Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors

Fluorescence resonance energy transfer (FRET)-based molecular biosensors serve as important tools for studying protein activity in live cells and have been widely used for this purpose over the past decade. However, FRET biosensors are rarely used in the context of the live organism because of the inherent high cellular complexity and imaging challenges associated with the three-dimensional environment. Here we provide a protocol for using single-chain intramolecular FRET-based biosensors in early development. We provide a general protocol for FRET ratio imaging in embryos, including the data-acquisition conditions and the algorithm for ratio image generation. We then use the pRaichu RacFRET biosensor to exemplify the adaptation and optimization of a particular biosensor for use in live zebrafish embryos. Once an optimized biosensor is available, the complete procedure, including introduction of the probes into embryos, imaging and data analysis, requires 2-3 d.     

Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics

Genetically-coded, fluorescence resonance energy transfer (FRET) biosensors are widely used to study molecular events from single cells to whole organisms. They are unique among biosensors because of their spontaneous fluorescence and targeting specificity to both organelles and tissues. In this review, we discuss the theoretical basis of FRET with a focus on key parameters responsible for designing FRET biosensors that have the highest sensitivity. Next, we discuss recent applications that are grouped into four common biosensor design patterns—intermolecular FRET, intramolecular FRET, FRET from substrate cleavage and FRET using multiple colour fluorescent proteins. Lastly, we discuss recent progress in creating fluorescent proteins suitable for FRET purposes. Together these advances in the development of FRET biosensors are beginning to unravel the interconnected and intricate signalling processes as they are occurring in living cells and organisms.     

A further insight into the biosorption mechanism of Au(III) by infrared spectrometry

Background

Fluorescent protein (FP)-based biosensors based on the principle of intramolecular Förster resonance energy transfer (FRET) enable the visualization of a variety of biochemical events in living cells. The construction of these biosensors requires the genetic insertion of a judiciously chosen molecular recognition element between two distinct hues of FP. When the molecular recognition element interacts with the analyte of interest and undergoes a conformational change, the ratiometric emission of the construct is altered due to a change in the FRET efficiency. The sensitivity of such biosensors is proportional to the change in ratiometric emission, and so there is a pressing need for methods to maximize the ratiometric change of existing biosensor constructs in order to increase the breadth of their utility.

Results

To accelerate the development and optimization of improved FRET-based biosensors, we have developed a method for function-based high-throughput screening of biosensor variants in colonies of Escherichia coli. We have demonstrated this technology by undertaking the optimization of a biosensor for detection of methylation of lysine 27 of histone H3 (H3K27). This effort involved the construction and screening of 3 distinct libraries: a domain library that included several engineered binding domains isolated by phage-display; a lower-resolution linker library; and a higher-resolution linker library.

Conclusion

Application of this library screening methodology led to the identification of an optimized H3K27-trimethylation biosensor that exhibited an emission ratio change (66%) that was 2.3 × improved relative to that of the initially constructed biosensor (29%).     

Revisiting the Ramachandran plot from a new angle

The pioneering work of Ramachandran and colleagues emphasized the dominance of steric constraints in specifying the structure of polypeptides. The ubiquitous Ramachandran plot of backbone dihedral angles (φ and ψ) defined the allowed regions of conformational space. These predictions were subsequently confirmed in proteins of known structure. Ramachandran and colleagues also investigated the influence of the backbone angle τ on the distribution of allowed φ/ψ combinations. The “bridge region” (φ ≤ 0° and −20° ≤ ψ ≤ 40°) was predicted to be particularly sensitive to the value of τ. Here we present an analysis of the distribution of φ/ψ angles in 850 non-homologous proteins whose structures are known to a resolution of 1.7 Å or less and sidechain B-factor less than 30 Ų. We show that the distribution of φ/ψ angles for all 87,000 residues in these proteins shows the same dependence on τ as predicted by Ramachandran and colleagues. Our results are important because they make clear that steric constraints alone are sufficient to explain the backbone dihedral angle distributions observed in proteins. Contrary to recent suggestions, no additional energetic contributions, such as hydrogen bonding, need be invoked.     

The Hot (Invisible?) Hand: Can Time Sequence Patterns of Success/Failure in Sports Be Modeled as Repeated Random Independent Trials?

