Bioconjugation of Proteins with a Paramagnetic NMR and Fluorescent Tag

Site‐specific labeling of proteins with lanthanide ions offers great opportunities for investigating the structure, function, and dynamics of proteins by virtue of the unique properties of lanthanides. Lanthanide‐tagged proteins can be studied by NMR, X‐ray, fluorescence, and EPR spectroscopy. However, the rigidity of a lanthanide tag in labeling of proteins plays a key role in the determination of protein structures and interactions. Pseudocontact shift (PCS) and paramagnetic relaxation enhancement (PRE) are valuable long‐range structure restraints in structural‐biology NMR spectroscopy. Generation of these paramagnetic restraints generally relies on site‐specific tagging of the target proteins with paramagnetic species. To avoid nonspecific interaction between the target protein and paramagnetic tag and achieve reliable paramagnetic effects, the rigidity, stability, and size of lanthanide tag is highly important in paramagnetic labeling of proteins. Here 4′‐mercapto‐2,2′: 6′,2′′‐terpyridine‐6,6′′‐dicarboxylic acid (4MTDA) is introduced as a a rigid paramagnetic and fluorescent tag which can be site‐specifically attached to a protein by formation of a disulfide bond. 4MTDA can be readily immobilized by coordination of the protein side chain to the lanthanide ion. Large PCSs and RDCs were observed for 4MTDA‐tagged proteins in complexes with paramagnetic lanthanide ions. At an excitation wavelength of 340 nm, the complex formed by protein–4MTDA and Tb³⁺ produces high fluorescence with the main emission at 545 nm. These interesting features of 4MTDA make it a very promising tag that can be exploited in NMR, fluorescence, and EPR spectroscopic studies on protein structure, interaction, and dynamics.     

3‐Mercapto‐2,6‐Pyridinedicarboxylic Acid: A Small Lanthanide‐Binding Tag for Protein Studies by NMR Spectroscopy

Paramagnetic effects from lanthanide ions present powerful tools for protein studies by nuclear magnetic resonance (NMR) spectroscopy provided that the lanthanide can be site‐specifically and rigidly attached to the protein. A new, particularly small and rigid lanthanide‐binding tag, 3‐mercapto‐2,6‐pyridinedicarboxylic acid (3MDPA), was synthesized and attached to two different proteins via a disulfide bond. The complexes of the N‐terminal domain of the E. coli arginine repressor (ArgN) with seven different paramagnetic lanthanide ions and Co²⁺ were analyzed in detail by NMR spectroscopy. The magnetic susceptibility anisotropy (Δχ) tensors and metal position were determined from pseudocontact shifts. The 3MDPA tag generated very different Δχ tensor orientations compared to the previously studied 4‐mercaptomethyl‐DPA tag, making it a highly complementary and useful tool for protein NMR studies.     

4,4'-dithiobisdipicolinic acid: a small and convenient lanthanide binding tag for protein NMR spectroscopy

Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structure restraints for studies of proteins by nuclear magnetic resonance spectroscopy. To elicit PCSs, the lanthanide must be attached site-specifically to the target protein. In addition, it needs to be attached rigidly to avoid averaging of the PCSs due to mobility with respect to the protein and it must not interfere with the function of the protein. Here, we present a dipicolinic acid reagent that spontaneously forms a disulfide bond with thiol groups of accessible cysteine residues. A minimal number of rotatable bonds between the cysteine side chain and the tag helps to minimise mobility. Combined with the small size of the tag and quantitative tagging yields, these features make it a highly attractive tool for generating structure restraints by paramagnetic lanthanides.     

New chemistry for the study of multiprotein complexes: the six-histidine tag as a receptor for a protein crosslinking reagent

Background: To study very large macromolecular complexes, it would be useful to be able to incorporate probe molecules, such as fluorescent tags or photoactivatable crosslinkers, into specific sites on proteins. Current methods for doing this use relatively large amounts of highly purified protein, limiting the general utility of these approaches. The need for covalent posttranslational chemistry also makes it extremely difficult to use modified proteins in studies of native complexes in crude lysates or in living cells. We set out to develop a protein tag that would circumvent these problems.Results: A very simple type of molecular recognition, metal-ligand complexation, can be used to deliver a nickel-based crosslinking reagent to proteins containing a six-histidine (His₆) tag. When activated with a peracid, the His₆-Ni complex mediates oxidative crosslinking of nearby proteins. The crosslinking reaction does not involve freely diffusible intermediates, and thus only those proteins in close proximity to the His₆-tagged polypeptide are crosslinked.Conclusions: The His₆ tag, commonly used as an affinity handle for the purification of recombinant proteins, can also be used as an internal receptor for an oxidative protein-crosslinking reagent. No covalent protein modifications are necessary, since the Hiss tag is introduced at the DNA level. The crosslinking reaction is fast, efficient in most cases, and provides products that are easily separated from most other proteins present. This methodology should find widespread use in the study of multiprotein complexes.     

