Immunoglobulin-binding Domain Research Articles

Selective correlations between aliphatic 13C nuclei in protein solid-state NMR

Solid-state nuclear magnetic resonance (NMR) is a potent tool for studying the structures and dynamics of insoluble proteins. It starts with signal assignment through multi-dimensional correlation experiments, where the aliphatic 13Cα-13Cβ correlation is indispensable for identifying specific residues. However, developing efficient methods for achieving this correlation is a challenge in solid-state NMR. We present a simple band-selective zero-quantum (ZQ) recoupling method, named POST-C4161 (PC4), which enhances 13Cα-13Cβ correlations under moderate magic-angle spinning (MAS) conditions. PC4 requires minimal 13C radio-frequency (RF) field and proton decoupling, exhibits high stability against RF variations, and achieves superior efficiency. Comparative tests on various samples, including the formyl-Met-Leu-Phe (fMLF) tripeptide, microcrystalline β1 immunoglobulin binding domain of protein G (GB1), and membrane protein of mechanosensitive channel of large conductance from Methanosarcina acetivorans (MaMscL), demonstrate that PC4 selectively enhances 13Cα-13Cβ correlations by up to 50 % while suppressing unwanted correlations, as compared to the popular dipolar-assisted rotational resonance (DARR). It has addressed the long-standing need for selective 13C–13C correlation methods. We anticipate that this simple but efficient PC4 method will have immediate applications in structural biology by solid-state NMR.

Multi-eGO: Model Improvements toward the Study of Complex Self-Assembly Processes.

Structure-based models have been instrumental in simulating protein folding and suggesting hypotheses about the mechanisms involved. Nowadays, at least for fast-folding proteins, folding can be simulated in explicit solvent using classical molecular dynamics. However, other self-assembly processes, such as protein aggregation, are still far from being accessible. Recently, we proposed that a hybrid multistate structure-based model, multi-eGO, could help to bridge the gap toward the simulation of out-of-equilibrium, concentration-dependent self-assembly processes. Here, we further improve the model and show how multi-eGO can effectively and accurately learn the conformational ensemble of the amyloid β42 intrinsically disordered peptide, reproduce the well-established folding mechanism of the B1 immunoglobulin-binding domain of streptococcal protein G, and reproduce the aggregation as a function of the concentration of the transthyretin 105-115 amyloidogenic peptide. We envision that by learning from the dynamics of a few minima, multi-eGO can become a platform for simulating processes inaccessible to other simulation techniques.

Characterization of SpsQ from Staphylococcus pseudintermedius as an affinity chromatography ligand for canine therapeutic antibodies.

Coagulase-positive Staphylococci express protein A, which binds to host antibodies, to evade the immune system. Taking advantage of its specific binding to antibodies, protein A from Staphylococcus aureus, which is called SpA, is commonly used as an affinity chromatography ligand for human therapeutic antibodies. However, among four canine IgG subclasses (A, B, C, and D), only IgG-B binds to SpA strongly and establishing an efficient and robust purification scheme for canine therapeutic antibodies whose IgG subclass is A, C, or D remains difficult and depends on finding a suitable substitute to SpA. S. pseudintermedius, a major coagulase-positive Staphylococci found in dogs, expresses spsQ gene which is orthologous to S. aureus spa. We hypothesized that to serve S. pseudintermedius to better adapt to the dog immune system, SpsQ would bind to canine IgGs stronger than SpA, making it a better affinity chromatography ligand for canine therapeutic antibodies. To characterize SpsQ, we first determined the spsQ nucleotide sequence from S. pseudintermedius isolates. Based on the identified sequence, we prepared recombinant proteins containing the immunoglobulin-binding domains of SpA (r-SpA) and SpsQ (r-SpsQ) and determined their binding capacity for each canine IgG subclass. The binding capacity of r-SpsQ for IgG-B was almost as high as that of r-SpA. Interestingly, while both r-SpsQ and r-SpA showed no binding to IgG-C, the binding capacity of r-SpsQ for IgG-A and IgG-D was significantly higher than that of r-SpA. Finally, we performed affinity chromatography using r-SpsQ- or r-SpA-immobilized resin and revealed that the recovery rates of IgG-A and IgG-D using r-SpsQ were significantly higher than those using r-SpA. Our findings indicate that SpsQ has a strong potential to be used as an affinity chromatography ligand for canine therapeutic antibodies of subclass A, B, and D.