The long lasting debate initiated by Gilovich, Vallone and Tversky in is revisited: does a “hot hand” phenomenon exist in sports? Hereby we come back to one of the cases analyzed by the original study, but with a much larger data set: all free throws taken during five regular seasons () of the National Basketball Association (NBA). Evidence supporting the existence of the “hot hand” phenomenon is provided. However, while statistical traces of this phenomenon are observed in the data, an open question still remains: are these non random patterns a result of “success breeds success” and “failure breeds failure” mechanisms or simply “better” and “worse” periods? Although free throws data is not adequate to answer this question in a definite way, we speculate based on it, that the latter is the dominant cause behind the appearance of the “hot hand” phenomenon in the data.     

A comparison of donor-acceptor pairs for genetically encoded FRET sensors: application to the Epac cAMP sensor as an example

We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors.     

Plasticity of Human Protein Disulfide Isomerase: EVIDENCE FOR MOBILITY AROUND THE X-LINKER REGION AND ITS FUNCTIONAL SIGNIFICANCE*

Protein disulfide isomerase (PDI), which consists of multiple domains arranged as abb′xa′c, is a key enzyme responsible for oxidative folding in the endoplasmic reticulum. In this work we focus on the conformational plasticity of this enzyme. Proteolysis of native human PDI (hPDI) by several proteases consistently targets sites in the C-terminal half of the molecule (x-linker and a′ domain) leaving large fragments in which the N terminus is intact. Fluorescence studies on the W111F/W390F mutant of full-length PDI show that its fluorescence is dominated by Trp-347 in the x-linker which acts as an intrinsic reporter and indicates that this linker can move between “capped” and “uncapped” conformations in which it either occupies or exposes the major ligand binding site on the b′ domain of hPDI. Studies with a range of constructs and mutants using intrinsic fluorescence, collision quenching, and extrinsic probe fluorescence (1-anilino-8-naphthalene sulfonate) show that the presence of the a′ domain in full-length hPDI moderates the ability of the x-linker to generate the capped conformation (compared with shorter fragments) but does not abolish it. Hence, unlike yeast PDI, the major conformational plasticity of full-length hPDI concerns the mobility of the a′ domain “arm” relative to the bb′ “trunk” mediated by the x-linker. The chaperone and enzymatic activities of these constructs and mutants are consistent with the interpretation that the reversible interaction of the x-linker with the ligand binding site mediates access of protein substrates to this site.     

Direct interaction of eag domains and cyclic nucleotide–binding homology domains regulate deactivation gating in hERG channels

Human ether-á-go-go (eag)-related gene (hERG) potassium channels play a critical role in cardiac repolarization and are characterized by unusually slow closing (deactivation) kinetics. The N-terminal “eag” domain and a C-terminal C-linker/cyclic nucleotide–binding homology domain (CNBHD) are required for regulation of slow deactivation. The region between the S4 and S5 transmembrane domains (S4–S5 linker) is also implicated in this process, but the mechanism for regulation of slow deactivation is unclear. Here, using an eag domain–deleted channel (hERG Δeag) fused to Citrine fluorescent protein, we found that most channels bearing individual alanine mutations in the S4–S5 linker were directly regulated by recombinant eag domains fused to a cyan fluorescent protein (N-eag-CFP) and had robust Förster resonance energy transfer (FRET). Additionally, a channel bearing a group of eight alanine residues in the S4–S5 linker was not measurably regulated by N-eag-CFP domains, but robust FRET was measured. These findings demonstrate that the eag domain associated with all of the S4–S5 linker mutant channels. In contrast, channels that also lacked the CNBHD (hERG Δeag ΔCNBHD-Citrine) were not measurably regulated by N-eag-CFP nor was FRET detected, suggesting that the C-linker/CNBHD was required for eag domains to directly associate with the channel. In a FRET hybridization assay, N-eag-CFP had robust FRET with a C-linker/CNBHD-Citrine, suggesting a direct and specific interaction between the eag domain and the C-linker/CNBHD. Lastly, coexpression of a hERG subunit lacking the CNBHD and the distal C-terminal region (hERG ΔpCT-Citrine) with hERG Δeag-CFP subunits had FRET and partial restoration of slow deactivation. Collectively, these findings reveal that the C-linker/CNBHD, but not the S4–S5 linker, was necessary for the eag domain to associate with the channel, that the eag domain and the C-linker/CNBHD were sufficient for a direct interaction, and that an intersubunit interaction between the eag domain and the C-linker/CNBHD regulated slow deactivation in hERG channels at the plasma membrane.     

