Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging

A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescence labeling with high efficiency, specificity and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ～100% efficiency while maintaining the biological function. We show that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis. We also show the unique power of our method in single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor.     

Site-specific in vitro and in vivo incorporation of molecular probes to study G-protein-coupled receptors

Chemical modification of proteins has a rich history in biochemistry and chemical biology. However, studies of membrane protein function, especially in cases where functional expression is low and purification and reconstitution are not feasible, present unique challenges. Heptahelical G-protein-coupled receptors (GPCRs) are a particularly important class of cell-surface receptors that represent targets of more than a quarter of all therapeutic drugs. Understanding with chemical precision how GPCRs function in biological membranes remains a central problem in biology. Recently a number of creative strategies have been developed that allow site-specific attachment of chemical probes or tags directly on expressed receptors or on biologically active peptide ligands or substrates. One particularly important advance is the genetic encoding of unnatural amino acids (UAAs) with unique small bioorthogonal tags using amber codon suppression in mammalian cells. This method should allow site-specific labeling of GPCRs with various molecular probes to facilitate cell-based studies of protein-protein or protein-ligand interactions and the visualization of conformational changes using fluorescence spectroscopy or single-molecule imaging.     

基于TurboID的植物蛋白邻近标记实验方法

邻近标记作为近些年发展起来的一项检测活细胞内蛋白互作关系和亚细胞结构蛋白组的新型技术,已成功应用于多种动植物体系的研究。该技术通过给诱饵蛋白融合一个具有特定催化连接活性的酶,在酶的催化作用下将小分子底物(如生物素)共价连接到酶邻近的内源蛋白,通过富集和分析被标记的蛋白可获得与诱饵互作的蛋白组。经定向进化产生的生物素连接...     

Kinetics and inhibition of glutamate carboxypeptidase II using a microplate assay

Proteomics, the study of protein function on a global scale, will play an important role in furthering our understanding of gene functions, complex biological pathways, and discovery of novel drug targets. A number of techniques have been developed for proteomic studies to identify and analyze proteins, compare protein expression levels, and study protein-protein interactions. Recent developments have applied a DNA array-type approach to immobilize proteins on a surface for high-throughput analysis. Here we report the development and construction of protein chips using derivatized glass and nitrocellulose-coated slides and the employment of recombinant proteins fused with green and red fluorescent proteins for detection. Fluorescent signals were found to be proportional to the amount of arrayed proteins and could be readily detected with a conventional fluorescence slide scanner. This technique allows the investigation of protein-protein interactions without the need for additional labeling steps of probe proteins.     

一种利用荧光蛋白mNeonGreen2鉴定EI24蛋白拓扑结构的方法

方便且精准地检测跨膜蛋白拓扑结构,尤其是跨膜片段的氨基(N-)和羧基(C-)端的朝向,有利于发现新的蛋白质与蛋白质之间的相互作用,并进一步揭示蛋白质重要的生物学功能．自组装荧光蛋白已被广泛用于观察蛋白质与蛋白质之间的相互作用、标记细胞内源蛋白质并实现mRNA定位的可视化．本文扩展了自组装荧光蛋白的应用,将自组装荧光蛋白mNeonGreen2与定点标记技术相结合,以确定跨膜蛋白的拓扑结构．通过该方法,第一次清楚地证明了EI24的N端和C端均朝向细胞质方向．此外,该方法可用于确定定位于其他细胞器且结构尚未解析的跨膜蛋白的拓扑结构．     

Rapid functional analysis of protein-protein interactions by fluorescent C-terminal labeling and single-molecule imaging

Detection of protein-protein interactions is a fundamental step to understanding gene function. Here we report a sensitive and rapid method for assaying protein-protein interactions at the single-molecule level. Protein molecules were synthesized in a cell-free translation system in the presence of Cy5-puro, a fluorescent puromycin, using mRNA without a stop codon. The interaction of proteins thus prepared was visualized using a single-molecule imaging technique. As a demonstration of this method, a motor protein, kinesin, was labeled with Cy5-puro at an efficiency of about 90%, and the processive movement of kinesin along microtubules was observed by using total internal reflection microscopy. It took only 2 h from the synthesis of proteins to the functional analysis. This method is applicable to the functional analysis of various kinds of proteins.     