The impact of fluorinated ionic liquids aggregation in the interactions with proteins

Proteins have wide biological activities facilitating their employment in several applications from academia to industry. Their implementation depends on costly downstream processes yielding proteins with high purity, stability, and activity. Ionic liquids (ILs) have emerged in the field of protein extraction and purification as an asset due to the possibility to fine-tune the ILs properties to a specific process. However, there is a lack of information concerning the interactions between ILs and proteins to guide the selection of the best ILs for these bottom-line applications. In this work, fluorinated ionic liquids (FILs) were used to infer the interactions with different proteins. FILs have enhanced solubilization mechanisms as a consequence of their rich aggregation behavior due to their amphiphilic character, which can aid the performance of FILs in protein extraction and purification. In this work, the determination of the FILs diffusion coefficients using NMR experiments has provided new information on the size and structure of the different aggregates in their entire range of concentration with water. Therefore, B1 immunoglobulin binding domain of streptococcal protein G (GB1), Bacillus subtilis lipase A (BSLA), and interferon-alpha 2b (IFN-α 2b) were selected to study the interactions between FILs and proteins. From a broad screening of FILs, it was possible to conclude which structural features are contributing to the interaction with the three proteins. This study constitutes an important first step in guiding the selection of appropriate FILs for protein applications.

Pharmacokinetic Engineering of OX40-Blocking Anticalin Proteins Using Monomeric Plasma Half-Life Extension Domains.

Anticalin® proteins have been proven as versatile clinical stage biotherapeutics. Due to their small size (∼20 kDa), they harbor a short intrinsic plasma half-life which can be extended, e.g., by fusion with IgG or Fc. However, for antagonism of co-immunostimulatory Tumor Necrosis Factor Receptor Superfamily (TNFRSF) members in therapy of autoimmune and inflammatory diseases, a monovalent, pharmacokinetically optimized Anticalin protein format that avoids receptor clustering and therefore potential activation is favored. We investigated the suitability of an affinity-improved streptococcal Albumin-Binding Domain (ABD) and the engineered Fab-selective Immunoglobulin-Binding Domain (IgBD) SpGC3Fab for plasma Half-Life Extension (HLE) of an OX40-specific Anticalin and bispecific Duocalin proteins, neutralizing OX40 and a second co-immunostimulatory TNFRSF member. The higher affinity of ABD fusion proteins to human serum albumin (HSA) and Mouse Serum Albumin (MSA), with a 4 to 5-order of magnitude lower KD compared with the binding affinity of IgBD fusions to human/mouse IgG, translated into longer terminal plasma half-lives (t 1/2). Hence, the anti-OX40 Anticalin-ABD protein reached t 1/2 values of ∼40 h in wild-type mice and 110 h in hSA/hFcRn double humanized mice, in contrast to ∼7 h observed for anti-OX40 Anticalin-IgBD in wild-type mice. The pharmacokinetics of an anti-OX40 Anticalin-Fc fusion protein was the longest in both models (t 1/2 of 130 h and 146 h, respectively). Protein formats composed of two ABDs or IgBDs instead of one single HLE domain clearly showed longer presence in the circulation. Importantly, Anticalin-ABD and -IgBD fusions showed OX40 receptor binding and functional competition with OX40L-induced cellular reactivity in the presence of albumin or IgG, respectively. Our results suggest that fusion to ABD or IgBD can be a versatile platform to tune the plasma half-life of Anticalin proteins in response to therapeutic needs.

The Penn State Protein Ladder system for inexpensive protein molecular weight markers

We have created the Penn State Protein Ladder system to produce protein molecular weight markers easily and inexpensively (less than a penny a lane). The system includes plasmids which express 10, 15, 20, 30, 40, 50, 60, 80 and 100 kD proteins in E. coli. Each protein migrates appropriately on SDS-PAGE gels, is expressed at very high levels (10–50 mg per liter of culture), is easy to purify via histidine tags and can be detected directly on Western blots via engineered immunoglobulin binding domains. We have also constructed plasmids to express 150 and 250 kD proteins. For more efficient production, we have created two polycistronic expression vectors which coexpress the 10, 30, 50, 100 kD proteins or the 20, 40, 60, 80 kD proteins. 50 ml of culture is sufficient to produce 20,000 lanes of individual ladder protein or 3750 lanes of each set of coexpressed ladder proteins. These Penn State Protein Ladder expression plasmids also constitute useful reagents for teaching laboratories to demonstrate recombinant expression in E. coli and affinity protein purification, and to research laboratories desiring positive controls for recombinant protein expression and purification.

