Chemical biology of protein lipidation: semi-synthesis and structure elucidation of prenylated RabGTPases

Rab/Ypt guanosine triphosphatases (GTPases) represent a family of key membrane traffic regulators in eukaryotic cells. For their function Rab/Ypt proteins require double modification with two covalently bound geranylgeranyl lipid moieties at the C-terminus. Generally, prenylated proteins are very difficult to obtain by recombinant or enzymatic methods. We generated prenylated RabGTPases using a combination of chemical synthesis and protein engineering. This semi-synthesis depends largely on the availability of functionalized prenylated peptides corresponding to the proteins' native structure or modifications. We developed solution phase and solid phase strategies for the generation of peptides corresponding to the prenylated C-terminus of Rab7 GTPase in preparative amounts enabling us to crystallize the mono-prenylated Ypt1:RabGDI complex. The structure of the complex provides a structural basis for the ability of RabGDI to inhibit the release of nucleotide by Rab proteins and a molecular basis for understanding a RabGDI mutant that causes mental retardation in humans.     

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.     

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.     

A photoactivatable prenylated cysteine designed to study isoprenoid recognition.

Protein prenylation, involving the alkylation of a specific C-terminal cysteine with a C(15) or C(20) isoprenoid unit, is an essential posttranslational modification required by most GTP-binding proteins for normal biological activity. Despite the ubiquitous nature of this modification and numerous efforts aimed at inhibiting prenylating enzymes for therapeutic purposes, the function of prenylation remains unclear. To explore the role the isoprenoid plays in mediating protein-protein recognition, we have synthesized a photoactivatable, isoprenoid-containing cysteine analogue (2) designed to act as a mimic of the C-terminus of prenylated proteins. Photolysis experiments with 2 and RhoGDI (GDI), a protein which interacts with prenylated Rho proteins, suggest that the GDI is in direct contact with the isoprenoid moiety. These results, obtained using purified GDI as well as Escherichia coli (E. coli) crude extract containing GDI, suggest that this analogue will be an effective and versatile tool for the investigation of putative isoprenoid binding sites in a variety of systems. Incorporation of this analogue into peptides or proteins should allow for even more specific interactions between the photoactivatable isoprenoid and any number of isoprenoid binding proteins.     

One‐Pot N2C/C2C/N2N Ligation To Trap Weak Protein–Protein Interactions

Weak transient protein–protein interactions (PPIs) play an essential role in cellular dynamics. However, it is challenging to obtain weak protein complexes owing to their short lifetime. Herein we present a general and facile method for trapping weak PPIs in an unbiased manner using proximity‐induced ligations. To expand the chemical ligation spectrum, we developed novel N2N (N‐terminus to N‐terminus) and C2C (C‐terminus to C‐terminus) ligation approaches. By using N2C (N‐terminus to C‐terminus), N2N, and C2C ligations in one pot, the interacting proteins were linked. The weak Ypt1:GDI interaction drove C2C ligation with t_1/2 of 4.8 min and near quantitative conversion. The Ypt1‐GDI conjugate revealed that binding of Ypt1 G‐domain causes opening of the lipid‐binding site of GDI, which can accommodate one prenyl group, giving insights into Rab membrane recycling. Moreover, we used this strategy to trap the KRas homodimer, which plays an important role in Ras signaling.     

Solid-phase organic tagging resins for labeling biomolecules by 1,3-dipolar cycloaddition: application to the synthesis of a fluorescent non-peptidic vasopressin receptor ligand

Two novel solid-phase organic tagging (SPOrT) resins were synthesized to facilitate the labeling of peptides and small organic compounds with a fluorescent probe. Both resins were obtained from the commercially available backbone amide linker (BAL) resin. Following the solid-phase synthesis of model compounds, a tripeptide and benzazepine, the fluorescent probe derived from Lissamine Rhodamine B was incorporated through CuI-catalyzed 1,3-dipolar cycloaddition. Final cleavage in acidic media enabled access to both types of molecules in good yield with high purity. The SPOrT resin was successfully applied to the preparation of the first non-peptidic fluorescent compound with a nanomolar affinity for the human vasopressin V2 receptor (V2R) subtype. This molecule will find application in binding assays that use polarization or fluorescence resonance energy-transfer (FRET) techniques. The SPOrT resins are also well suited for other tags and the parallel synthesis of a fluorescently tagged library for protein screening.     