A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR,EPR and Time-Resolved Luminescence Studies

A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral Ln^III tag, referred to as C12 , is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with Tb^III or Tm^III ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron–electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the Tb^III complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.     

Specific ion effects at protein surfaces: a molecular dynamics study of bovine pancreatic trypsin inhibitor and horseradish peroxidase in selected salt solutions

The distribution of sodium, choline, sulfate, and chloride ions around two proteins, horseradish peroxidase (HRP) and bovine pancreatic trypsin inhibitor (BPTI), is investigated by means of molecular dynamics simulations with the aim to elucidate ion adsorption at the protein surface. Although the two proteins under investigation are very different from each other, the ion distributions around them are remarkably similar. Sulfate is always strongly attached to the proteins, choline shows a significant, but unspecific, propensity for the protein surfaces, and sodium ions have a weak surface affinity, while chloride has virtually no preference for the protein surface. In mixtures of all four ion species in protein solutions, the resulting distributions are almost a superposition of the distributions of sodium sulfate and choline chloride, except that sodium partially replaces choline close to the proteins. The present simulations support a picture of ions interacting with individual ionic and polar amino acid groups rather than with an averaged protein surface. The results thus show how subtle the so-called Hofmeister and electroselectivity effects are in salt solution of proteins, making all simplified interaction models questionable.     

Convenient synthesis of multifunctional EDTA-based chiral metal chelates substituted with an S-mesylcysteine

We describe the synthetic route to ethylenediaminetetraacetic acid (EDTA) derivatives that can be attached to surface-exposed thiol functional groups of cysteine residues in proteins, via a methylthiosulfonate moiety that is connected in a stereochemically unique way to the C-1 carbon atom of EDTA. Such compounds can be used to align proteins in solution without the need to add liquid crystalline media, and are, therefore, of great interest for the NMR spectroscopic analysis of biomolecules. The binding constant for the paramagnetic tag to lanthanide ions was determined by measuring luminescence. For the Tb(+3)-ligand complex, a K(b) value of 6.5 x 10(17) M(-1) was obtained. This value is in excellent agreement with literature values for the related EDTA compound. In addition, it could be shown that there is no significant reduction in the luminescence intensity upon addition of a 10(4) excess of Ca2+ ions, indicating that this paramagnetic tag is compatible with buffers containing high concentrations of divalent alkaline earth ions.     

Structure determination of protein-ligand complexes by transferred paramagnetic shifts

Rational drug design depends on the knowledge of the three-dimensional (3D) structure of complexes between proteins and lead compounds of low molecular weight. A novel nuclear magnetic resonance (NMR) spectroscopy strategy based on the paramagnetic effects from lanthanide ions allows the rapid determination of the 3D structure of a small ligand molecule bound to its protein target in solution and, simultaneously, its location and orientation with respect to the protein. The method relies on the presence of a lanthanide ion in the protein target and on fast exchange between bound and free ligand. The binding affinity of the ligand and the paramagnetic effects experienced in the bound state are derived from concentration-dependent (1)H and (13)C spectra of the ligand at natural isotopic abundance. Combined with prior knowledge of the crystal or solution structure of the protein and of the magnetic susceptibility tensor of the lanthanide ion, the paramagnetic data define the location and orientation of the bound ligand molecule with respect to the protein from simple 1D NMR spectra. The method was verified with the ternary 30 kDa complex between the lanthanide-labeled N-terminal domain of the epsilon exonuclease subunit from the Escherichia coli DNA polymerase III, the subunit theta, and thymidine. The binding mode of thymidine was found to be very similar to that of thymidine monophosphate present in the crystal structure.     