The leukemia-specific fusion gene ETV6/RUNX1 perturbs distinct key biological functions primarily by gene repression

Background

ETV6/RUNX1 (E/R) (also known as TEL/AML1) is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL) and also most likely the crucial factor for disease initiation; its role in leukemia propagation and maintenance, however, remains largely elusive. To address this issue we performed a shRNA-mediated knock-down (KD) of the E/R fusion gene and investigated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions in two E/R-positive leukemic cell lines.

Findings

Microarray analyses identified 777 genes whose expression was substantially altered. Although approximately equal proportions were either up- (KD-UP) or down-regulated (KD-DOWN), the effects on biological processes and pathways differed considerably. The E/R KD-UP set was significantly enriched for genes included in the “cell activation”, “immune response”, “apoptosis”, “signal transduction” and “development and differentiation” categories, whereas in the E/R KD-DOWN set only the “PI3K/AKT/mTOR signaling” and “hematopoietic stem cells” categories became evident. Comparable expression signatures obtained from primary E/R-positive ALL samples underline the relevance of these pathways and molecular functions. We also validated six differentially expressed genes representing the categories “stem cell properties”, “B-cell differentiation”, “immune response”, “cell adhesion” and “DNA damage” with RT-qPCR.

Conclusion

Our analyses provide the first preliminary evidence that the continuous expression of the E/R fusion gene interferes with key regulatory functions that shape the biology of this leukemia subtype. E/R may thus indeed constitute the essential driving force for the propagation and maintenance of the leukemic process irrespective of potential consequences of associated secondary changes. Finally, these findings may also provide a valuable source of potentially attractive therapeutic targets.     

Crystal Structure of the Src Family Kinase Hck SH3-SH2 Linker Regulatory Region Supports an SH3-dominant Activation Mechanism

Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity. However, the amino acid sequence of the Hck linker represents a suboptimal ligand for the isolated SH3 domain, suggesting that it may form the polyproline type II helical conformation required for SH3 docking only in the context of the intact structure. To test this hypothesis directly, we determined the crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker. Despite the absence of the kinase domain, the structures and relative orientations of the SH2 and SH3 domains in this shorter protein were very similar to those observed in near full-length, down-regulated Hck. However, the SH2 kinase linker adopted a modified topology and failed to engage the SH3 domain. This new structure supports the idea that these noncatalytic regions work together as a “conformational switch” that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hck protein. Our results also provide fresh structural insight into the facile induction of Hck activity by HIV-1 Nef and other Hck SH3 domain binding proteins and implicate the existence of innate conformational states unique to individual Src family members that “fine-tune” their sensitivities to activation by SH3-based ligands.     

论我国国家植物园体系建设: 以任务带学科构建国家植物园迁地保护综合体系

植物园诞生的原初是“皇家”或“国家”意志的产物, 植物园的概念从公元前2,800年我国的“神农本草园”起源, 至今已历经沧桑巨变, 而西方文艺复兴后演替出了现代植物园。科研、保护、教育与示范四大功能始终是植物园的主线。植物园作为专门从事野生植物收集、科学研究、引种驯化和保护利用的专业研究机构, 始终肩负着国家的重要使命。本文系统综述了植物园的起源与演变, 并对世界各国的国家植物园与国家植物园体系进行了系统梳理和分析。在对我国植物园历史与发展概况总结的基础上, 论述了我国国家植物园体系建设的定位与目标、区域布局、科学研究、人才队伍、基础设施等五个方面的思考, 以任务带学科构建我国国家植物园迁地保护综合体系。     

