Second-Generation Covalent TMP-Tag for Live Cell Imaging

Chemical tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with organic fluorophores that have improved brightness and other specialized properties. Recently, we successfully rendered our TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here we report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical synthesis for routine production of a variety of A-TMP-probe v2.0 labels. We then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, we demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited number of chemical tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and organic chemistry as tools not only for chemical tag engineering but also more broadly for synthetic biology.     

Insights into the mechanism and catalysis of the native chemical ligation reaction

Native chemical ligation of unprotected peptide segments involves reaction between a peptide-alpha-thioester and a cysteine-peptide, to yield a product with a native amide bond at the ligation site. Peptide-alpha-thioalkyl esters are commonly used because of their ease of preparation. These thioalkyl esters are rather unreactive so the ligation reaction is catalyzed by in situ transthioesterification with thiol additives. The most common thiol catalysts used to date have been either a mixture of thiophenol/benzyl mercaptan, or the alkanethiol MESNA. Despite the use of these thiol catalysts, ligation reactions typically take 24-48 h. To gain insight into the mechanism of native chemical ligaton and in order to find a better catalyst, we investigated the use of a number of thiol compounds. Substituted thiophenols with pK(a) > 6 were found to best combine the ability to exchange rapidly and completely with thioalkyl esters, and to then act as effective leaving groups in reaction of the peptide-thioester with the thiol side chain of a cysteine-peptide. A highly effective and practical catalyst was (4-carboxylmethyl)thiophenol ('MPAA'), a nonmalodorous, water-soluble thiol. Use of MPAA gave an order of magnitude faster reaction in model studies of native chemical ligation and in the synthesis of a small protein, turkey ovomucoid third domain (OMTKY3). MPAA should find broad use in native chemical ligation and in the total synthesis of proteins.     

Simulation of electron transfer between cytochrome C2 and the bacterial photosynthetic reaction center: Brownian dynamics analysis of the native proteins and double mutants

Electron transfer is essential for bacterial photosynthesis which converts light energy into chemical energy. This paper theoretically studies the interprotein electron transfer from cytochrome c(2) of Rhodobacter capsulatus to the photosynthetic reaction center of Rhodobacter sphaeroides in native and mutated systems. Brownian dynamics is used with an exponential distance-dependent electron-transfer rate model to compute bimolecular rate constants, which are consistent with experimental data when reasonable prefactors and decay constants are used. Interestingly, switching of the reaction mechanism from the diffusion-controlled limit in the native proteins to the activation-controlled limit in one of the mutants (DK(L261)/KE(C99)) was found. We also predict that the second-order rate for the native reaction center/cytochrome c(2) system will decrease with increasing ionic strength, a characteristic of electrostatically controlled docking.     

Staudinger ligation: a peptide from a thioester and azide

[reaction: see text] The technique of native chemical ligation enables the total chemical synthesis of proteins. This method is limited, however, by an absolute requirement for a cysteine residue at the ligation juncture. Here, this restriction is overcome with a new chemical ligation method in which a phosphinobenzenethiol is used to link a thioester and azide. The product is an amide with no residual atoms.     

Chances and pitfalls of chemical cross-linking with amine-reactive N-hydroxysuccinimide esters

In this report we summarize our experiences with the reaction products of N-hydroxysuccinimide (NHS) esters, which are widely used for chemical cross-linking of lysine residues in proteins. We describe the products, which should be scrutinized during data analysis using customized software when NHS esters are employed for chemical cross-linking. Reaction products of NHS esters were observed not only with lysines, but also with serines, tyrosines, and threonines. This report is intended to be a practical guide for those working in the field of chemical cross-linking and mass spectrometry.     

Kinetic Assay of the Michael Addition‐Like Thiol–Ene Reaction and Insight into Protein Bioconjugation

The chemical modification of proteins is a valuable technique in understanding the functions, interactions, and dynamics of proteins. Reactivity and selectivity are key issues in current chemical modification of proteins. The Michael addition‐like thiol–ene reaction is a useful tool that can be used to tag proteins with high selectivity for the solvent‐exposed thiol groups of proteins. To obtain insight into the bioconjugation of proteins with this method, a kinetic analysis was performed. New vinyl‐substituted pyridine derivatives were designed and synthesized. The reactivity of these vinyl tags with L ‐cysteine was evaluated by UV absorption and high‐resolution NMR spectroscopy. The results show that protonation of pyridine plays a key role in the overall reaction rates. The kinetic parameters were assessed in protein modification. The different reactivities of these vinyl tags with solvent‐exposed cysteine is valuable information in the selective labeling of proteins with multiple functional groups.     

Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry

Guanidination of the epsilon-amino group of lysine-terminated tryptic peptides can be accomplished selectively in one step with O-methylisourea hydrogen sulfate. This reaction converts lysine residues into more basic homoarginine residues. It also protects the epsilon-amino groups against unwanted reaction with sulfonation reagents, which can then be used to selectively modify the N-termini of tryptic peptides. The combined reactions convert lysine-terminated tryptic peptides into modified peptides that are suitable for de novo sequencing by postsource decay matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The guanidination reaction is very pH dependent. Product yields and reaction kinetics were studied in aqueous solution using either NaOH or diisopropylethylamine as the base. Methods are reported for derivatizing and sequencing lysine-terminated tryptic peptides at low pmole levels. The postsource decay (PSD) MALDI tandem mass spectra of a model peptide (VGGYGYGAK), the homoarginine analog and the sulfonated homoarginine analog are compared. These spectra show the influence that each chemical modification has on the peptide fragmentation pattern. Finally, we demonstrate that definitive protein identifications can be achieved by PSD MALDI sequencing of derivatized peptides obtained from solution digests of model proteins and from in-gel digests of 2D-gel separated proteins.     

Single-Step N-Terminal Modification of Proteins via a Bio-Inspired Copper(II)-Mediated Aldol Reaction

The chemical modification of proteins is an effective technique for manipulating the properties and functions of proteins, and for creating protein-based materials. The N-terminus is a promising target for single-site modification that provides modified proteins with uniform structures and properties. In this paper, a copper(II)-mediated aldol reaction with 2-pyridinecarboxaldehyde (2-PC) derivatives is proposed as an operationally simple method to selectively modify the N-terminus of peptides and proteins at room temperature and physiological pH. The copper(II) ion activates the N-terminal amino acids by complexation with an imine of the N-terminal amino acid and 2-PCs, realizing the selective formation of the nucleophilic intermediate at the N-terminus. This results in a stable carbon-carbon bond between the 2-PCs and the α-carbon of various N-terminal amino acids. The reaction is applied to four different proteins, including biopharmaceuticals such as filgrastim and trastuzumab. The modified trastuzumab retains the human epidermal growth factor receptor 2 recognition activity.     

Combination of two-dimensional gel electrophoresis with microsequencing and amino acid composition analysis: improvement of speed and sensitivity in protein characterization

High-resolution two-dimensional polyacrylamide electrophoresis (2-DE) is commonly used as an analytical approach to resolve and detect most of the numerous protein species of an organism. However, the isolation of microgram amounts of protein in a 2-DE spot in a form suitable for microsequence analysis and amino acid composition analysis is a key step in the chemical characterization of these proteins. With the development of chemically inert membranes it is now possible to retain proteins present in low quantities from the polyacrylamide matrix with high yields. The immobilized proteins are suitable for direct sequence analysis and amino acid composition analysis. The combination of protein chemical and electrophoretic techniques makes it possible to obtain chemical information from subpicomole quantities of protein, resulting in the availability of a new set of biologically important proteins for structural analysis. This paper summarizes the methods and strategies for the chemical protein analysis of 2-DE spots in our laboratory.     

A synthetic reaction network: chemical amplification using nonequilibrium autocatalytic reactions coupled in time

This article reports a functional chemical reaction network synthesized in a microfluidic device. This chemical network performs chemical 5000-fold amplification and shows a threshold response. It operates in a feedforward manner in two stages: the output of the first stage becomes the input of the second stage. Each stage of amplification is performed by a reaction autocatalytic in Co(2+). The microfluidic network is used to maintain the two chemical reactions away from equilibrium and control the interactions between them in time. Time control is achieved as described previously (Angew. Chem., Int. Ed. 2003, 42, 768) by compartmentalizing the reaction mixture inside plugs which are aqueous droplets carried through a microchannel by an immiscible fluorinated fluid. Autocatalytic reaction displayed sensitivity to mixing; more rapid mixing corresponded to slower reaction rates. Synthetic chemical reaction networks may help understand the function of biochemical reaction networks, the goal of systems biology. They may also find practical applications. For example, the system described here may be used to detect visually, in a simple format, picoliter volumes of nanomolar concentrations of Co(2+), an environmental pollutant.     

