Unfolding of Green Fluorescent Protein mut2 in wet nanoporous silica gels

Many of the effects exerted on protein structure, stability, and dynamics by molecular crowding and confinement in the cellular environment can be mimicked by encapsulation in polymeric matrices. We have compared the stability and unfolding kinetics of a highly fluorescent mutant of Green Fluorescent Protein, GFPmut2, in solution and in wet, nanoporous silica gels. In the absence of denaturant, encapsulation does not induce any observable change in the circular dichroism and fluorescence emission spectra of GFPmut2. In solution, the unfolding induced by guanidinium chloride is well described by a thermodynamic and kinetic two-state process. In the gel, biphasic unfolding kinetics reveal that at least two alternative conformations of the native protein are significantly populated. The relative rates for the unfolding of each conformer differ by almost two orders of magnitude. The slower rate, once extrapolated to native solvent conditions, superimposes to that of the single unfolding phase observed in solution. Differences in the dependence on denaturant concentration are consistent with restrictions opposed by the gel to possibly expanded transition states and to the conformational entropy of the denatured ensemble. The observed behavior highlights the significance of investigating protein function and stability in different environments to uncover structural and dynamic properties that can escape detection in dilute solution, but might be relevant for proteins in vivo.     

Tracking unfolding and refolding of single GFPmut2 molecules

The unfolding and refolding kinetics of >600 single GFPmut2 molecules, entrapped in wet nanoporous silica gels, were followed by monitoring simultaneously the fluorescence emission of the anionic and neutral state of the chromophore, primed by two-photon excitation. The rate of unfolding, induced by guanidinium chloride, was determined by counting the number of single molecules that disappear in fluorescence images, under conditions that do not cause bleaching or photoinduced conversion between chromophore protonation states. The unfolding rate is of the order of 0.01 min(-1), and its dependence on denaturant concentration is very similar to that previously reported for high protein load gels. Upon rinsing the gels with denaturant-free buffer, the GFPmut2 molecules refold with rates >10 min(-1), with an apparently random distribution between neutral and anionic states, that can be very different from the preunfolding equilibrium. A subsequent very slow (lifetime of approximately 70 min) relaxation leads to the equilibrium distribution of the protonation states. This mechanism, involving one or more native-like refolding intermediates, is likely rate limited by conformational rearrangements that are undetectable in circular dichroism experiments. Several unfolding/refolding cycles can be followed on the same molecules, indicating full reversibility of the process and, noticeably, a bias of denaturated molecules toward refolding in the original protonation state.     

Role of histidine 148 in stability and dynamics of a highly fluorescent GFP variant

The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pK_a values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein.     

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.     

Fluorescence labeling, purification, and immobilization of a double cysteine mutant calmodulin fusion protein for single-molecule experiments

We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring of protein dynamics. The small size of CaM hinders its immobilization in low-weight-percentage agarose gels; however, fusion of CaM to MBP via a flexible linker provides sufficient restriction of translational mobility in 1% agarose gels. Cysteine residues were engineered into MBP.CaM (MBP-T34C,T110C-CaM) and labeled with donor and acceptor fluorescent probes yielding a construct (MBP.CaM-DA) which can be used for single-molecule single-pair fluorescence resonance energy transfer (spFRET) experiments. Mass spectrometry was used to verify the mass of MBP.CaM-DA. Assays measuring the activity of CaM reveal minimal activity differences between wild-type CaM and MBP.CaM-DA. Single-molecule fluorescence images of the donor and acceptor dyes were fit to a two-dimensional Gaussian function to demonstrate colocalization of donor and acceptor dyes. FRET is demonstrated both in bulk fluorescence spectra and in fluorescence trajectories of single MBP.CaM-DA molecules. The extension of this method to other biomolecules is also proposed.     

Structure and dynamics of proteins encapsulated in silica hydrogels by Trp phosphorescence

This report establishes the conditions for monitoring the intrinsic Trp phosphorescence of proteins encapsulated in silica hydrogels and demonstrates the usefulness of the delayed emission for examining potential perturbations of protein structure-dynamics by the silica matrix. Phosphorescence measurements were conducted both in low temperature (140 K) glasses and at ambient temperature on the proteins apo- and Cd-azurin, alkaline phosphatase and liver alcohol dehydrogenase together with the complexes of liver alcohol dehydrogenase with coenzyme analogs ADPR and H(2)NADH. While spectral shifts and broadening indicate that alterations of the Trp microenvironment are more marked on superficial regions of the macromolecule the decay kinetics of deeply buried chromophores show that the internal flexibility of the polypeptide in two out of three cases is significantly affected by silica entrapment. Both the intrinsic lifetime and the bimolecular acrylamide quenching constant confirm that, relative to the aqueous solution, in hydrogels the globular fold is more rigid with azurin, looser with alcohol dehydrogenase and substantially unaltered with alkaline phosphatase. It was also noted that large amplitude structural fluctuations, as those involved in coenzyme binding to alcohol dehydrogenase or thermally activated in alkaline phosphatase, were not restricted by gelation. Common features of the three silica entrapped proteins are pronounced conformational heterogeneity and immobilization of rotational motions of the macromolecule in the long time scale of seconds.     