A protein A based Staphylococcus aureus vaccine with improved safety

Exposure to Staphylococcus aureus does not lead to immunity as evidenced by the persistent colonization of one third of the human population. S. aureus immune escape is mediated by factors that preempt complement activation, destroy phagocytes, and modify B and T cell responses. One such factor, Staphylococcal protein A (SpA) encompasses five Immunoglobulin binding domains (IgBDs) that associate with the Fcγ domain to block phagocytosis. IgBDs also associate with Fab encoded by VH3 clan related genes. SpA binding to VH3-IgM that serves as a B cell receptor results in B cell expansion and secretion of antibodies with no specificity for S. aureus. SpA crosslinking of VH3-IgG and VH3-IgE bound to cognate receptors of mast cells and basophils promotes histamine release and anaphylaxis. Earlier work developed a prototype variant SpAKKAA with four amino acid substitutions in each IgBD. When tested in animal models, SpAKKAA elicited neutralizing antibodies and protection against infection. We show here that SpAKKAA retains crosslinking activity for VH3-IgG and VH3-IgE. We use a rational approach to design and test 67 new SpA variants for loss of VH3 binding and anaphylactic activities. We identify two detoxified candidates that elicit SpA-neutralizing antibodies and protect animals from S. aureus colonization and bloodstream infection. The new detoxified SpA candidates bear three instead of four amino acid substitutions thus increasing the development of SpA-specific antibodies. We propose that detoxified SpA variants unable to crosslink VH3-idiotypic immunoglobulin may be suitably developed as clinical-grade vaccines for safety and efficacy testing in humans.

Estimating the Directional Flexibility of Proteins from Equilibrium Thermal Fluctuations.

The directional flexibility of proteins is an equilibrium molecular property which is accessible to both experiment and computation. Single molecule force spectroscopy (SMFS) experiments report effective directional spring constants to describe the collective anisotropic response of a protein structure to mechanical pulling forces applied along selected axes. On the other hand, computational methods have thus far employed either indirect force based nonequilibrium simulations or coarse-grained elastic network models (ENM) to predict protein directional spring constants. Here, we examine the ability of equilibrium atomistic Molecular Dynamics (MD) simulations to estimate the directional flexibility and mechanical anisotropy of proteins. MD-derived effective directional spring constants are found to correlate well with SMFS spring constants (ρ2 = 0.97-0.99; Adj R2 = 0.92-0.99) and unfolding forces (ρ2 = 0.85-0.97; Adj R2 = 0.63-0.91) for five different globular proteins. Specifically, the computed spring constants reproduce the mechanical anisotropy reported by SMFS along five different directions of green fluorescence protein (GFP) and six directions of the immunoglobulin-binding B1 domain of streptococcal protein G (GB1). Further, protein dynamics as captured in MD can be translated into spring constants which can distinguish the N-C directional flexibility of ubiquitin (Ub) from two structurally homologous small ubiquitin-like modifier (SUMO1 and SUMO2) isoforms. We apply our computational framework to study the mechanical anisotropy of Ub along the seven lysine-C-term directions which are functionally relevant. We show that Ub possesses two distinct flexibility scales along these directions which roughly differ by an order of magnitude. Further, our studies reveal that the mechanical anisotropy of Ub is modified in contrasting ways by the binding of two partner proteins (UBCH5A and UEV) which attach and recognize these biomolecular tag proteins. On the basis of equilibrium MD benchmarks for flexibility along 2485 bond vectors in Ub, we propose and validate a new covariance-propagation scheme to extract spring constants from ENM normal modes. We also critically examine the ability of ENM to predict directional flexibility of proteins and suggest modifications to improve these intuitive and scalable descriptions.

Method for efficient soluble expression and purification of recombinant hyperactive Tn5 transposase

Efficient preparation of libraries is the key step of next-generation sequencing (NGS) methods. Tn5 transposase enables simple, robust and highly efficient tagmentation-based library construction. Here, we report a simple and reliable expression and purification strategy based on fusing Tn5 to the small B1 immunoglobulin binding domain of Streptococcal protein G (GB1) and high affinity 10× His tag. The purified recombinant Tn5 showed high DNA tagmentation activity and ultra-low nucleic acid contamination. This method greatly cuts the costs of Tn5-based NGS library construction and is beneficial to the development of new NGS methods.