Ethanol Oxidation Reaction Mechanism on Gold Nanowires from Density Functional Theory

Thin gold nanowires (NWs) are materials that could be used as support in different chemical reactions. Using density functional theory (DFT) it was shown that NWs that form linear atomic chains (LACs) are suitable for stimulating chemical reactions. To this end, the oxidation reaction of ethanol supported on the LACs of Au−NWs was investigated. Two types of LACs were used for the study, one pure and the other with an oxygen impurity. The results showed that the oxygen atom in the LAC fulfills important functions throughout the reaction pathway. Before the chemical reaction, it was observed that the LAC with impurity gains structural stability, that is, the oxygen acts as an anchor for the gold atoms in the LAC. In addition, the LAC was shown to be sensitive to disturbances in its vicinity, which modifies its nucleophilic character. During the chemical reaction, the oxidation of ethanol occurs through two different reaction paths and in two stages, both producing acetaldehyde (CH₃CHO). The different reaction pathways are a consequence of the presence of oxygen in the LAC (oxygen conditions the formation of reaction intermediates). In addition, the oxygen in the LAC also modifies the kinetic behavior in both reaction stages. It was observed that, by introducing an oxygen impurity in the LAC, the activation energy barriers decrease ∼69 % and ∼97 % in the first and second reaction stages, respectively.     

Reagentless fluorescent biosensors based on proteins for continuous monitoring systems

There is a lack of commercially available efficient and autonomous systems capable of continuous monitoring of (bio)chemical data for clinical, environmental, food, or industrial samples. The weakest link in the design of these systems is the (bio)chemical receptor (bCR). The bCR should have transducer ability, the recognition event should be a single reaction, and the bCR should be easily regenerated. Transport proteins and enzymes are well placed as bCR for optical continuous monitoring systems (OCMS). In this paper we review quantitative aspects and the main transducer strategies which have been developed for transport proteins, using periplasmic binding proteins (linking an environmentally sensitive fluorophore or FRET between two fluorophores) and concanavalin A (competitive reversible assays) as representative examples. Efficient immobilization systems and implementation in OCMS are also reviewed. Some kinds of enzymes can fulfil the necessary requirements to be appropriate bCR. Strategies using flavoenzymes chemically modified with fluorophores can be successfully implemented in OCMS and they are, in our opinion, the most appropriate option.     

A Type of Auxiliary for Native Chemical Peptide Ligation beyond Cysteine and Glycine Junctions

Native chemical ligation enables the chemical synthesis of proteins. Previously, thiol‐containing auxiliary groups have been used to extend the reaction scope beyond N‐terminal cysteine residues. However, the N‐benzyl‐type auxiliaries used so far result in rather low reaction rates. Herein, a new N^α‐auxiliary is presented. Consideration of a radical fragmentation for cleavage led to the design of a new auxiliary group which is selectively removed under mildly basic conditions (pH 8.5) in the presence of TCEP and morpholine. Most importantly and in contrast to previously described auxiliaries, the 2‐mercapto‐2‐phenethyl auxiliary is not limited to Gly‐containing sites and ligations succeed at sterically demanding junctions. The auxiliary is introduced in high yield by on‐resin reductive amination with commercially available amino acid building blocks. The synthetic utility of the method is demonstrated by the synthesis of two antimicrobial proteins, DCD‐1L and opistoporin‐2.     

Intein-mediated synthesis of geranylgeranylated Rab7 protein in vitro

Production of recombinant proteins is an important prerequisite for biotechnology and life sciences in general. However, there is a paucity of methods for production of posttranslationally modified recombinant proteins or proteins with non-native functional groups, such as fluorophores, spin labels, and so forth. In this work we have used a combination of organic synthesis and in vitro protein ligation to construct monoprenylated Rab7 GTPase. The protein was prepared from a recombinant N-terminal portion and a peptide mimicking the C terminus of Rab7. For construction of a synthetic six-amino-acid-long fluorescent monoprenylated peptide, we used a block condensation strategy. Ligation was achieved with a yield of >70%. The resulting protein was purified from the unligated peptide by a combination of organic extraction and phase partitioning and refolding. The refolded monoprenylated semisynthetic Rab7 protein (Rab7GG) formed a stable complex with its natural chaperone REP-1 (Rab escort protein 1) and could serve as an acceptor of the second prenyl group in the enzymatic prenylation reaction. Using fluorescence spectroscopy, we characterized the interaction of the Rab7GG:REP-1 complex with Rab geranylgeranyl transferase and came to the conclusion that it functioned as a genuine intermediate of the prenylation reaction. Thus, we present the first example of the in vitro generation of a semisynthetic lipidated protein using the native chemical ligation method.     