Tyrosine phenol-lyase and tryptophan indole-lyase encapsulated in wet nanoporous silica gels: Selective stabilization of tertiary conformations

The pyridoxal 5'-phosphate-dependent enzymes tyrosine phenol-lyase and tryptophan indole-lyase were encapsulated in wet nanoporous silica gels, a powerful method to selectively stabilize tertiary and quaternary protein conformations and to develop bioreactors and biosensors. A comparison of the enzyme reactivity in silica gels and in solution was carried out by determining equilibrium and kinetic parameters, exploiting the distinct spectral properties of catalytic intermediates and reaction products. The encapsulated enzymes exhibit altered distributions of ketoenamine and enolimine tautomers, increased values of inhibitors dissociation constants, slow attaining of steady-state in the presence of substrate and substrate analogs, modified steady-state distribution of catalytic intermediates, and a sixfold-eightfold decrease of specific activities. This behavior can be rationalized by a reduced conformational flexibility for the encapsulated enzymes and a selective stabilization of either the open (inactive) or the closed (active) form of the enzymes. Despite very similar structures and catalytic mechanisms, the influence of encapsulation is more pronounced for tyrosine phenol-lyase than tryptophan indole-lyase. This finding indicates that subtle structural and dynamic differences can lead to distinct interactions of the protein with the gel matrix.     

Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles

Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.     

Evidence of discrete substates and unfolding pathways in green fluorescent protein

We present evidence of conformational substates of a green fluorescent protein mutant, GFPmut2, and of their relationship with the protein behavior during chemical unfolding. The fluorescence of single molecules, excited by two infrared photons from a pulsed laser, was detected in two separate channels that simultaneously collected the blue or the green emission from the protein chromophore chemical states (anionic or neutral, respectively). Time recording of the fluorescence signals from molecules in the native state shows that the chromophore, an intrinsic probe sensitive to conformational changes, switches between the two states with average rates that are found to assume distinct values, thereby suggesting a multiplicity of protein substates. Furthermore, under denaturing conditions, the chromophore switching rate displays different and reproducible time evolutions that are characterized by discrete unfolding times. The correlation that is found between native molecules' switching rate values and unfolding times appears as direct evidence that GFPmut2 can unfold only along distinct paths that are determined by the initial folded substate of the protein.     

Purification of green fluorescent protein using a two-intein system

A two-intein purification system was developed for the affinity purification of GFPmut3*, a mutant of green fluorescent protein. The GFPmut3* was sandwiched between two self-cleaving inteins. This approach avoided the loss of the target protein which may result from in vivo cleavage of a single intein tag. The presence of N- and C-terminal chitin-binding domains allowed the affinity purification by a single-affinity chitin column. After the fusion protein was expressed and immobilized on the affinity column, self-cleavage of the inteins was sequentially induced to release the GFPmut3*. The yield was 2.41 mg from 1 l of bacterial culture. Assays revealed that the purity was up to 98% of the total protein. The fluorescence and circular dichroism spectrum of GFPmut3* demonstrated that the purified protein retains the correctly folded structure and function.     

Analysis of time-resolved fluorescence anisotropy in lipid-protein systems

The subnanosecond fluorescence and motional dynamics of the tryptophan residue in the bacteriophage M13 coat protein incorporated within pure dioleoylphosphatidylcholine (DOPC) as well as dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) and dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bilayers (80/20 w/w) with various L/P ratio have been investigated. The fluorescence decay is decomposed into four components with lifetimes of about 0.5, 2.0, 4.5 and 10.0 ns, respectively. In pure DOPC and DOPC/DOPG lipid bilayers, above the phase transition temperature, the rotational diffusion of the protein molecules contributes to the depolarization and the anisotropy of tryptophan is fitted to a dual exponential function. The longer correlation time, describing the rotational diffusion of the whole protein, shortens with increasing temperature and decreasing protein aggregation number. In DMPC/DMPG lipid bilayers, below the phase transition, the rotational diffusion of the protein is slowed down such that the subnanosecond anisotropy decay of tryptophan in this system reflects only the segmental motion of the tryptophan residue. Because of a heterogeneous microenvironment, the anisotropy decay must be described by three exponentials with a constant term, containing a negative coefficient and a negative decay time constant. From such a decay, the tryptophan residue within the aggregate undergoes a more restricted motion than the one exposed to the lipids. At 20 degrees C, the order parameter of the transition moment of the isolated tryptophan is about 0.9 and that for the exposed one is about 0.5.     