DNA数据存储的科研概况国际对比与分析

全球数据量快速增长,成为数字经济发展的核心引擎,但传统数据存储介质受到功耗、体积、成本等限制,难以满足不断增长的数据存储需求。以脱氧核糖核酸(deoxyribonucleic acid,DNA)分子作为存储介质的新型存储方式引起了国内外高度重视,世界主要国家均对其研究进行了顶层规划,部署了一系列重要科研计划。但是,DNA数据存储作为一个新兴交叉研究领域,其发展的“源”与“流”仍存在需要深入分析的问题。针对该问题,从信息、半导体与合成生物学交叉融合的角度深入挖掘DNA数据存储发展的源头,对近年来国际上主要国家与地区在DNA数据存储领域的发展规划进行分析归纳,梳理国内外的科研项目规划布局,尤其是美国“半导体合成生物学联盟”推动的基础研究项目、美国国防部高级研究计划局(Defense Advanced Research Projects Agency,DARPA)与美国情报高级研究计划局(Intelligence Advanced Research Projects Activity,IARPA)推动的面向应用的集中攻关项目、欧盟的地平线2020计划以及我国的重点研发计划等。通过比较可发现,美国主要采用政府部门主导、应用目标导向的研究模式,欧盟与我国在“十三五”期间及时跟进;我国在“十四五”期间设立了重点研发计划“生物与信息融合(BT与IT融合)”,致力于推动DNA数据存储等领域的发展,实现DNA数据存储发展带动生化仪器乃至生物经济、数字经济的发展。探索DNA数据存储发展的“源”和“流”,为从事该领域的研究者识别真正制约该领域发展的“真问题”提供参考,也为科技管理部门研判DNA数据存储的国际发展趋势提供参考。     

Metal ion dependence, thermodynamics, and kinetics for intramolecular docking of a GAAA tetraloop and receptor connected by a flexible linker

The GAAA tetraloop-receptor motif is a commonly occurring tertiary interaction in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interaction makes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A(7) linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking {[Co(NH(3))(6)(3+)] < [Ca(2+)], [Mg(2+)], [Mn(2+)] < [Na(+)], [K(+)]}. Analysis of metal ion cooperativity yielded Hill coefficients of approximately 2 for Na(+)- or K(+)-dependent docking versus approximately 1 for the divalent ions and Co(NH(3))(6)(3+). Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U(7) and A(14) single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.     

Pandemics in the age of Twitter: content analysis of Tweets during the 2009 H1N1 outbreak

Background

Surveys are popular methods to measure public perceptions in emergencies but can be costly and time consuming. We suggest and evaluate a complementary “infoveillance” approach using Twitter during the 2009 H1N1 pandemic. Our study aimed to: 1) monitor the use of the terms “H1N1” versus “swine flu” over time; 2) conduct a content analysis of “tweets”; and 3) validate Twitter as a real-time content, sentiment, and public attention trend-tracking tool.

Methodology/Principal Findings

Between May 1 and December 31, 2009, we archived over 2 million Twitter posts containing keywords “swine flu,” “swineflu,” and/or “H1N1.” using Infovigil, an infoveillance system. Tweets using “H1N1” increased from 8.8% to 40.5% (R ² = .788; p<.001), indicating a gradual adoption of World Health Organization-recommended terminology. 5,395 tweets were randomly selected from 9 days, 4 weeks apart and coded using a tri-axial coding scheme. To track tweet content and to test the feasibility of automated coding, we created database queries for keywords and correlated these results with manual coding. Content analysis indicated resource-related posts were most commonly shared (52.6%). 4.5% of cases were identified as misinformation. News websites were the most popular sources (23.2%), while government and health agencies were linked only 1.5% of the time. 7/10 automated queries correlated with manual coding. Several Twitter activity peaks coincided with major news stories. Our results correlated well with H1N1 incidence data.

Conclusions

This study illustrates the potential of using social media to conduct “infodemiology” studies for public health. 2009 H1N1-related tweets were primarily used to disseminate information from credible sources, but were also a source of opinions and experiences. Tweets can be used for real-time content analysis and knowledge translation research, allowing health authorities to respond to public concerns.     

