Olesya V. Stepanenko

Fluorescent Proteins as Biomarkers and Biosensors: Throwing Color Lights on Molecular and Cellular Processes

Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.

Modern fluorescent proteins: from chromophore formation to novel intracellular applications

The diverse biochemical and photophysical properties of fluorescent proteins (FPs) have enabled the generation of a growing palette of colors, providing unique opportunities for their use in a variety of modern biology applications. Modulation of these FP characteristics is achieved through diversity in both the structure of the chromophore as well as the contacts between the chromophore and the surrounding protein barrel. Here we review our current knowledge of blue, green, and red chromophore formation in permanently emitting FPs, photoactivatable FPs, and fluorescent timers. Progress in understanding the interplay between FP structure and function has allowed the engineering of FPs with many desirable features, and enabled recent advances in microscopy techniques such as super-resolution imaging of single molecules, imaging of protein dynamics, photochromic FRET, deep-tissue imaging, and multicolor two-photon microscopy in live animals.

Beta-Barrel Scaffold of Fluorescent Proteins: Folding, Stability and Role in Chromophore Formation

This review focuses on the current view of the interaction between the β-barrel scaffold of fluorescent proteins and their unique chromophore located in the internal helix. The chromophore originates from the polypeptide chain and its properties are influenced by the surrounding protein matrix of the β-barrel. On the other hand, it appears that a chromophore tightens the β-barrel scaffold and plays a crucial role in its stability. Furthermore, the presence of a mature chromophore causes hysteresis of protein unfolding and refolding. We survey studies measuring protein unfolding and refolding using traditional methods as well as new approaches, such as mechanical unfolding and reassembly of truncated fluorescent proteins. We also analyze models of fluorescent protein unfolding and refolding obtained through different approaches, and compare the results of protein folding in vitro to co-translational folding of a newly synthesized polypeptide chain.

Hydrophobic interactions and ionic networks play an important role in thermal stability and denaturation mechanism of the porcine odorant-binding protein.

Despite the fact that the porcine odorant‐binding protein (pOBP) possesses a single tryptophan residue (Trp 16) that is characterized by a high density microenvironment (80 atoms in a sphere with radius 7 Å) with only one polar group (Lys 120) and three bound water molecules, pOBP displayed a red shifted fluorescence emission spectrum (λmax = 340 nm). The protein unfolding in 5M GdnHCl was accompanied by the red shift of the fluorescence emission spectrum (λmax = 353 nm), by the increase of fluorescence quantum yield, and by the decrease of lifetime of the excited state (from 4.25 ns in native state to 3.15 ns in the presence of 5M GdnHCl). Taken together these data indicate the existence of an exciplex complex (Trp 16 with Lys 120 and/or with bound molecules of water) in the protein native state. Heat‐induced denaturation of pOBP resulted in significant red shifts of the fluorescence emission spectra: the value of the ratio (I320/I365) upon excitation at λex = 297 nm (parameter A) decreases from 1.07 to 0.64 passing from 60 to 85°C, and the calculated midpoint of transition was centered at 70°C. Interestingly, even at higher temperature, the values of the parameter A both in the absence and in the presence of GdnHCl did not coincide. This suggests that a portion of the protein structure is still preserved upon the temperature‐induced denaturation of the protein in the absence of GdnHCl. CD experiments performed on pOBP in the absence and in the presence of GdnHCl and at different temperatures were in agreement with the fluorescence results. In addition, the obtained experimental data were corroborated by the analysis of the 3D structure of pOBP which revealed the amino acid residues that contribute to the protein dynamics and stability. Finally, molecular dynamics simulation experiments pointed out the important role of ion pair interactions as well as the molecular motifs that are responsible for the high thermal stability of pOBP, and elucidated the reasons of the protein aggregation that occurred at high temperature. Proteins 2008.

Allosteric effects of chromophore interaction with dimeric near-infrared fluorescent proteins engineered from bacterial phytochromes.