The synthesis of acid- and base-labile lipopeptides on solid support

Lipidated peptides, including characteristic partial structures of human Ras proteins, were synthesized by means of a new solid-phase technique in 22-68 % yield. This technique gives access to farnesylated, palmitoylated, and doubly lipidated peptides as methyl esters or carboxylic acids carrying a fluorescent tag or a maleimide moiety for coupling to proteins. The peptide backbones were built up on the resin by using 9-fluorenylmethoxycarbonyl chemistry together with the oxidatively cleavable hydrazide linker. As a key step, the acid-labile farnesyl and basic-labile palmitoyl lipid groups were introduced onto the resin after the cleavage of appropriate acid- or reduction-sensitive protecting groups from the cysteine residues. Optional introduction of different fluorescent tags or a maleimide group into the peptide was followed by release of the resin-bound target peptide as the methyl ester or carboxylic acid by very mild copper(II)-mediated oxidation in slightly acidic or basic media. This new methodology should substantially facilitate the access to lipidated peptides for the study of important biological phenomena like biological signal transduction, localization, and vesicular transport.     

Solid-phase synthesis of lipidated peptides

A new flexible and efficient methodology for the solid-phase synthesis of lipidated peptides has been developed. The approach is based on the use of previously synthesized building blocks and overcomes the limitations of previously reported methods, since long doubly lipidated peptides can be synthesized by using this route. Furthermore, it was thus possible to prepare a large number of N- and H-Ras peptides bearing a wide range of reporter and/or linking groups--efficient tools for the investigation of biological processes. In terms of efficiency and flexibility this solid-phase method is superior to the solution-phase synthesis. It gives pure peptides in multimilligram amounts within a much shorter time and with superior overall yield.     

Traceless Purification and Desulfurization of Tau Protein Ligation Products

We present a novel strategy for the traceless purification and synthetic modification of peptides and proteins obtained by native chemical ligation. The strategy involves immobilization of a photocleavable semisynthetic biotin–protein conjugate on streptavidin‐coated agarose beads, which eliminates the need for tedious rebuffering steps and allows the rapid removal of excess peptides and additives. On‐bead desulfurization is followed by delivery of the final tag‐free protein product. The strategy is demonstrated in the isolation of a tag‐free Alzheimer's disease related human tau protein from a complex EPL mixture as well as a triphosphorylated peptide derived from the C‐terminus of tau.     

The (2-phenyl-2-trimethylsilyl)ethyl-(PTMSEL)-linker in the synthesis of glycopeptide partial structures of complex cell surface glycoproteins

The (2-phenyl-2-trimethylsilyl)ethyl-(PTMSEL) linker represents a novel fluoride-sensitive anchor for the solid-phase synthesis of protected peptides and glycopeptides. Its cleavage is achieved under almost neutral conditions using tetrabutylammonium fluoride trihydrate in dichloromethane thus allowing the construction of complex molecules sensitive to basic and acidic media commonly required for the cleavage of standard linker systems. The advantages of the PTMSEL linker are demonstrated in the synthesis of glycopeptides from the liver intestine (LI)-cadherin and the mucin MUC1, bearing carbohydrate moieties such as N-linked chitobiose or O-linked sialyl-T(N)-residues. The synthesis of these types of glycopeptides is difficult because they are prone to secondary structure formation during the synthesis on the solid phase as well as in the completely deprotected form. Using the PTMSEL linker these molecules are accessible by automated synthesis according to the Fmoc strategy without frequently observed side reactions such as aspartimide or diketopiperazine formation.     

A New Highly Versatile Handle for Chemistry on a Solid Support: The Pipecolic Linker

The design, synthesis, and potential application of the pipecolic linker is presented. This new versatile handle can immobilize primary, secondary, and aromatic amines, as well as alcohols, phenols, and hydrazides, on a solid support. Compared with other linkers, the anchoring step is easy and efficient. The release of final products from the resin proceeds upon acidic treatment with high purities. The pipecolic linker offers the promise of being using in peptide chemistry to produce peptides modified at the N and C terminus, peptidomimetics, as well as small organic molecules.     

Solid-phase synthesis of DOTA-peptides

A general synthetic route to two DOTA-linked N-Fmoc amino acids (DOTA-F and DOTA-K) is described that allows insertion of DOTA at any endo-position within a peptide sequence. Three model pentapeptides were prepared to test the general utility of these derivatives in solid-phase peptide synthesis. Both DOTA derivatives reacted smoothly by means of standard HBTU activation chemistry to the point of insertion of the DOTA amino acid, but extension of the peptide chain beyond the DOTA-amino acid insertion required the use of pre-activated C-pentafluorophenyl ester N-alpha-Fmoc amino acids. Three Gal-80 binding peptides (12-mers) were then prepared by using this methodology with DOTA positioned either at the N terminus or at one of two different internal positions;the binding of the resulting GdDOTA-12-mers to Gal-80 were compared. The methodology described here allows versatile, controlled introduction of DOTA into any location within a peptide sequence. This provides a potential method for the screening of libraries of DOTA-linked peptides for optimal targeting properties.     