Design, synthesis, and evaluation of a lanthanide chelating protein probe: CLaNP-5 yields predictable paramagnetic effects independent of environment

Immobilized lanthanide ions offer the opportunity to refine structures of proteins and the complexes they form by using restraints obtained from paramagnetic NMR experiments. We report the design, synthesis, and spectroscopic evaluation of the lanthanide chelator, Caged Lanthanide NMR Probe 5 (CLaNP-5) readily attachable to a protein surface via two cysteine residues. The probe causes tunable pseudocontact shifts, alignment, paramagnetic relaxation enhancement, and luminescence, by chelating it to the appropriate lanthanide ion. The observation of single shifts and the finding that the magnetic susceptibility tensors obtained from shifts and alignment analyses are highly similar strongly indicate that the probe is rigid with respect to the protein backbone. By placing the probe at various positions on a model protein it is demonstrated that the size and orientation of the magnetic susceptibility tensor of the probe are independent of the local protein environment. Consequently, the effects of the probe are readily predictable using a protein structure only. These findings designate CLaNP-5 as a protein probe to deliver unambiguous high quality structural restraints in studies on protein-protein and protein-ligand interactions.     

Immobilization of histidine-tagged recombinant proteins onto micropatterned surfaces for cell-based functional assays

This letter describes a method for preparing protein microarrays that allow the functional analysis of proteins at a cellular level. This method involves the utilization of recombinant proteins genetically engineered to carry a fusion tag that has an affinity for metal ions. A micropatterned alkanethiol monolayer was used to prepare a microarray having multiple spots with immobilized metal ions. The fusion protein was chelated to the spots under physiological conditions. The feasibility of the method was demonstrated by culturing neural stem cells on the microarray that displayed oligohistidine-tagged epidermal growth factor.     

Conformational study of L-lysine in aqueous solution by lanthanide shift probes

Conformation of L-lysine in aqueous solution was investigated by lanthanide shift probes (Dy, Ho, Er, Tm and Yb). Reilley's method was employed to separate the contact and dipolar components of the ¹³C paramagnetic shifts. This study reveals that Cα shift has the largest contact contribution while the other carbon shifts are dominated by the dipolar contribution. The average overall conformation of L-lysine in aqueous solution is extended with the molecular backbone in trans form. In the complex, lanthanide ion coordinates to the carboxyl group with Ln—O bond length 2.2 Å and the whole ligand is located outside the zero-dipolar shift cone of the lanthanide ion. The electronic spin density distribution on the ligand nuclei shows that the spin polarization is the predominant mechanism of the contact interaction for nuclei in close proximity to the bound lanthanide ion.     

Backbone-only protein solution structures with a combination of classical and paramagnetism-based constraints: a method that can be scaled to large molecules.

Herein, it is shown that a medium-resolution solution structure of a protein can be obtained with the sole assignment of the protein backbone and backbone-related constriants if a derivative with a firmly bound paramagnetic metal is available. The proof-of-concept is provided on calbindin D9k, a calcium binding protein in which one of the two calcium ions can be selectively substituted by a paramagnetic lanthanide ion. The constraints used are HN (and Ha) nuclear Overhauser effects (NOEs), hydrogen bonds, dihedral angle constriants from chemical shifts, and the following paramagnetism-based constraints: 1) pseudocontact shifts, acquired by substituting one (or more) lanthanide(s) in the C-terminal calcium binding site; 2) N-HN residual dipolar couplings due to self-orientation induced by the paramagnetic lanthanide(s); 3) cross-correlations between the Curie and internuclear dipole-dipole interactions; and 4) paramagnetism-induced relaxation rate enhancements. An upper distance limit for internuclear distances between any two backbone atoms was also given according to the molecular weight of the protein. For this purpose, the paramagnetism-based constraints were collectively implemented in the program CYANA for solution structure determinations, similarly to what was previously done for the program DYANA. The method is intrinsically suitable for large molecular weight proteins.     

Synthesis and characterization of a 'fluorous' (fluorinated alkyl) affinity reagent that labels primary amine groups in proteins/peptides

Strong non-covalent interactions such as biotin-avidin affinity play critical roles in protein/peptide purification. A new type of 'fluorous' (fluorinated alkyl) affinity approach has gained popularity due especially to its low level of non-specific binding to proteins/peptides. We have developed a novel water-soluble fluorous labeling reagent that is reactive (via an active sulfo-N-hydroxylsuccinimidyl ester group) to primary amine groups in proteins/peptides. After fluorous affinity purification, the bulky fluorous tag moiety and the long oligoethylene glycol (OEG) spacer of this labeling reagent can be trimmed via the cleavage of an acid labile linker. Upon collision-induced dissociation, the labeled peptide ion yields a characteristic fragment that can be retrieved from the residual portion of the fluorous affinity tag, and this fragment ion can serve as a marker to indicate that the relevant peptide has been successfully labeled. As a proof of principle, the newly synthesized fluorous labeling reagent was evaluated for peptide/protein labeling ability in phosphate-buffered saline (PBS). Results show that both the aqueous environment protein/peptide labeling and the affinity enrichment/separation process were highly efficient.     