Fluorescent proteins (FPs) engineered from bacterial phytochromes attract attention as probes for in vivo imaging due to their near-infrared (NIR) spectra and use of available in mammalian cells biliverdin (BV) as chromophore. We studied spectral properties of the iRFP670, iRFP682 and iRFP713 proteins and their mutants having Cys residues able to bind BV either in both PAS (Cys15) and GAF (Cys256) domains, in one of these domains, or without these Cys residues. We show that the absorption and fluorescence spectra and the chromophore binding depend on the location of the Cys residues. Compared with NIR FPs in which BV covalently binds to Cys15 or those that incorporate BV noncovalently, the proteins with BV covalently bound to Cys256 have blue-shifted spectra and higher quantum yield. In dimeric NIR FPs without Cys15, the covalent binding of BV to Сys256 in one monomer allosterically inhibits the covalent binding of BV to the other monomer, whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers. The NIR FPs with both Cys residues have the narrowest blue-shifted spectra and the highest quantum yield. Our analysis resulted in the iRFP713/Val256Cys protein with the highest brightness in mammalian cells among available NIR FPs.

Distinct Effects of Guanidine Thiocyanate on the Structure of Superfolder GFP

Having a high folding efficiency and a low tendency to aggregate, the superfolder GFP (sfGFP) offers a unique opportunity to study the folding of proteins that have a β-barrel topology. Here, we studied the unfolding–refolding of sfGFP that was induced by guanidine thiocyanate (GTC), which is a stronger denaturing agent than GdnHCl or urea. Structural perturbations of sfGFP were studied by spectroscopic methods (absorbance, fluorescence, and circular dichroism), by acrylamide quenching of tryptophan and green chromophore fluorescence, and by size-exclusion chromatography. Low concentrations of GTC (up to 0.1 M) induce subtle changes in the sfGFP structure. The pronounced changes in the visible absorption spectrum of sfGFP which are accompanied by a dramatic decrease in tryptophan and green chromophore fluorescence was recorded in the range 0–0.7 M GNC. These alterations of sfGFP characteristics that erroneously can be mixed up with appearance of intermediate state in fact have pure spectroscopic but not structural nature. Higher concentrations of GTC (from 0.7 to 1.7 M), induce a disruption of the sfGFP structure, that is manifested in simultaneous changes of all of the detected parameters. Full recovery of native properties of denaturated sfGFP was observed after denaturant removal. The refolding of sfGFP passes through the accumulation of the off-pathway intermediate state, in which inner alpha-helix and hence green chromophore and Trp57 are still not tuned up to (properly integrated into) the already formed β-barrel scaffold of protein. Incorporation of the chromophore in the β-barrel in the pathway of refolding and restoration of its ability to fluoresce occur in a narrow range of GTC concentrations from 1.0 to 0.7 M, and a correct insertion of Trp 57 occurs at concentrations ranging from 0.7 to 0 M GTC. These two processes determine the hysteresis of protein unfolding and refolding.

New insight in protein-ligand interactions. 2. Stability and properties of two mutant forms of the d-galactose/D-glucose-binding protein from E. coli

The galactose/glucose-binding protein from E. coli (GGBP) is a 32 kDa protein possessing the typical two-domains structure of the ligand-binding proteins family. GGBP is characterized by low dissociation constant values with respect to glucose binding, displaying an affinity constant for glucose in micromolar range. This feature makes GGBP unsuitable as a sensitive probe for continuous glucose monitoring in blood of diabetic patients. In this work we designed, produced, and characterized two mutant forms of GGBP carrying the following amino acid substitutions in the active center of the protein: W183A or F16A. The two mutant GGBP forms retained a globular structure similar to that of the wild-type GGBP and displayed an affinity for glucose lower than the wild-type GGBP. A deep inspection of the entire set of the obtained results pointed out that the N- and C-terminal domains of GGBP-W183A in the absence of glucose have a stability lower than that of the wild-type protein. In the presence of glucose, the two domains of GGBP-W183A were tightly bound, making the protein structure more stable to the action of denaturing agents. On the contrary, the mutant form GGBP-F16A possesses a very restricted structural stability both in the absence and in the presence of glucose. In this work the role of Phe 16 and W 183 are discussed with regard to the structural and functional features of GGBP. In addition, some general guidelines are reported for the design of a novel glucose biosensor based on the use of GGBP.