Calmodulin binding transcription activators: An interplay between calcium signalling and plant stress tolerance

In plants, next to the secondary messengers lies an array of signal relaying molecules among which Calmodulins convey the unequivocal alarms of calcium influxes to Calmodulin-Binding Transcription Activators (CAMTA). Upon reception, CAMTA transcription factors decode the calcium signatures by transcribing the genes corresponding to the specific stimulus, thus have direct/indirect engagement in the complex signalling crosstalk. CAMTA transcription factors make an important contribution to the genome of all eukaryotes, including plants, from Brassica napus (18) to Carica papaya (2), the number of CAMTA genes varies across the plant species, however they exhibit a similar evolutionarily conserved domain organization including a DNA-Binding Domain (CG-1), a Transcription Factor Immunoglobulin Binding Domain (TIG), a Calmodulin-Binding Domain (CaMBD/IQ) and several Ankyrin repeats. The regulatory region of CAMTA genes possess multiple stress-responsive cis motifs including ABRE, SARE, G-box, W-box, AuXRE, DRE and others. CAMTA TFs in Arabidopsis have been studied extensively, however in other plants (with a few exceptions), the evidence merely bases upon expression analyses. CAMTAs are reported to orchestrate biotic as well as abiotic stresses including those occurring due to water and temperature fluctuations as well as heavy metals, light and salinity. Through CG-1 domain, CAMTA TFs bind the CG-box in the promoter of their target genes and modulate their expression under adverse conditions. Here we present a glimpse of how calcium signatures are coded and decoded and translated into necessary responses. In addition, we have emphasized on exploitation of the multiple-stress responsive nature of CAMTAs in engineering plants with desired traits.

The Hybrid Protein ZZ–OL as an Analytical Tool for Biotechnology Research

The gene of the hybrid protein that encodes the double synthetic fragment proZZ of the immunoglobulin-binding domain of protein A of Staphylococcus aureus and apo-obelin joined by a short linker has been cloned. The corresponding hybrid protein has been obtained by expression in Esсheriсhia coli cells. The protein activated with a substrate (coelenterazine) possesses the bioluminescent Са2+-dependent activity of the photoprotein close to that of recombinant wild-type obelin, and the immunoglobulin-binding ability of protein A. It has been shown that the hybrid can be used as a highly sensitive label to detect antibodies and estimate their affinity and interaction with recombinant proteins, as well as in investigations of other kinds.

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site-Specific Elucidation of Hidden Dynamics in Enzymes.

Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis-CuII NTA label to obtain site-specific information on dynamics for both an α-helix and β-sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW-EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary α-helix in human glutathione transferase A1-1 (hGSTA1-1). We observe two dynamical modes for the helix. The addition of the inhibitor GS-Met and GS-Hex resulted in hGSTA1-1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis-based labelling approach to measure site-specific dynamics using room temperature lineshape analysis.

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site‐Specific Elucidation of Hidden Dynamics in Enzymes

AbstractProtein dynamics is at the heart of all cellular processes. Here, we utilize the dHis‐CuIINTA label to obtain site‐specific information on dynamics for both an α‐helix and β‐sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW‐EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary α‐helix in human glutathione transferase A1‐1 (hGSTA1‐1). We observe two dynamical modes for the helix. The addition of the inhibitor GS‐Met and GS‐Hex resulted in hGSTA1‐1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis‐based labelling approach to measure site‐specific dynamics using room temperature lineshape analysis.

Accelerated Protein Folding Using Greedy-Proximal A.

The protein folding problem has been studied in the field of molecular biophysics and biochemistry for many years. Even small changes in folding patterns may lead to serious diseases such as Alzheimer's or Parkinson's where proteins are folded either too quickly or too slowly. Molecular dynamics (MD) is one of the tools used to understand how proteins fold into native conformations. While it captures sequences of conformations that lead over time to the folded state, limitations in simulation timescales remain problematic. Although many approaches have been suggested to speed up the simulation process using rapid changes in temperature or pressure, we propose a rational approach, Greedy-proximal A* (GPA*), derived from path finding algorithms to explore the supposed shortest path folding pathway from the unfolded to a given folded conformation. We introduce several new protein structure comparison metrics based on the contact map distance to help mitigate the challenges faced by "standard" metrics. We test our approach on proteins which represent the two main types of secondary structure: (a) the Trp-cage miniprotein construct TC5b (1L2Y) which is a short, fast-folding protein that represents an α-helical secondary structure formed because of a locked tryptophan in the middle, (b) the immunoglobulin binding domain of the streptococcal protein G (1GB1), containing an α-helix and several β-sheets, and (c) the chicken villin subdomain HP-35, N68H protein (1YRF)-one of the fastest folding proteins which forms three α-helices. We compare our algorithm to replica-exchange MD and steered MD methods which represent the main algorithms used for accelerating folding proteins with MD. We find that GPA* not only reduces the computational time needed to obtain the folded conformation without adding artificial energy bias but also makes it possible to generate trajectories which contain minimal motions needed for the folding transition.