DNA-binding orientation and domain conformation of the E. coli rep helicase monomer bound to a partial duplex junction: single-molecule studies of fluorescently labeled enzymes

The SF1 DNA helicases are multi-domain proteins that can unwind duplex DNA in reactions that are coupled to ATP binding and hydrolysis. Crystal structures of two such helicases, Escherichia coli Rep and Bacillus stearothermophilus PcrA, show that the 2B sub-domain of these proteins can be found in dramatically different orientations (closed versus open) with respect to the remainder of the protein, suggesting that the 2B domain is highly flexible. By systematically using fluorescence resonance energy transfer at the single-molecule level, we have determined both the orientation of an E.coli Rep monomer bound to a 3'-single-stranded-double-stranded (ss/ds) DNA junction in solution, as well as the relative orientation of its 2B sub-domain. To accomplish this, we developed a highly efficient procedure for site-specific fluorescence labeling of Rep and a bio-friendly immobilization scheme, which preserves its activities. Both ensemble and single-molecule experiments were carried out, although the single-molecule experiments proved to be essential here in providing quantitative distance information that could not be obtained by steady-state ensemble measurements. Using distance-constrained triangulation procedures we demonstrate that in solution the 2B sub-domain of a Rep monomer is primarily in the "closed" conformation when bound to a 3'-ss/ds DNA, similar to the orientation observed in the complex of PcrA bound to a 3'-ss/ds DNA. Previous biochemical studies have shown that a Rep monomer bound to such a 3'-ss/ds DNA substrate is unable to unwind the DNA and that a Rep oligomer is required for helicase activity. Therefore, the closed form of Rep bound to a partial duplex DNA appears to be an inhibited form of the enzyme.     

Expression of proteins with dimethylarginines in Escherichia coli for protein-protein interaction studies

Protein arginine methylation often modulates protein-protein interactions. To isolate a sufficient quantity of proteins enriched in methyl arginine(s) from natural sources for biochemical studies is laborious and difficult. We describe here an expression system that produces recombinant proteins that are enriched in omega-N(G),N(G)-asymmetry dimethylarginines. A yeast type I arginine methyltransferase gene (HMT1) is put on a plasmid under the control of the Escherichia coli methionine aminopeptidase promoter for constitutive expression. The protein targeted for post-translational modification is put on the same plasmid behind a T7 promoter for inducible expression of His(6)-tagged proteins. Sbp1p and Stm1p were used as model proteins to examine this expression system. The 13 arginines within the arginine-glycine-rich motif of Sbp1p and the RGG sequence near the C terminus of Stm1p were methylated. Unexpectedly, the arginine residue on the thrombin cleavage site (LVPRGS) of the fusion proteins can also be methylated by Hmt1p. Sbp1p and Sbp1p/hmt1 were covalently attached to solid supports for the isolation of interacting proteins. The results indicate that arginine methylation on Sbp1p exerts both positive and negative effects on protein-protein interaction.     

Determining protease activity in vivo by fluorescence cross-correlation analysis

To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions.     

Robust co-regulation of tyrosine phosphorylation sites on proteins reveals novel protein interactions