Classification and analysis of regulatory pathways using graph property, biochemical and physicochemical property, and functional property

Given a regulatory pathway system consisting of a set of proteins, can we predict which pathway class it belongs to? Such a problem is closely related to the biological function of the pathway in cells and hence is quite fundamental and essential in systems biology and proteomics. This is also an extremely difficult and challenging problem due to its complexity. To address this problem, a novel approach was developed that can be used to predict query pathways among the following six functional categories: (i) “Metabolism”, (ii) “Genetic Information Processing”, (iii) “Environmental Information Processing”, (iv) “Cellular Processes”, (v) “Organismal Systems”, and (vi) “Human Diseases”. The prediction method was established trough the following procedures: (i) according to the general form of pseudo amino acid composition (PseAAC), each of the pathways concerned is formulated as a 5570-D (dimensional) vector; (ii) each of components in the 5570-D vector was derived by a series of feature extractions from the pathway system according to its graphic property, biochemical and physicochemical property, as well as functional property; (iii) the minimum redundancy maximum relevance (mRMR) method was adopted to operate the prediction. A cross-validation by the jackknife test on a benchmark dataset consisting of 146 regulatory pathways indicated that an overall success rate of 78.8% was achieved by our method in identifying query pathways among the above six classes, indicating the outcome is quite promising and encouraging. To the best of our knowledge, the current study represents the first effort in attempting to identity the type of a pathway system or its biological function. It is anticipated that our report may stimulate a series of follow-up investigations in this new and challenging area.     

Development of FRET biosensors for mammalian and plant systems

Genetically encoded biosensors are increasingly used in visualising signalling processes in different organisms. Sensors based on green fluorescent protein technology are providing a great opportunity for using Förster resonance energy transfer (FRET) as a tool that allows for monitoring dynamic processes in living cells. The development of these FRET biosensors requires careful selection of fluorophores, substrates and recognition domains. In this review, we will discuss recent developments, strategies to create and optimise FRET biosensors and applications of FRET-based biosensors for use in the two major eukaryotic kingdoms and elaborate on different methods for FRET detection.     

Do Facial Expressions Develop before Birth?

Background

Fetal facial development is essential not only for postnatal bonding between parents and child, but also theoretically for the study of the origins of affect. However, how such movements become coordinated is poorly understood. 4-D ultrasound visualisation allows an objective coding of fetal facial movements.

Methodology/Findings

Based on research using facial muscle movements to code recognisable facial expressions in adults and adapted for infants, we defined two distinct fetal facial movements, namely “cry-face-gestalt” and “laughter- gestalt,” both made up of up to 7 distinct facial movements. In this conceptual study, two healthy fetuses were then scanned at different gestational ages in the second and third trimester. We observed that the number and complexity of simultaneous movements increased with gestational age. Thus, between 24 and 35 weeks the mean number of co-occurrences of 3 or more facial movements increased from 7% to 69%. Recognisable facial expressions were also observed to develop. Between 24 and 35 weeks the number of co-occurrences of 3 or more movements making up a “cry-face gestalt” facial movement increased from 0% to 42%. Similarly the number of co-occurrences of 3 or more facial movements combining to a “laughter-face gestalt” increased from 0% to 35%. These changes over age were all highly significant.

Significance

This research provides the first evidence of developmental progression from individual unrelated facial movements toward fetal facial gestalts. We propose that there is considerable potential of this method for assessing fetal development: Subsequent discrimination of normal and abnormal fetal facial development might identify health problems in utero.     

Gum arabic glycoprotein is a twisted hairy rope : a new model based on o-galactosylhydroxyproline as the polysaccharide attachment site