Production of glycosylphosphatidylinositol-anchored proteins for vaccines and directed binding of immunoliposomes to specific cell types.

Background:Liposomes are highly useful carriers for delivering drugs or antigens. The association of glycosylphosphatidylinositol (GPI)-anchored proteins to liposomes potentially enhances the immunogenic effect of vaccine antigens by increasing their surface concentration. Furthermore, the introduction of a universal immunoglobulin-binding domain can make liposomes targetable to virtually any desired receptor for which antibodies exist.Methods:We developed a system for the production of recombinant proteins with GPI anchors and histidine tags and Strep-tags for simplified purification from cells. This system was applied to i) the green fluorescent protein (GFP) as a reporter, ii) the promising Plasmodium falciparum vaccine antigen PfRH5 and iii) a doubled immunoglobulin Fc-binding domain termed ZZ from protein A of Staphylococcus aureus. As the GPI-attachment domain, the C-terminus of murine CD14 was used. After the recovery of these three recombinant proteins from Chinese hamster ovary (CHO) cells and association with liposomes, their vaccine potential and ability to target the CD4 receptor on lymphocytes in ex vivo conditions were tested.Results:Upon immunization in mice, the PfRH5-GPI-loaded liposomes generated antibody titers of 103 to 104, and showed a 45% inhibitory effect on in vitro growth at an IgG concentration of 600 µg/mL in P. falciparum cultures. Using GPI-anchored ZZ to couple anti-CD4 antibodies to liposomes, we created immunoliposomes with a binding efficiency of 75% to CD4+ cells in splenocytes and minimal off-target binding. Conclusions:Proteins are very effectively associated with liposomes via a GPI-anchor to form proteoliposome particles and these are useful for a variety of applications including vaccines and antibody-mediated targeting of liposomes. Importantly, the CHO-cell and GPI-tagged produced PfRH5 elicited invasion-blocking antibodies qualitatively comparable to other approaches.

Using Dimensionality Reduction to Analyze Protein Trajectories.

In recent years the analysis of molecular dynamics trajectories using dimensionality reduction algorithms has become commonplace. These algorithms seek to find a low-dimensional representation of a trajectory that is, according to a well-defined criterion, optimal. A number of different strategies for generating projections of trajectories have been proposed but little has been done to systematically compare how these various approaches fare when it comes to analysing trajectories for biomolecules in explicit solvent. In the following paper, we have thus analyzed a molecular dynamics trajectory of the C-terminal fragment of the immunoglobulin binding domain B1 of protein G of Streptococcus modeled in explicit solvent using a range of different dimensionality reduction algorithms. We have then tried to systematically compare the projections generated using each of these algorithms by using a clustering algorithm to find the positions and extents of the basins in the high-dimensional energy landscape. We find that no algorithm outshines all the other in terms of the quality of the projection it generates. Instead, all the algorithms do a reasonable job when it comes to building a projection that separates some of the configurations that lie in different basins. Having said that, however, all the algorithms struggle to project the basins because they all have a large intrinsic dimensionality.

Optimization of band-selective homonuclear dipolar recoupling in solid-state NMR by a numerical phase search.

Spin polarization transfers among aliphatic 13C nuclei, especially 13Cα-13Cβ transfers, permit correlations of their nuclear magnetic resonance (NMR) frequencies that are essential for signal assignments in multidimensional solid-state NMR of proteins. We derive and demonstrate a new radio-frequency (RF) excitation sequence for homonuclear dipolar recoupling that enhances spin polarization transfers among aliphatic 13C nuclei at moderate magic-angle spinning (MAS) frequencies. The phase-optimized recoupling sequence with five π pulses per MAS rotation period (denoted as PR5) is derived initially from systematic numerical simulations in which only the RF phases are varied. Subsequent theoretical analysis by average Hamiltonian theory explains the favorable properties of numerically optimized phase schemes. The high efficiency of spin polarization transfers in simulations is preserved in experiments, in part because the RF field amplitude in PR5 is only 2.5 times the MAS frequency so that relatively low 1H decoupling powers are required. Experiments on a microcrystalline sample of the β1 immunoglobulin binding domain of protein G demonstrate an average enhancement factor of 1.6 for 13Cα → 13Cβ polarization transfers, compared to the standard 13C-13C spin-diffusion method, implying a two-fold time saving in relevant 2D and 3D experiments.

