Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein‐based biosensors for Ca2+ and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)‐based biosensors by providing a means for varying the critical dipole–dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th β‐strand of mCherry. These new variants may ultimately prove useful for the creation of single FP‐based Ca2+ biosensors. 相似文献
Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue‐shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP‐labeled nucleus and tdTomato‐labeled plasma membrane, time‐lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two‐color protein labeling in living cells and in two‐color tumor labeling in mice. 相似文献
An Escherichia coli hygromycin B phosphotransferase (HPH) and its thermostabilized mutant protein, HPH5, containing five amino acid substitutions, D20G, A118V, S225P, Q226L, and T246A (Nakamura et al., J. Biosci. Bioeng., 100, 158–163 (2005)), obtained by an in vivo directed evolution procedure in Thermus thermophilus, were produced and purified from E. coli recombinants, and enzymatic comparisons were performed. The optimum temperatures for enzyme activity were 50 and 55 °C for HPH and HPH5 respectively, but the thermal stability of the enzyme activity and the temperature for protein denaturation of HPH5 increased, from 36 and 37.2 °C of HPH to 53 and 58.8 °C respectively. Specific activities and steady-state kinetics measured at 25 °C showed only slight differences between the two enzymes. From these results we concluded that HPH5 was thermostabilized at the protein level, and that the mutations introduced did not affect its enzyme activity, at least under the assay conditions. 相似文献
We present one‐ and two‐photon‐absorption fluorescence spectroscopic analysis of biliverdin (BV) chromophore–based single‐domain near‐infrared fluorescent proteins (iRFPs). The results of these studies are used to estimate the internal electric fields acting on BV inside iRFPs and quantify the electric dipole properties of this chromophore, defining the red shift of excitation and emission spectra of BV‐based iRFPs. The iRFP studied in this work is shown to fit well the global diagram of the red‐shift tunability of currently available BV‐based iRFPs as dictated by the quadratic Stark effect, suggesting the existence of the lower bound for the strongest red shifts attainable within this family of fluorescent proteins. The absolute value of the two‐photon absorption (TPA) cross section of a fluorescent calcium sensor based on the studied iRFP is found to be significantly larger than the TPA cross sections of other widely used genetically encodable fluorescent calcium sensors. 相似文献
Discoveries of major importance in life sciences and preclinical research are linked to the invention of microscopes that enable imaging of cells and their microstructures. Imaging technologies involving in vivo procedures using fluorescent dyes that permit labelling of cells have been developed over the last two decades. Fibered confocal fluorescence microscopy (FCFM) is an imaging technology equipped with fiber‐optic probes to deliver light to organs and tissues of live animals. This enables not only in vivo detection of fluorescent signals and visualization of cells, but also the study of dynamic processes, such cell proliferation, apoptosis and angiogenesis, under physiological and pathological conditions. This will allow the diagnosis of diseased organs and tissues and the evaluation of the efficacy of new therapies in animal models of human diseases. The aim of this report is to shed light on FCFM and its potential medical applications and discusses some factors that compromise the reliability and reproducibility of monitoring biological processes by FCFM. This report also highlights the issues concerning animal experimentation and welfare, and the contributions of FCFM to the 3Rs principals, replacement, reduction and refinement. 相似文献
The potential use of proteins in device applications has advanced in large part due to significant advances in the methods
and procedures of protein engineering, most notably, directed evolution. Directed evolution has been used to tailor a broad
range of enzymatic proteins for pharmaceutical and industrial applications. Thermal stability, chemical stability, and substrate
specificity are among the most common phenotypes targeted for optimization. However, in vivo screening systems for photoactive
proteins have been slow in development. A high-throughput screening system for the photokinetic optimization of photoactive
proteins would promote the development of protein-based field-effect transistors, artificial retinas, spatial light modulators,
photovoltaic fuel cells, three-dimensional volumetric memories, and optical holographic processors. This investigation seeks
to optimize the photoactive protein bacteriorhodopsin (BR) for volumetric optical and holographic memories. Semi-random mutagenesis
and in vitro screening were used to create and analyze nearly 800 mutants spanning the entire length of the bacterio-opsin (bop) gene. To fully realize the potential of BR in optoelectronic environments, future investigations will utilize global mutagenesis
and in vivo screening systems. The architecture for a potential in vivo screening system is explored in this study. We demonstrate
the ability to measure the formation and decay of the red-shifted O-state within in vivo colonies of Halobacterium salinarum, and discuss the implications of this screening method to directed evolution.