Structural stability of green fluorescent proteins entrapped in polyelectrolyte nanocapsules

Molecules of a green fluorescent protein mutant, GFPmut2, have been immobilized in nanocapsules, assemblies of charged polyelectrolyte multilayers, with the aim to study the effect of protein‐polyelectrolyte interactions on the protein stability against chemical denaturation. GFPmut2 proteins turn out to be stabilized and protected against the denaturating action of small chemical compounds. The nanocapsule protective effect on GFPmut2 is likely due to protein interactions with the negatively charged polymers, that induce an increase in the local rigidity of the protein nano‐environment. This suggestion is supported by Fluorescence Polarization measurements on GFPmut2 proteins bound to the NC layers. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Physical properties of the fluorescent sterol probe dehydroergosterol

Spectroscopic studies were performed on the fluorescent sterol probes ergosta-5,7,9(11),22-tetraen-3 beta-ol (dehydroergosterol) and cholesta-5,7,9(11)-trien-3 beta-ol (cholestatrienol). In most isotropic solvents, these molecules exhibited a single lifetime near 300 ps. Fluorescence lifetimes in 2-propanol were independent of emission wavelength and independent of excitation wavelength. Excited state behavior of these probes appears relatively simple. In isotropic solvents, dehydroergosterol fluorescence emission underwent at most a small Stokes shift as solvent polarity was modified. Time-resolved anisotropy decays indicated that dehydroergosterol decay was monoexponential, with rotational correlation times dependent on solvent viscosity. When incorporated into L-alpha-dimyristoylphosphatidylcholine liposomes at a concentration of 0.9 mol%, dehydroergosterol fluorescence lifetime decreased at the phase transition of this phospholipid indicating that the sterol probe was detecting physical changes of the bulk phospholipids. Furthermore, total fluorescence decays and anisotropy decays were sensitive to the environment of the sterol. Dehydroergosterol and cholestatrienol are thus useful probes for monitoring sterol behavior in biological systems.     

Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo H NMR and fluorescence spectroscopy

This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo ¹H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by ∼5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1 ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.     

Hyper-mobility of water around actin filaments revealed using pulse-field gradient spin-echo 1H NMR and fluorescence spectroscopy

This paper reports that water molecules around F-actin, a polymerized form of actin, are more mobile than those around G-actin or in bulk water. A measurement using pulse-field gradient spin-echo (1)H NMR showed that the self-diffusion coefficient of water in aqueous F-actin solution increased with actin concentration by ～5%, whereas that in G-actin solution was close to that of pure water. This indicates that an F-actin/water interaction is responsible for the high self-diffusion of water. The local viscosity around actin was also investigated by fluorescence measurements of Cy3, a fluorescent dye, conjugated to Cys 374 of actin. The steady-state fluorescence anisotropy of Cy3 attached to F-actin was 0.270, which was lower than that for G-actin, 0.334. Taking into account the fluorescence lifetimes of the Cy3 bound to actin, their rotational correlation times were estimated to be 3.8 and 9.1ns for F- and G-actin, respectively. This indicates that Cy3 bound to F-actin rotates more freely than that bound to G-actin, and therefore the local water viscosity is lower around F-actin than around G-actin.     

Dynamic fluorescence properties of bacterial luciferase intermediates

Three fluorescent species produced by the reaction of bacterial luciferase from Vibrio harveyi with its substrates have the same dynamic fluorescence properties, namely, a dominant fluorescence decay of lifetime of 10 ns and a rotational correlation time of 100 ns at 2 degrees C. These three species are the metastable intermediate formed with the two substrates FMNH2 and O2, both in its low-fluorescence form and in its high-fluorescence form following light irradiation, and the fluorescent transient formed on including the final substrate tetradecanal. For native luciferase, the rotational correlation time is 62 or 74 ns (2 degrees C) derived from the decay of the anisotropy of the intrinsic fluorescence at 340 nm or the fluorescence of bound 8-anilino-1-naphthalenesulfonic acid (470 nm), respectively. The steady-state anisotropy of the fluorescent intermediates is 0.34, and the fundamental anisotropy from a Perrin plot is 0.385. The high-fluorescence intermediate has a fluorescence maximum at 500 nm, and its emission spectrum is distinct from the bioluminescence spectrum. The fluorescence quantum yield is 0.3 but decreases on dilution with a quadratic dependence on protein concentration. This, and the large value of the rotational correlation time, would be explained by protein complex formation in the fluorescent intermediate states, but no increase in protein molecular weight is observed by gel filtration or ultracentrifugation. The results instead favor a proposal that, in these intermediate states, the luciferase undergoes a conformational change in which its axial ratio increases by 50%.     

