Selective Tryptophan Modification with Rhodium Carbenoids in Aqueous Solution

A new transition metal-based reaction has been developed for the selective modification of tryptophan residues on protein substrates. After activation of vinyl-substituted diazo compounds by Rh2(OAc)4, the resulting metallocarbenoid intermediates were found to modify indoles in aqueous media despite competing reactions with water. Both N- and 2-substituted indole products were observed in the reaction. Following initial small-molecule studies, the reaction was performed on two protein substrates. Both myoglobin and subtilisin Carlsberg were modified readily in aqueous solution, and the tryptophan selectivity of the reactions was confirmed through MS analyses of trypsin digest fragments. It was also demonstrated that myoblobin concentrations as low as 10 μM still led to appreciable levels of modification. Reconstitution experiments confirmed that myoglobin retained its ability to bind heme following modification

Chemoselective Tryptophan Labeling with Rhodium Carbenoids at Mild pH

Significant improvements have been made to a previously reported tryptophan modification method using rhodium carbenoids in aqueous solution, allowing the reaction to proceed at pH 6−7. This technique is based on the discovery that N-(tert-butyl)hydroxylamine promotes indole modification with rhodium carbenoids over a broad pH range (2−7). This methodology was demonstrated on peptide and protein substrates, generally yielding 40−60% conversion with excellent tryptophan chemoselectivity. The solvent accessibility of the indole side chains was found to be a key factor in successful carbenoid addition, as demonstrated by conducting the reaction at temperatures high enough to cause thermal denaturation of the protein substrate. Progress toward the expression of proteins bearing solvent accessible tryptophan residues as reactive handles for modification with rhodium carbenoids is also reported

Lipid Modification of Proteins through Sortase-Catalyzed Transpeptidation

A general chemoenzymatic method for the site-specific attachment of lipids to protein substrates is described. Sortase A is used to append short lipid-modified oligoglycine peptides to the C terminus of protein substrates bearing a five amino acid sortase A recognition sequence (LPETG). We demonstrate the attachment of a range of hydrophobic modifications in excellent yield (60−90%), including a simple step for removing the sortase enzyme postreaction. Lipoproteins prepared using these procedures were subsequently shown to associate with mammalian cells in a lipid tail-dependent fashion and localized to the plasma membrane and endosomes

Sustained Delivery of Chemokine CXCL12 from Chemically Modified Silk Hydrogels

A delivery platform was developed using silk-based hydrogels, and sustained delivery of the cationic chemokine CXCL12 at therapeutically relevant doses is demonstrated. Hydrogels were prepared from plain silk and silk that had been chemically modified with sulfonic acid groups. CXCL12 was mixed with the silk solution prior to gelation, resulting in 100% encapsulation efficiency, and both hydrated and lyophilized gels were compared. By attaching a fluorescein tag to CXCL12 using a site-specific sortase-mediated enzymatic ligation, release was easily quantified in a high-throughput manner using fluorescence spectroscopy. CXCL12 continually eluted from both plain and acid-modified silk hydrogels for more than 5 weeks at concentrations ranging from 10 to 160 ng per day, depending on the gel preparation method. Notably, acid-modified silk hydrogels displayed minimal burst release yet had higher long-term release rates compared to those of plain silk hydrogels. Similar release profiles were observed over a range of loading capacities, allowing dosage to be easily varied

Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using Sortases of Different Specificity

Site-Specific N- and C-Terminal Labeling of a Single Polypeptide Using Sortases of Different Specificit

Development of an <i>Influenza virus</i> Protein Array Using Sortagging Technology

Protein array technology is an emerging tool that enables high-throughput screening of protein–protein or protein–lipid interactions and identification of immunodominant antigens during the course of a bacterial or viral infection. In this work, we developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as “Sortagging”. LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular extracts were immobilized at their carboxyl-termini onto a preactivated amine-glass slide coated with a Gly3 linker. Immobilized proteins were revealed by specific antibodies, and the newly generated Sortag-protein chip can be used as a device for antigen and/or antibody screening. The specificity of the Sortase A (SrtA) reaction avoids purification steps in array building and allows immobilization of proteins in an oriented fashion. Previously, this versatile technology has been successfully employed for protein labeling and protein conjugation. Here, the tool is implemented to covalently link proteins of a viral genome onto a solid support. The system could readily be scaled up to proteins of larger genomes in order to develop protein arrays for high-throughput screening

Sequences of protein constructs used in this study.