Identification of formylglycine in sulfatases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

C(alpha)-Formylglycine, the catalytic amino acid residue in the active site of sulfatases, is generated by post-translational modification of a cysteine or serine residue. We describe a highly sensitive procedure for the detection of C(alpha)-formylglycine-containing peptides in tryptic digests of sulfatase proteins. The protocol is based on the formation of hydrazone derivatives of C(alpha)-formylglycine-containing peptides when using dinitrophenylhydrazine as a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives desorb and ionize with high efficiency and can be detected in the sub-femtomole range. The presence of C(alpha)-formylglycine is indicated by a mass increment of 180.13 u, corresponding to the hydrazone moiety, and also by a unique C-terminal fragment ion, characteristic of sulfatases, that becomes prominent in MALDI post-source decay mass spectra of the hydrazone derivatives.     

Labeling and Protecting N‐Terminal Protein Positions by β‐Peptidyl Aminopeptidase‐Catalyzed Attachment of β‐Amino‐Acid Residues – Insulin as a First Example

We have shown for the first time that a natural protein (human insulin) can be acylated at the N‐terminus with a β‐amino acid (H‐β³hAla‐), in a process catalyzed by the β‐peptidyl aminopeptidase 3‐2W4‐BapA. This selective modification, which could also be applied for protein labeling and tagging, should be generally useful, also to protect peptides and proteins from attack by common aminopeptidases.     

Chemo‐Enzymatic Synthesis of Fluorescent Rab 7 Proteins: Tools to Study Vesicular Trafficking in Cells

The N ‐methylanthraniloylisoprenoid diphosphate derivatives 1 and 2 bind to RabGGTase II and are enzymatically transferred to Rab 7 (the Rab proteins are small G‐proteins that control events of docking and fusion of intracellular vesicles). The fluorescent Rab 7 proteins thus obtained may become important tools for further biological studies on vesicular trafficking in cells.     

Preparation of tripeptide-bridged dicatechol ligands and their macrocyclic molybdenum(VI) complexes: fixation of the RGD sequence and the WKY sequence of urotensin ii in a cyclic conformation

Dicatechol ligands were prepared with caprylic acid (6-H(4)) or the naturally occurring RGD (23-H(4)) or WKY sequences (32-H(4)) as spacers. 6-H(4) was prepared by solution-phase amide coupling chemistry, while 16, the precursor of 23-H(4), was obtained by solution-phase and solid-phase preparation. In the latter case, a polystyrene resin with a hydrazine benzoate linker was used as the solid support. The last coupling step was performed simultaneously with cleavage of the peptide from the resin. The protecting groups of 16 were all removed in one step to yield the free ligand 23-H(4). The WKY-bridged derivative 32-H(4) was obtained by a similar solid-phase synthesis followed by deprotection. The reaction of all three ligands with dioxomolybdenum(VI) bis(acetylacetonate) afforded 19-membered metallamacrocycles in which the short peptides are conformationally fixed in a turn-type structure. Hereby, the side-chain functionalities of the peptides do not interfere in the metal complexation.     

Isotope-coded dimethyl tagging for differential quantification of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-product of lipid peroxidation

Peroxidation of cellular membrane lipids, rich in polyunsaturated fatty acids, generates electrophilic, α, β-unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE). HNE is a highly reactive and cytotoxic molecule that can react with the nucleophilic sites in proteins causing posttranslational modification. The identification of protein targets is an important first step; however, quantitative profiling of site-specific modifications is necessary to understand the biological impact of HNE-induced carbonylation. We report a method that uses light (H(12)CHO) and heavy (D(13)CDO) isotopic variant of formaldehyde to differentially label primary amines (N-termini and ε-amino group of lysines) in peptides through reductive methylation and, combined with selective enrichment of modified peptides, permits comparison of the extent of carbonylation in two samples after mixing for simultaneous liquid chromatography-mass spectrometry. Specifically, dimethyl-labeled peptide carbonyls were fractionated from unmodified peptides using solid-phase hydrazide chemistry to immobilize them to porous glass beads and, after removing the unmodified peptides by thoroughly washing the beads, subsequently recover them by acid-catalyzed hydrolysis. The method was developed using HNE-modified synthetic peptides and also showing enrichment from a complex matrix of digested human plasma proteins. Applicability was confirmed using apomyoglobin as an analyte, implicating thereby its potential value to proteome-wide identification and relative quantification of posttranslational protein carbonylation with residue-specific information. Because HNE attachment may not necessarily cause change in protein abundance, this modification-focused quantification should facilitate the characterization of accompanied changes in protein function and, also, provide important insights into molecular signaling mechanisms and a better understanding of cellular processes associated with oxidative stress.     