Cell signaling networks propagate information from extracellular cues via dynamic modulation of protein-protein interactions in a context-dependent manner. Networks based on receptor tyrosine kinases (RTKs), for example, phosphorylate intracellular proteins in response to extracellular ligands, resulting in dynamic protein-protein interactions that drive phenotypic changes. Most commonly used methods for discovering these protein-protein interactions, however, are optimized for detecting stable, longer-lived complexes, rather than the type of transient interactions that are essential components of dynamic signaling networks such as those mediated by RTKs. Substrate phosphorylation downstream of RTK activation modifies substrate activity and induces phospho-specific binding interactions, resulting in the formation of large transient macromolecular signaling complexes. Since protein complex formation should follow the trajectory of events that drive it, we reasoned that mining phosphoproteomic datasets for highly similar dynamic behavior of measured phosphorylation sites on different proteins could be used to predict novel, transient protein-protein interactions that had not been previously identified. We applied this method to explore signaling events downstream of EGFR stimulation. Our computational analysis of robustly co-regulated phosphorylation sites, based on multiple clustering analysis of quantitative time-resolved mass-spectrometry phosphoproteomic data, not only identified known sitewise-specific recruitment of proteins to EGFR, but also predicted novel, a priori interactions. A particularly intriguing prediction of EGFR interaction with the cytoskeleton-associated protein PDLIM1 was verified within cells using co-immunoprecipitation and in situ proximity ligation assays. Our approach thus offers a new way to discover protein-protein interactions in a dynamic context- and phosphorylation site-specific manner.     

Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification

An often limiting factor for studying protein folding by single-molecule fluorescence resonance energy transfer (FRET) is the ability to site-specifically introduce a photostable organic FRET donor (D) and a complementary acceptor (A) into a polypeptide chain. Using alternating-laser excitation and chymotrypsin inhibitor 2 as a model, we show that chemical labeling of a unique cysteine, followed by enzymatic modification of a reactive glutamine in an N-terminally appended substrate sequence recognition tag for transglutaminase (TGase) affords stoichiometrically D-/A-labeled protein suitable for single-molecule FRET experiments. Thermodynamic data indicate that neither the presence of the TGase tag nor D/A labeling perturbs protein stability. As the N terminus in proteins is typically solvent accessible, a TGase tag can (in principle) be appended to any protein of interest by genetic engineering. Two-step chemical/enzymatic labeling may thus represent a simple, low-cost, and widely available strategy for D/A labeling of proteins for FRET-based single-molecule protein folding studies, even for non-protein-experts laboratories.     

The thermal adaptation of the nitrogenase Fe protein from thermophilic Methanobacter thermoautotrophicus

The nitrogenase Fe protein is a key component of the biochemical machinery responsible for the process of biological nitrogen fixation. The Fe protein is a member of a class of nucleotide-binding proteins that couple the binding and hydrolysis of nucleoside triphosphates to conformational changes. The nucleotide-dependent conformational changes modulate the formation of a macromolecular complex, and some members of the class include Galpha, EF-Tu, and myosin. The members of this class are highly interesting model systems for the analysis of aspects of thermal adaptability, since their mechanisms involve protein conformational change and protein-protein interactions. In this study, we have used our extensive knowledge of the structure of the Azotobacter vinelandii nitrogenase Fe protein in multiple structural conformations, and standard homology modeling approaches have been used to generate reliable models of the Fe protein from thermophilic Methanobacter thermoautotrophicus in the analogous structural conformations. The resulting structural comparison reveals that thermal adaptation of the M. thermoautotrophicus Fe protein is conferred by a number of factors, including increased structural rigidity that results from various structural changes within the protein interior. The analysis of hypothetical docking models and nitrogenase complex structures provides insights into the thermal adaptation of the protein-protein interactions that support macromolecular complex formation and catalysis at higher temperatures.     

Modelling protein functional domains in signal transduction using Maude

Modelling of protein-protein interactions in signal transduction is receiving increased attention in computational biology. This paper describes recent research in the application of Maude, a symbolic language founded on rewriting logic, to the modelling of functional domains within signalling proteins. Protein functional domains (PFDs) are a critical focus of modern signal transduction research. In general, Maude models can simulate biological signalling networks and produce specific testable hypotheses at various levels of abstraction. Developing symbolic models of signalling proteins containing functional domains is important because of the potential to generate analyses of complex signalling networks based on structure-function relationships.     