Separation of the wound exudate from Acacia senegal (L.) Willd., “gum arabic,” on a preparative Superose-6 column gave two major fractions: a high molecular weight gum arabic glycoprotein (GAGP) containing about 90% carbohydrate and a lower molecular weight heterogenous gum arabic polysaccharide fraction. Hydrogen fluoride-deglycosylation of GAGP gave a large (~400 residue) hydroxyproline-rich polypeptide backbone (dGAGP). Alkaline hydrolysis of GAGP showed that most of the carbohydrate was attached to the polypeptide backbone as small (~30 residue) hydroxyproline (Hyp)-polysaccharide substituents. After partial acid hydrolysis of the Hyp-polysaccharide fraction we identified O-galactosylhydroxyproline as the glycopeptide linkage, identical with that of hydroxyproline-rich arabinogalactan-proteins (AGPs). However, unlike the acidic alanine-rich AGPs, GAGP is basic and notably deficient in alanine. Thus, while the GAGP polypeptide backbone more closely resembles that of the Hyp-rich cell wall protein extensin, the GAGP polysaccharide sidechains resemble AGPs. Possibly all three proteins comprise a phylogenetically related extensin superfamily of extended rod-like macromolecules. The “wattle-blossom” model for AGP and gum arabic predicts a few large polysaccharide substituents along the polypeptide backbone of a spheroidal macromolecule. On the contrary, our data imply a rodlike molecule with numerous small polysaccharide substituents (attached to 24% of the Hyp residues), regularly arranged along a highly periodic polypeptide backbone based, hypothetically, on a 10 to 12 residue repetitive peptide motif. Thus, a simple statistical model of the gum arabic glycoprotein predicts a repeating polysaccharide-peptide subunit of about 7 kilodaltons. The small polysaccharide substituents will maximize intramolecular hydrogen bonding if aligned along the long axis of the molecule, forming in effect a twisted hairy rope. Electron micrographs of rotary shadowed GAGP molecules support that prediction and may also explain how such apparently large molecules can exit the cell by endwise reptation through the small pores of the primary cell wall.     

Intrinsic α‐helical and β‐sheet conformational preferences: A computational case study of alanine

A fundamental question in protein science is what is the intrinsic propensity for an amino acid to be in an α-helix, β-sheet, or other backbone dihedral angle (-ψ) conformation. This question has been hotly debated for many years because including all protein crystal structures from the protein database, increases the probabilities for α-helical structures, while experiments on small peptides observe that β-sheet-like conformations predominate. We perform molecular dynamics (MD) simulations of a hard-sphere model for Ala dipeptide mimetics that includes steric interactions between nonbonded atoms and bond length and angle constraints with the goal of evaluating the role of steric interactions in determining protein backbone conformational preferences. We find four key results. For the hard-sphere MD simulations, we show that (1) β-sheet structures are roughly three and half times more probable than α-helical structures, (2) transitions between α-helix and β-sheet structures only occur when the backbone bond angle τ (N–C_α–C) is greater than 110°, and (3) the probability distribution of τ for Ala conformations in the “bridge” region of-ψ space is shifted to larger angles compared to other regions. In contrast, (4) the distributions obtained from Amber and CHARMM MD simulations in the bridge regions are broader and have increased τ compared to those for hard sphere simulations and from high-resolution protein crystal structures. Our results emphasize the importance of hard-sphere interactions and local stereochemical constraints that yield strong correlations between -ψ conformations and τ.     

827Spatio-Temporal Quantification of FRET in Living Cells by Fast Time-Domain FLIM: A Comparative Study of Non-Fitting Methods

Förster Resonance Energy Transfer (FRET) measured with Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate spatio-temporal regulation of protein-protein interactions in living cells. When using standard fitting methods to analyze time domain FLIM, the correct estimation of the FRET parameters requires a high number of photons and therefore long acquisition times which are incompatible with the observation of dynamic protein-protein interactions. Recently, non-fitting strategies have been developed for the analysis of FLIM images: the polar plot or “phasor” and the minimal fraction of interacting donor mf_D. We propose here a novel non-fitting strategy based on the calculation of moments. We then compare the performance of these three methods when shortening the acquisition time: either by reducing the number of counted photons N or the number of temporal channels N_ch, which is particularly adapted for the original fast-FLIM prototype presented in this work that employs the time gated approach. Based on theoretical calculations, Monte Carlo simulations and experimental data, we determine the domain of validity of each method. We thus demonstrate that the polar approach remains accurate for a large range of conditions (low N, N_ch or small fractions of interacting donor f_D). The validity domain of the moments method is more restricted (not applicable when f_D<0.25 or when N_ch = 4) but it is more precise than the polar approach. We also demonstrate that the mf_D is robust in all conditions and it is the most precise strategy; although it does not strictly provide the fraction of interacting donor. We show using the fast-FLIM prototype (with an acquisition rate up to 1 Hz) that these non-fitting strategies are very powerful for on-line analysis on a standard computer and thus for quantifying automatically the spatio-temporal activation of Rac-GTPase in living cells by FRET.