, electron microscopy and immunochemical observations of full-length integrin ectodomains and intact integrins, it has been agreed that unactivated integrins are in the bent conformation, both isolated in solution and in living cells. However, consensus is yet to emerge on the bent or extended conformation of activated integrins and on their mechanism of activation (the switchblade, the deadbolt and the S-S reduction models), which require further experimental tests at the cell level to become established facts. Here, we tested the proposed structural rearrangements undergone by integrin alpha(IIb)beta(3) after cell activation, by using Förster-type fluorescence resonance energy transfer (FRET) and attached fluorescent labels to Fab fragments of monoclonal antibodies directed to the betaA domain of the beta(3) subunit (donor, Alexa488-P97 Fab) and to the Calf-2 domain of the alpha(IIb) subunit (acceptor, Cy3-M3 Fab or Cy3-M10 Fab). The FRET efficiencies observed after ADP or TRAP platelet activation changed less than 20% from the resting values, showing that the distance between the labeled Fab fragments changes only modestly after platelet activation by physiological agonists. This observation is consistent with a conformational model of the activated integrin in the cell less extended than in the switchblade model.

Two-Photon Fluorescence Anisotropy Imaging to Elucidate the Dynamics and the Stability of Immobilized Proteins.

Time/spatial-resolved fluorescence determines anisotropy values of supported-fluorescent proteins through different immobilization chemistries, evidencing some of the molecular mechanisms that drive the stabilization of proteins at the interfaces with solid surfaces. Fluorescence anisotropy imaging provides a normalized protein mobility parameter that serves as a guide to study the effect of different immobilization parameters (length and flexibility of the spacer arm and multivalency of the protein-support interaction) on the final stability of the supported proteins. Proteins in a more constrained environment correspond to the most thermostable ones, as was shown by thermal inactivation studies. This work contributes to explain the experimental evidence found with conventional methods based on observable measurements; thus this advanced characterization technique provides reliable molecular information about the immobilized proteins with sub-micrometer spatial resolution. Such information has been very useful for fabricating highly stable heterogeneous biocatalysts with high interest in industrial developments.