Intrasequence GFP in class I MHC molecules,a rigid probe for fluorescence anisotropy measurements of the membrane environment

Fluorescence anisotropy measurements can elucidate the microenvironment of a membrane protein in terms of its rotational diffusion, interactions, and proximity to other proteins. However, use of this approach requires a fluorescent probe that is rigidly attached to the protein of interest. Here we describe the use of one such probe, a green fluorescent protein (GFP) expressed and rigidly held within the amino acid sequence of a major histocompatibility complex (MHC) class I molecule, H2L(d). We contrast the anisotropy of this GFP-tagged MHC molecule, H2L(d)GFPout, with that of an H2L(d) that was GFP-tagged at its C-terminus, H2L(d)GFPin. Both molecules fold properly, reach the cell surface, and are recognized by specific antibodies and T-cell receptors. We found that polarized fluorescence images of H2L(d)GFPout in plasma membrane blebs show intensity variations that depend on the relative orientation of the polarizers and the membrane normal, thus demonstrating that the GFP is oriented with respect to the membrane. These variations were not seen for H2L(d)GFPin. Before transport to the membrane surface, MHC class I associates with the transporter associated with antigen processing complex in the endoplasmic reticulum. The intensity-dependent steady-state anisotropy in the ER of H2L(d)GFPout was consistent with FRET homotransfer, which indicates that a significant fraction of these molecules were clustered. After MCMV-peptide loading, which supplies antigenic peptide to the MHC class I releasing it from the antigen processing complex, the anisotropy of H2L(d)GFPout was independent of intensity, suggesting that the MHC proteins were no longer clustered. These results demonstrate the feasibility and usefulness of a GFP moiety rigidly attached to the protein of interest as a probe for molecular motion and proximity in cell membranes.     

Probing the Ca(2+) switch of the neuronal Ca(2+) sensor GCAP2 by time-resolved fluorescence spectroscopy

We report fluorescence lifetime and rotational anisotropy measurements of the fluorescent dye Alexa647 attached to the guanylate cyclase-activating protein 2 (GCAP2), an intracellular myristoylated calcium sensor protein operating in photoreceptor cells. By linking the dye to different protein regions critical for monitoring calcium-induced conformational changes, we could measure fluorescence lifetimes and rotational correlation times as a function of myristoylation, calcium, and position of the attached dye, while GCAP2 was still able to regulate guanylate cyclase in a Ca(2+)-sensitive manner. We observe distinct site-specific variations in the fluorescence dynamics when externally changing the protein conformation. A clear reduction in fluorescence lifetime suggests that in the calcium-free state a dye marker in amino acid position 131 senses a more hydrophobic protein environment than in position 111. Saturating GCAP2 with calcium increases the fluorescence lifetime and hence leads to larger exposure of position 111 to the solvent and at the same time to a movement of position 131 into a hydrophobic protein cleft. In addition, we find distinct, biexponential anisotropy decays reflecting the reorientational motion of the fluorophore dipole and the dye/protein complex, respectively. Our experimental data are well described by a "wobbling-in-a-cone" model and reveal that for dye markers in position 111 of the GCAP2 protein both addition of calcium and myristoylation results in a pronounced increase in orientational flexibility of the fluorophore. Our results provide evidence that the up-and-down movement of an α-helix that is situated between position 111 and 131 is a key feature of the dynamics of the protein-dye complex. Operation of this piston-like movement is triggered by the intracellular messenger calcium.     

The effects of Ca2+ on lipid diffusion

The effects of Ca2+ on rotational and translational diffusion of lipids in multilamellar dimyristoylphosphatidylcholine (DMPC)-water systems were investigated by time-resolved phosphorescence anisotropy steady-state fluorescence polarization and fluorescence recovery after photobleaching (FRAP) experiments. Above the phase transition temperature (Tm), addition of Ca2+ caused a steady increase in the segmental motion of the phosphorescent probe, but resulted in slower diffusion of the fluorescent and lateral diffusion probes. The former result is attributed to changes in the structure of the lipid/water interface that affects the chromophore mobility on the phosphorescence time scale but does not reflect lipid motion. Below the phase transition temperature, slower diffusion of all probes were observed with increasing concentrations of Ca2+, with sudden large changes occurring at [Ca2+] approximately 500 mM. This behaviour is attributed to association of Ca2+ with the lipid phosphate groups and the exclusion of water molecules which results in tighter packing of lipids and smaller segmental motion, leading eventually to the immobilization of lipid molecules.