Aryldithioethyloxycarbonyl (Ardec): a new family of amine protecting groups removable under mild reducing conditions and their applications to peptide synthesis

The development of phenyldithioethyloxycarbonyl (Phdec) and 2-pyridyldithioethyloxycarbonyl (Pydec) protecting groups, which are thiol-labile urethanes, is described. These new disulfide-based protecting groups were introduced onto the epsilon-amino group of L-lysine; the resulting amino acid derivatives were easily converted into N alpha-Fmoc building blocks suitable for both solid- and solution-phase peptide synthesis. Model dipeptide(Ardec)s were prepared by using classical peptide couplings followed by standard deprotection protocols. They were used to optimize the conditions for complete thiolytic removal of the Ardec groups both in aqueous and organic media. Phdec and Pydec were found to be cleaved within 15 to 30 min under mild reducing conditions: i) by treatment with dithiothreitol or beta-mercaptoethanol in Tris.HCl buffer (pH 8.5-9.0) for deprotection in water and ii) by treatment with beta-mercaptoethanol and 1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) in N-methylpyrrolidinone for deprotection in an organic medium. Successful solid-phase synthesis of hexapeptides Ac-Lys-Asp-Glu-Val-Asp-Lys(Ardec)-NH2 has clearly demonstrated the full orthogonality of these new amino protecting groups with Fmoc and Boc protections. The utility of the Ardec orthogonal deprotection strategy for site-specific chemical modification of peptides bearing several amino groups was illustrated firstly by the preparation of a fluorogenic substrate for caspase-3 protease containing the cyanine dyes Cy 3.0 and Cy 5.0 as FRET donor/acceptor pair, and by solid-phase synthesis of an hexapeptide bearing a single biotin reporter group.     

Synthesis and characterization of a new modification of PtAl

A new PtAl phase, which is stable at room temperature under oxidizing conditions, has been synthesized at 200 °C and 5 MPa. X‐ray studies reveal it to be different from the known polymorphs of PtAl, which crystallize in the FeSi and CsCl structure types. The crystal structure of the new PtAl modification is found to be isotypical with the low‐temperature modification of PdAl. In situ high‐temperature X‐ray diffraction experiments in air were performed to study the thermal behavior of the new PtAl alloy. In the temperature range between 400 and 700 °C, Pt₅Al₃ forms as an intermediate phase. At higher temperature the alloy decomposes, resulting in the formation of platinum and Al₂O₃. The thermodynamic instability at high temperatures explains why this new modification has not been observed using contemporary metallurgic processes. Copyright © 2003 John Wiley & Sons, Ltd.     

Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation

BACKGROUND: The site-specific chemical modification of proteins has proved to be extremely powerful for generating tools for the investigation of biological processes. Although a few elegant methods exist for engineering a recombinant protein at a unique position, these techniques cannot be easily extended to allow several different chemical probes to be specifically introduced into a target sequence. As such multiply labeled proteins could be used to study many biological processes, and in particular biomolecular interactions, we decided to investigate whether such protein reagents could be generated using an extension of the semisynthesis technique known as expressed protein ligation. RESULTS: A solid-phase expressed protein ligation (SPPL) technology is described that enables large semisynthetic proteins to be assembled on a solid support by the controlled sequential ligation of a series of recombinant and synthetic polypeptide building blocks. This modular approach allows multiple, different chemical modifications to be introduced site-specifically into a target protein. This process, which is analogous to solid-phase peptide synthesis, was used to dual-label the amino and carboxyl termini of the Crk-II adapter protein with the fluorescence resonance energy transfer pair tetramethylrhodamine and fluorescein, respectively. The resulting construct reports (through a fluorescence change) the phosphorylation of Crk-II by the nonreceptor protein tyrosine kinase, c-Abl, and was used to probe the protein-protein interactions that regulate this important post-translational process. CONCLUSIONS: SPPL provides a powerful method for specifically modifying proteins at multiple sites, as was demonstrated by generating a protein-based biosensor for Crk-II phosphorylation. Such protein derivatives are extremely useful for investigating protein function in vitro and potentially in vivo. This modular approach should be applicable to many different protein systems.