A knot in the protein structure - probing the near-infrared fluorescent protein iRFP designed from a bacterial phytochrome.

The possibility of engineering near‐infrared fluorescent proteins and biosensors from bacterial phytochrome photoreceptors (BphPs) has led to substantial interest in this family of proteins. The near‐infrared fluorescent proteins have allowed non‐invasive bio‐imaging of deep tissues and whole organs in living animals. BphPs and derived near‐infrared fluorescent proteins contain a structural element, called a knot, in their polypeptide chains. The formation of knot structures in proteins was refuted for a long time. Here, we studied the denaturation and renaturation processes of the near‐infrared fluorescent probe iRFP, engineered from RpBphP2, which utilizes a heme‐derived tetrapyrrole compound biliverdin as a chromophore. iRFP contains a unique figure‐of‐eight knot. The denaturation and renaturation curves of the iRFP apoform coincided well, suggesting efficient refolding. However, the iRFP holoform exhibited irreversible unfolding and aggregation associated with the bound chromophore. The knot structure in the apoform did not prevent subsequent binding of biliverdin, resulting in the functional iRFP holoform. We suggest that the irreversibility of protein unfolding is caused by post‐translational protein modifications, such as chromophore binding, rather than the presence of the knot. These results are essential for future design of BphP‐based near‐infrared probes, and add important features to our knowledge of protein folding.

New Insight into Protein−Ligand Interactions. The Case of the d-Galactose/d-Glucose-Binding Protein from Escherichia coli

In this work we have shown that the unfolding-refolding process of the D-galactose/D-glucose-binding protein (GGBP) in the presence of glucose (Glc) induced by the chemical denaturant Gdn-HCI is reversible. In addition, Glc binding does not only stabilize GGBP structure but it also considerably slows down the achievement of the equilibrium between the native protein in GGBP/Glc complex and the unfolded protein. The limiting step of the unfolding-refolding process of the complex GGBP/Glc is the arrangement/de-arrangement of the configuration fit between the protein in the native state and the ligand. The rate of these processes increases/decreases with the increase/decrease of the denaturant concentration. Calcium depletion had a pronounced destabilizing effect on the structure of GGBP but did not affect the stability of GGBP/Glc complex. Unfolding of GGBP/Ca complex is reversible. Only incubation of the unfolded protein at high temperature leads to an irreversible process due to the aggregation of the protein. The amount of protein aggregation is determined by the protein concentration, the temperature and the duration of the incubation.

Understanding the role of Arg96 in structure and stability of green fluorescent protein

Arg96 is a highly conservative residue known to catalyze spontaneous green fluorescent protein (GFP) chromophore biosynthesis. To understand a role of Arg96 in conformational stability and structural behavior of EGFP, the properties of a series of the EGFP mutants bearing substitutions at this position were studied using circular dichroism, steady state fluorescence spectroscopy, fluorescence lifetime, kinetics and equilibrium unfolding analysis, and acrylamide‐induced fluorescence quenching. During the protein production and purification, high yield was achieved for EGFP/Arg96Cys variant, whereas EGFP/Arg96Ser and EGFP/Arg96Ala were characterized by essentially lower yields and no protein was produced when Arg96 was substituted by Gly. We have also shown that only EGFP/Arg96Cys possessed relatively fast chromophore maturation, whereas it took EGFP/Arg96Ser and EGFP/Arg96Ala about a year to develop a noticeable green fluorescence. The intensity of the characteristic green fluorescence measured for the EGFP/Arg96Cys and EGFP/Arg96Ser (or EGFP/Arg96Ala) was 5‐ and 50‐times lower than that of the nonmodified EGFP. Intriguingly, EGFP/Arg96Cys was shown to be more stable than EGFP toward the GdmCl‐induced unfolding both in kinetics and in the quasi‐equilibrium experiments. In comparison with EGFP, tryptophan residues of EGFP/Arg96Cys were more accessible to the solvent. These data taken together suggest that besides established earlier crucial catalytic role, Arg96 is important for the overall folding and conformational stability of GFP. Proteins 2008.