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ESI-MS and SDS-PAGE characterization of segmentally labeled FH8-IDR-HP63 and IDR-HP63.

(A) Characterization data for FH8-IDR-HP63 with selective incorporation of 15N in the HP63 segment (calcd MW assuming 100% 15N incorporation in HP63 = 20797 Da). (B) Characterization data for FH8-IDR-HP63 with selective incorporation of 15N in the FH8-IDR segment (calcd MW assuming 100% 15N incorporation in FH8-IDR = 20859 Da). (C) Characterization data for IDR-HP63 (FH8 tag removed) with selective incorporation of 15N in the IDR segment (calcd MW assuming 100% 15N incorporation in IDR = 12202 Da). Calculated MW values are average molecular weight predicted using the BMRB Molecular Mass Calculator. * = MeCN adducts from LC-ESI-MS mobile phase (calcd Δmass for MeCN adduct = +41 Da). All gels were visualized by Coomassie staining, and molecular weight standards (kDa) are indicated to the left of each gel image. The original, uncropped gel images are also provided in S14 Fig. (PDF)</p

Overlaid ¹⁵N-HSQC spectra of FH8-IDR-HP63 and IDR-HP(877–974).

Superimposed 15N-HSQC spectra for FH8-IDR-HP63 segmentally labeled with 13C/15N in the FH8-IDR portion (red, recorded on a 600 MHz 1H frequency instrument) and uniformly 15N-labeled IDR-HP(877–974) (native sequence control, open blue contours, same spectrum as S9 Fig, recorded at 500 MHz 1H frequency). The filled red contours indicate segmentally assigned FH8-IDR-HP63 resonances (see Fig 5 in main text). An “X” signifies the eight FH8-IDR-HP63 resonances for which there are no overlapping or adjacent resonances in the IDR-HP(877–974) control. Seven out of these eight FH8-IDR-HP63 resonances were assigned to the introduced TEV site (L74Y75F76Q77G78), the single glycine spacer (G114), or the leucine (L115) of the sortase ligation site. Double arrows indicate four segmentally assigned resonances (solid red ovals) for which there are nearby matching signals in the native control (open blue contours). The length of every arrow is equal or smaller than 0.05 ppm in the 1H dimension. Primary structure diagrams are shown below the spectra for segmentally labeled FH8-IDR-H63 (sequence position numbering begins with the first residue of the FH8 domain) and uniformly labeled IDR-HP(877–974) (residue numbering corresponds to the sequence of native villin 4). Non-black coloration in the structure diagrams (red or blue) indicates the position of isotopic labels (13C and/or 15N). (PDF)</p

¹⁵N-HSQC NMR spectra (25°C) of segmentally labeled FH8-IDR-HP63 and IDR-HP63.

(A) Spectrum recorded at 600 MHz (1H frequency) with 256 indirect increments using a FH8-IDR-HP63 sample with the FH8-IDR portion segmentally labeled with 13C/15N isotopes. All observed resonances for the labeled FH8-IDR segment are represented by open red contours, and signals that have been segmentally assigned to the IDR linker portion are overlaid with solid red ovals. Residue-specific NMR assignments for 36 residues in the linker region (27 from the villin 4 IDR and 9 residues from the flanking TEV and sortase ligation sites) and four FH8 domain peaks (false positives from the segmental assignment) are indicated with residue numbers. The numbers for the TEV site (7 residues) are shown in grey, FH8 domain (4 residues) are shown in blue, and two residues corresponding to the SML ligation site (G114, L115) are shown in green. Seven segmentally assigned peaks for which no residue specific assignments were obtained are marked with “+”. Residue numbers shown correspond to the sequence of FH8-IDR-HP63 (see S2 Fig). To relate the assigned IDR residues to the corresponding positions in wild type villin 4, a value of 798 should be added. (B) Spectrum recorded at 500 MHz (1H frequency) with 196 indirect increments using an IDR-HP63 sample with the IDR segmentally labeled with 15N isotopes. All observed resonances for the labeled IDR segment are represented by solid red signals. Residue-specific NMR assignments for 25 residues in the linker region were unambiguously transferred from panel A (out of 27 listed on panel A). Three new resonances were observed (marked with “x”) following removal of FH8. Two of these are presumed to correspond to residues E79 and E80, which are adjacent to the newly formed N-terminal residue G78. The two residues corresponding to the SML ligation site (G114, L115) are shown in green. As in the case of FH8-IDR-HP63 (panel A), additional peaks (6 total) for which no residue specific assignments were obtained are marked with “+”.</p