These authors contributed equally to this work. 相似文献
Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (Tm) of 77.3 ± 0.1°C (4.6°C higher than the wild-type) and a half-life of over 13 hr at 65°C (7.9-fold better than the wild-type), with unchanged kinetic parameters. Stabilizing mutations Q11L and A191S were incorporated into PFEI variant L30P, previously described to be enantioselective in the hydrolysis of the (−)-enantiomer of the Vince lactam. The final variant Q11L/L30P/A191S showed a significant improvement in thermal stability (Tm of 80.8 ± 0.1°C and a half-life of 65 min at 75°C), while retaining enantioselectivity (E > 100). Structural studies revealed that A191S establishes a hydrogen bond network between a V-shaped hairpin and the α/β hydrolase domain that leads to higher rigidity and thus would contribute to explaining the increase in stability. 相似文献
Molecular biosensors are devices of molecular size that are designed for sensing different analytes on the basis of biospecific recognition. They should provide two coupled functions - the recognition (specific binding) of the target and the transduction of information about the recognition event into a measurable signal. The present review highlights the achievements and prospects in design and operation of molecular biosensors for which the transduction mechanism is based on fluorescence. We focus on the general strategy of fluorescent molecular sensing, construction of sensor elements, based on natural and designed biopolymers (proteins and nucleic acids). Particular attention is given to the coupling of sensing elements with fluorescent reporter dyes and to the methods for producing efficient fluorescence responses. 相似文献
The linker histone H1 has a fundamental role in DNA compaction. Although models for H1 binding generally involve the H1 C‐terminal tail and sites S1 and S2 within the H1 globular domain, there is debate about the importance of these binding regions and almost nothing is known about how they work together. Using a novel fluorescence recovery after photobleaching (FRAP) procedure, we have measured the affinities of these regions individually, in pairs, and in the full molecule to demonstrate for the first time that binding among several combinations is cooperative in live cells. Our analysis reveals two preferred H1 binding pathways and we find evidence for a novel conformational change required by both. These results paint a complex, highly dynamic picture of H1–chromatin binding, with a significant fraction of H1 molecules only partially bound in metastable states that can be readily competed against. We anticipate the methods we have developed here will be broadly applicable, particularly for deciphering the binding kinetics of other nuclear proteins that, similar to H1, interact with and modify chromatin. 相似文献
Cell death plays a critical role in health and homeostasis as well as in the pathogenesis and treatment of a broad spectrum of diseases and can be broadly divided into two main categories: apoptosis, or programmed cell death, and necrosis, or acute cell death. While these processes have been characterized extensively in vitro, label‐free detection of apoptosis and necrosis at the cellular level in vivo has yet to be shown. In this study, for the first time, fluorescence lifetime imaging microscopy (FLIM) of intracellular reduced nicotinamide adenine dinucleotide (NADH) was utilized to assess the metabolic response of in vivo mouse epidermal keratinocytes following induction of apoptosis and necrosis. Results show significantly elevated levels of both the mean lifetime of NADH and the intracellular ratio of protein bound‐to‐free NADH in the apoptotic compared to the necrotic tissue. In addition, the longitudinal profiles of these two cell death processes show remarkable differences. By identifying and extracting these temporal metabolic signatures, apoptosis in single cells can be studied in native tissue environments within the living organism.