Split intein facilitated tag affinity purification for recombinant proteins with controllable tag removal by inducible auto-cleavage

Purification tags are robust tools that can be used to purify a variety of target proteins. However, tag removal remains an expensive and significant issue that must be resolved. Based on the affinity and the trans-splicing activity between the two domains of Ssp DnaB split-intein, a novel approach for tag affinity purification of recombinant proteins with controllable tag removal by inducible auto-cleavage has been developed. This system provides a new affinity method and avoids premature splicing of the intein fused proteins expressed in host cells. The affinity matrix can be reused. In addition, this method is compatible with his-tag affinity purification technique. Our methods provide the insights for establishing a novel recombinant protein preparation system.     

Engineered Ferritin with Eu3+ as a Bright Nanovector: A Photoluminescence Study

Ferritin nanoparticles play many important roles in theranostic and bioengineering applications and have been successfully used as nanovectors for the targeted delivery of drugs due to their ability to specifically bind the transferrin receptor (TfR1, or CD71). They can be either genetically or chemically modified for encapsulating therapeutics or probes in their inner cavity. Here, we analyzed a new engineered ferritin nanoparticle, made of the H chain mouse ferritin (HFt) fused with a specific lanthanide binding tag (LBT). The HFt-LBT has one high affinity lanthanide binding site per each of the 24 subunits and a tryptophane residue within the tag that acts as an antenna able to transfer the energy to the lanthanide ions via a LRET process. In this study, among lanthanides, we selected europium for its red emission that allows to reduce overlap with tissue auto-fluorescence. Steady state emission measurements and time-resolved emission spectroscopy have been employed to investigate the interaction between the HFt-LBT and the Eu³⁺ ions. This allowed us to identify the Eu³⁺ energy states involved in the process and to pave the way for the future use of HFt-LBT Eu³⁺ complex in theranostics.     

Double-lanthanide-binding tags for macromolecular crystallographic structure determination

A double-lanthanide-binding tag (dLBT), a small peptide sequence engineered to bind two lanthanide ions (e.g., Tb3+) with high affinity, was used to solve the phase problem for the structure determination of ubiquitin by the single-wavelength anomalous diffraction (SAD) method. Since the dLBT is comprised exclusively of encoded amino acids, the necessity for the incorporation of unnatural amino acids or chemical modification of the protein as a prerequisite for X-ray structure determination is eliminated. A construct encoding the dLBT as an N-terminal fusion with ubiquitin provides for facile expression and purification using standard methods. Phasing of the single-wavelength X-ray data (at 2.6 A resolution) using only the anomalous signal from the two tightly bound Tb3+ ions in the dLBT led to clear electron-density maps. Nearly 75% of the ubiquitin structure was built using automated model-building software without user intervention. It is anticipated that this technique will be broadly applicable, complementing existing macromolecular phasing methodologies. The dLBT should be particularly useful in cases where protein derivatization with heavy atoms proves to be problematic or synchrotron facilities are unavailable.     

Synthesis of metal-chelating lipids to sensitize lanthanide ions

Sensitization of lanthanide ions is important for lanthanide ion-based assays and sensing. To the best of our knowledge, there are very few reports of lanthanide ion sensitization after it is incorporated into the liposome surface. This paper describes the syntheses of several saturated and polymerizable metal-chelating lipids based on chelidamic acid. The lipids are synthesized either from (S)-ornithine or racemic 2,3-diaminopropanoic acid. These lipids as well as polymerized liposomes incorporating these lipids sensitize lanthanide ions. Liposomes from the lipid 18-Eu(3+) provided a probe that relies not only on the emission wavelengths of Eu(3+) but also on a reproducible lifetime that can be used for protein identification.     