Evaluation of bicinchoninic acid as a ligand for copper(i)-catalyzed azide-alkyne bioconjugations

The Cu(i)-catalyzed cycloaddition of terminal azides and alkynes (click chemistry) represents a highly specific reaction for the functionalization of biomolecules with chemical moieties such as dyes or polymer matrices. In this study we evaluate the use of bicinchoninic acid (BCA) as a ligand for Cu(i) under physiological reaction conditions. We demonstrate that the BCA-Cu(i)-complex represents an efficient catalyst for the conjugation of fluorophores or biotin to alkyne- or azide-functionalized proteins resulting in increased or at least equal reaction yields compared to commonly used catalysts like Cu(i) in complex with TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) or BPAA (bathophenanthroline disulfonic acid). The stabilization of Cu(i) with BCA represents a new strategy for achieving highly efficient bioconjugation reactions under physiological conditions in many application fields.     

Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization.

The highly chemoselective reaction between unprotected peptides bearing an N-terminal Cys residue and a C-terminal thioester enables the total and semi-synthesis of complex polypeptides. Here we extend the utility of this native chemical ligation approach to non-cysteine containing peptides. Since alanine is a common amino acid in proteins, ligation at this residue would be of great utility. To achieve this goal, a specific alanine residue in the parent protein is replaced with cysteine to facilitate synthesis by native chemical ligation. Following ligation, selective desulfurization of the resulting unprotected polypeptide product with H(2)/metal reagents converts the cysteine residue to alanine. This approach, which provides a general method to prepare alanyl proteins from their cysteinyl forms, can be used to chemically synthesize a variety of polypeptides, as demonstrated by the total chemical syntheses of the cyclic antibiotic microcin J25, the 56-amino acid streptococcal protein G B1 domain, and a variant of the 110-amino acid ribonuclease, barnase.     

Chemical Synthesis of Proteins with Non‐Strategically Placed Cysteines Using Selenazolidine and Selective Deselenization

Although native chemical ligation has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. The challenging protein, 125‐residue human phosphohistidine phosphatase 1 (PHPT1), has three cysteines near the C‐terminus, which are not strategically placed for ligation. Herein, we report the first sequential native chemical ligation/deselenization reaction. PHPT1 was prepared from three unprotected peptide segments using two ligation reactions at cysteine and alanine junctions. Selenazolidine was utilized as a masked precursor for N‐terminal selenocysteine in the middle segment, and, following ligation, deselenization provided the native alanine residue. This approach was used to synthesize both the wild‐type PHPT1 and an analogue in which the active‐site histidine was substituted with the unnatural and isosteric amino acid β‐thienyl‐l ‐alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site.     

Reactive Chemical Probes: Beyond the Kinase Cysteinome

The reaction of small‐molecule chemical probes with proteins has been harnessed to develop covalent inhibitor drugs and protein‐profiling technologies. This Essay discusses some of the recent enhancements to the chemical biology toolkit that are enabling the study of previously unchartered areas of chemoproteomic space. An analysis of the kinome is used to illustrate the potential for these approaches enable the pursuit of new targets using reactive chemical probes.     

Synthesis of fluorescently labeled mono- and diprenylated Rab7 GTPase

Modification of proteins with isoprenoid lipids is a widespread phenomenon in eukaryotic organisms that has received much attention due to its involvement in the progression of several diseases including cancer. Progress in studies of prenylated proteins has been hampered by difficulties associated with isolation of these proteins from native or recombinant sources. Small GTPases of the Rab family represent a particularly difficult example since they are doubly C-terminally geranylgeranylated and in some cases methylated. Here, we report an efficient and versatile strategy for the synthesis of mono- and digeranylgeranylated fluorescent RabGTPases using a combination of chemical synthesis and expressed protein ligation. Using this approach we generated fluorescent mono- and diprenylated Rab7 proteins that display near-native properties and form stoichiometric complexes with their natural chaperone REP-1. We demonstrate that the complex formed from semisynthetic monoprenylated Rab7 and REP-1 represents a genuine intermediate of the Rab prenylation reaction and thus provides a unique tool for studies of the Rab prenylation mechanism. Semisynthetic Rab7 proteins were used to develop a novel fluorescence-based in vitro prenylation assay. Using this assay we dissected the mechanism of the Rab7 double-geranylgeranylation reaction mediated by Rab geranylgeranyl transferase. We conclude that the reaction follows a random sequential mechanism. These results highlight the usefulness of the semisynthetic reaction intermediates in the study of protein posttranslational modification.