邻近标记在蛋白质组学中的发展及应用

人体内各种复杂的生命活动离不开蛋白质之间的相互作用。这种相互作用具有瞬时性和结合力弱等特点,并受到多种动态调节,特别是蛋白质翻译后修饰（post-translation modifications, PTM）。传统的亲和质谱检测方法存在蛋白纯化的局限性,在高效检测到动态变化方面存在不足。邻近标记是一种能够给与靶蛋白质瞬时靠近,或者互作（邻近）的蛋白质加上生物素的技术,它与质谱检测技术的联合使用能检测细胞过程中弱的、瞬时的蛋白质相互作用,有效解决上述问题。本文综述了基于生物素的邻近标记方法的发展现状,从依赖于融合序列的生物素标记开始,依次介绍有关生物素连接酶、过氧化物酶及其进化后的2代标记方法等经典生物素标记的方法和原理,比较各个方法间的差异和优缺点;也列举了一些近年来新出现的标记方法,如将生物素连接酶进行拆分、鉴定蛋白质在不同复合物中功能的方法、抗体靶向的标记方法,以及其他来源的生物素连接酶突变体,例如枯草芽孢杆菌（Bacillus subtilis）的C端氨基酸突变的生物素连接酶,能够应用在苍蝇和蠕虫中的生物素连接酶突变体。本文对这些方法进行归纳总结,旨在为初步接触该领域的科研工作者提供参考,同时也希望能够提供一些新的思路,推动蛋白质相互作用组学的发展。     

Protein interaction network for Alzheimer's disease using computational approach

Alzheimer''s disease (AD) is the most common form of dementia. It is the sixth leading cause of death in old age people. Despite recent advances in the field of drug design, the medical treatment for the disease is purely symptomatic and hardly effective. Thus there is a need to understand the molecular mechanism behind the disease in order to improve the drug aspects of the disease. We provided two contributions in the field of proteomics in drug design. First, we have constructed a protein-protein interaction network for Alzheimer''s disease reviewed proteins with 1412 interactions predicted among 969 proteins. Second, the disease proteins were given confidence scores to prioritize and then analyzed for their homology nature with respect to paralogs and homologs. The homology persisted with the mouse giving a basis for drug design phase. The method will create a new drug design technique in the field of bioinformatics by linking drug design process with protein-protein interactions via signal pathways. This method can be improvised for other diseases in future.     

邻近标记在蛋白质组学中的发展及应用

人体内各种复杂的生命活动离不开蛋白质之间的相互作用。这种相互作用具有瞬时性和结合力弱等特点,并受到多种动态调节,特别是蛋白质翻译后修饰（post-translation modifications, PTM）。传统的亲和质谱检测方法存在蛋白纯化的局限性,在高效检测到动态变化方面存在不足。邻近标记是一种能够给与靶蛋白质瞬时靠近,或者互作（邻近）的蛋白质加上生物素的技术,它与质谱检测技术的联合使用能检测细胞过程中弱的、瞬时的蛋白质相互作用,有效解决上述问题。本文综述了基于生物素的邻近标记方法的发展现状,从依赖于融合序列的生物素标记开始,依次介绍有关生物素连接酶、过氧化物酶及其进化后的2代标记方法等经典生物素标记的方法和原理,比较各个方法间的差异和优缺点;也列举了一些近年来新出现的标记方法,如将生物素连接酶进行拆分、鉴定蛋白质在不同复合物中功能的方法、抗体靶向的标记方法,以及其他来源的生物素连接酶突变体,例如枯草芽孢杆菌（Bacillus subtilis）的C端氨基酸突变的生物素连接酶,能够应用在苍蝇和蠕虫中的生物素连接酶突变体。本文对这些方法进行归纳总结,旨在为初步接触该领域的科研工作者提供参考,同时也希望能够提供一些新的思路,推动蛋白质相互作用组学的发展。     

The use of immobilized ubiquitin for biosensor analysis of the mitochondrial subinteractome