Molecular Dynamics Simulations of Metal Ion Binding to the His‐tag Motif

In our previous study, we have observed that the chelation of various metal ions to the His‐tag motifs mostly involves the i and i+2 His residues for Ni²⁺, Cu²⁺, Zn²⁺ and Co²+. In the present study, various 200 ps molecular dynamics simulations were further conducted to investigate the chelating pathway of various metal ions to the His‐tag motif with 6 His residues (His‐tag6) and the binding affinities of these metal binding pockets towards these metal ions. The results indicate that His‐tag6 with the chelated metal ion located in positions His(2,4) or His(3,5) exhibits the strongest affinity for Ni²+ and Cu²+.K⁺ was found to be preferred to chelate in His(1,3) and His(3,5) coordinations. However, Fe³+ was found to have higher affinity towards His(1,3) and His(2,4) binding pockets. Our results also suggest that Ni²⁺ exhibits the highest binding affinity towards His‐tag6 over the other metal ions. Most of the structural variations of the His‐tag6 motif were from the Histidyl side chains during metal ion binding. In addition, there is an inverse linear correlation between the final chelated distance and the charge/volume ratio of metal ion. There is a negative correlation between the metal binding affinity and the averaged potential energy generated from the MD simulations.     

PHOTOOXIDATION OF LANTHANIDE ION-LYSOZYME COMPLEXES. A NEW APPROACH TO THE EVALUATION OF INTRAMOLECULAR DISTANCES IN PROTEINS

Abstract— Rare-earth metal ions give 1:1 complexes with hen's egg-white lysozyme. Spectroscopic and enzymic activity measurements suggest that the binding site consists of the side chains of glutamic-35 and aspartic-52. The spatial conformation of these complexes is practically identical to that of native lysozyme, especially as concerns the environment of the tryptophyl side chains. Irradiation of La³⁺-lysozyme by visible light, in the presence of proflavine as photosensitizer, causes the oxidative modification of all the tryptophyl and methionyl residues at almost the same rate as in uncomplexed protein. On the other hand, when lanthanide ions with nonvanishing magnetic moments were coordinated with lysozyme, at least some tryptophans and methionines were protected from photooxidative attack. The distance of the protected residues from the coordination site increased with increasing magnetic moment of the bound metal ion, which suggests that inhibition of the photoprocess was mainly due to perturbation of the lifetime of the electronically excited intermediate species. On the basis of the atomic coordinates of lysozyme in the crystal state, it is thus possible to define a "quenching radius" for the various lanthanide ions; these radii could in turn be used to evaluate intramolecular distances in proteins of unknown tertiary structure, by identifying the amino acid residues that are protected or photooxidized upon irradiation of complexes between the given protein and several different lanthanide ions. Our studieson lysozyme allow us to define five radii of protection, ranging from 6·7 Å for Sm³⁺ to over 17 Å for Dy³⁺, Ho³⁺, Er³⁺ and Tb³⁺. Therefore, this technique opens the possibility of mapping appreciably large regions of a protein molecule.     

Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic fragment ions for protein ubiquitination

In this work, synthetic peptides were used to determine the fragmentation behavior of ubiquitinated peptides and to find ions diagnostic for peptide ubiquitination. The ubiquitin-calmodulin peptide1 was chosen as the model peptide for naturally occurring ubiquitinated proteins cleaved with endoproteinase gluC. In addition, the fragmentation behavior of model ubiquitinated peptides produced by tryptic digestion was also of great interest since the standard protocols for proteomics-based protein identification use trypsin as the protease. Attachment of ubiquitin to a target protein results in a branched structure, but only ions from the ubiquitin side chain (and the lysine to which it is attached) can be used as diagnostic ions, since fragment ions that contain other amino acids from the parent protein will vary in mass. Characteristic b-type fragment ions from the gluC cleavage of the ubiquitin side chain (designated as b ions) were found which involve only the ubiquitin tail (b2, b3, b4, b5 and b6 ions at m/z 189.06, 302.12, 439.18, 552.30 and 651.30, respectively). Maximum production of these ions occurred at a collision energy of 45 eV in a Q-TOF instrument. Although a non-ubiquitinated peptide may produce isobaric fragment ions, it is unlikely that it can produce these ions in combination. With liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments, ubiquitinated peptides can readily be determined by surveying the reconstructed or extracted ion chromatograms of the diagnostic fragment ions for common peaks. Characteristic ions resulting from tryptic cleavage of the side chain were found in cleavage products with a missed cleavage, resulting in a LRGG- tag instead of a GG- tag. For the LRGG-tagged peptide, diagnostic MS/MS fragment ions (at m/z 270.17 and 384.21) from the ubiquitin tail (b2 and b4, respectively) were found, along with an internal fragment ion (LRGGK-28) at m/z 484.30. These ions should prove useful in precursor-ion scanning experiments for identifying peptides modified by attachment of ubiquitin, and for locating the site of ubiquitin attachment.