Protein ubiquitination is an important mechanism responsible not only for specific labeling of proteins for their subsequent degradation; it also determines localization of proteins in the cell and regulation of protein-protein interactions. In the context of protein-protein interactions binding of (mono/poly)ubiquitinated molecules to proteins containing specific ubiquitin binding domains plays the decisive role. Formation of the ubiquitin interactome has been demonstrated for cytosol. Involvement of mitochondria and associated extramitochondrial proteins into such interactions still requires detailed investigation. In this study using an optical biosensor we have demonstrated binding of proteins of mouse brain mitochondrial lysates to immobilized monomeric ubiquitin. Model purified proteins, which are known to be associated with the outer mitochondrial compartment (glyceraldehyde-3-phosphate dehydorgenase, creatine phosphokinase), interacted with immobilized ubiquitin as well as with each other. This suggests that (poly)ubiquitinated chains may be involved in protein-protein interactions between ubiquitinated and non-ubiquitinated proteins and thus may contribute to formation of (mitochondrial) ubiquitin subinteractome.     

Direct detection of caspase-3 activation in single live cells by cross-correlation analysis

Dual color fluorescence cross-correlation spectroscopy (FCCS) provides information about the coincidence of spectrally well-defined two fluorescent molecules in a small observation area at the single-molecule level. To evaluate the activity of caspase-3 in vivo directly, FCCS was applied to single live cells. We constructed chimeric proteins that consisted of tandemly fused enhanced green FP (EGFP) and monomeric red FP (mRFP). In control experiments, the protease reaction was monitored in solution, where a decrease in cross-correlation amplitude was observed due to specific cleavage of the amino acid sequence between EGFP and mRFP. Moreover, a decrease in cross-correlation amplitude could be detected in a live cell, where caspase-3 activation was induced by apoptosis. This is the first report of FP-based in vivo cross-correlation analysis. FP-based FCCS may become the most versatile method for analysis of protein-protein interactions in live cells.     

Computational approaches to protein-protein interaction

The interactions between proteins allow the cell's life. A number of experimental, genome-wide, high-throughput studies have been devoted to the determination of protein-protein interactions and the consequent interaction networks. Here, the bioinformatics methods dealing with protein-protein interactions and interaction network are overviewed. 1. Interaction databases developed to collect and annotate this immense amount of data; 2. Automated data mining techniques developed to extract information about interactions from the published literature; 3. Computational methods to assess the experimental results developed as a consequence of the finding that the results of high-throughput methods are rather inaccurate; 4. Exploitation of the information provided by protein interaction networks in order to predict functional features of the proteins; and 5. Prediction of protein-protein interactions.     

Protein stabilization by specific binding of guanidinium to a functional arginine-binding surface on an SH3 domain

Guanidinium hydrochloride (GuHCl) at low concentrations significantly stabilizes the Fyn SH3 domain. In this work, we have demonstrated that this stabilizing effect is manifested through a dramatic (five- to sixfold) decrease in the unfolding rate of the domain with the folding rate being affected minimally. This behavior contrasts to the effect of NaCl, which stabilizes this domain by accelerating the folding rate. These data imply that the stabilizing effect of GuHCl is not predominantly ionic in nature. Through NMR studies, we have identified a specific binding site for guanidinium, and we have determined a dissociation constant of 90 mM for this interaction. The guanidinium-binding site overlaps with a functionally important arginine-binding pocket on the domain surface, and we have shown that GuHCl is a specific inhibitor of the peptide-binding activity of the domain. A different SH3 domain possessing a similar arginine-binding pocket is also thermodynamically stabilized by GuHCl. These data suggest that many proteins that normally interact with arginine-containing ligands may also be able to specifically interact with guanidinium. Thus, some caution should be used when using GuHCl as a denaturant in protein folding studies. Since arginine-mediated interactions are often important in the energetics of protein-protein interactions, our observations could be relevant for the design of small molecule inhibitors of protein-protein interactions.