Fast NMR method to probe solvent accessibility and disordered regions in proteins

Understanding protein structure and dynamics, which govern key cellular processes, is crucial for basic and applied research. Intrinsically disordered protein (IDP) regions display multifunctionality via alternative transient conformations, being key players in disease mechanisms. IDP regions are abundant, namely in small viruses, allowing a large number of functions out of a small proteome. The relation between protein function and structure is thus now seen from a different perspective: as IDP regions enable transient structural arrangements, each conformer can play different roles within the cell. However, as IDP regions are hard and time-consuming to study via classical techniques (optimized for globular proteins with unique conformations), new methods are required. Here, employing the dengue virus (DENV) capsid (C) protein and the immunoglobulin-binding domain of streptococcal protein G, we describe a straightforward NMR method to differentiate the solvent accessibility of single amino acid N-H groups in structured and IDP regions. We also gain insights into DENV C flexible fold region biological activity. The method, based on minimal pH changes, uses the well-established 1H-15N HSQC pulse sequence and is easily implementable in current protein NMR routines. The data generated are simple to interpret, with this rapid approach being an useful first-choice IDPs characterization method.

Fluorescent, Recombinant-Protein-Conjugated, Near-Infrared-Emitting Quantum Dots for in Vitro and in Vivo Dual-Color Molecular Imaging.

Near-infrared (NIR)-emitting fluorescent probes are widely used for molecular imaging at the whole-body level. However, NIR-emitting fluorescent probes emitting over λ=700 nm are not suitable for molecular imaging at the cellular level, because most of the conventional fluorescence microscopes have very low optical sensitivity in the NIR region. Thus, to achieve fluorescence imaging at the cellular and whole-body levels by using single probes, visible and NIR-emitting dual-color fluorescent probes are desirable. For dual-color fluorescence molecular imaging, we synthesized fluorescent, recombinant-protein-conjugated, NIR-emitting quantum dots (QDs), in which the recombinant protein consists of enhanced green fluorescent protein (EGFP) and the immunoglobulin binding domain (B1) of protein G. This dual-color fluorescent QD probe binds the Fc region of immunoglobulin G (IgG) through its B1 domain at the QD surface and acts as a molecular-imaging probe at both the cellular and whole-body levels. In this paper, we present the synthesis of fluorescent, recombinant protein (HisEGFP-GB1)-conjugated, NIR-emitting QDs and their application to the dual-color molecular imaging of breast cancer cells in vitro and in vivo.

Efficient 15N-13C Polarization Transfer by Third-Spin-Assisted Pulsed Cross-Polarization Magic-Angle-Spinning NMR for Protein Structure Determination.

We introduce a pulsed third-spin-assisted recoupling experiment that produces high-intensity long-range 15N-13C cross peaks using low radiofrequency (rf) energy. This Proton-Enhanced Rotor-echo Short-Pulse IRradiATION Cross-Polarization (PERSPIRATIONCP) pulse sequence operates with the same principle as the Proton-Assisted Insensitive-Nuclei Cross-Polarization (PAINCP) experiment but uses only a fraction of the rf energy by replacing continuous-wave 13C and 15N irradiation with rotor-echo 90° pulses. Using formyl-Met-Leu-Phe (f-MLF) and β1 immunoglobulin binding domain of protein G (GB1) as model proteins, we demonstrate experimentally how PERSPIRATIONCP polarization transfer depends on the CP contact time, rf power, pulse flip angle, and 13C carrier frequency and compare the PERSPIRATIONCP performance with the performances of PAINCP, RESPIRATIONCP, and SPECIFICCP for measuring 15N-13C cross peaks. PERSPIRATIONCP achieves long-range 15N-13C transfer and yields higher cross peak-intensities than that of the other techniques. Numerical simulations reproduce the experimental trends and moreover indicate that PERSPIRATIONCP relies on 15N-1H and 13C-1H dipolar couplings rather than 15N-13C dipolar coupling for polarization transfer. Therefore, PERSPIRATIONCP is an rf-efficient and higher-sensitivity alternative to PAINCP for measuring long-range 15N-13C correlations, which are essential for protein resonance assignment and structure determination. Using cross peaks from two PERSPIRATIONCP 15N-13C correlation spectra as the sole distance restraints, supplemented with (φ, ψ) torsion angles obtained from chemical shifts, we calculated the GB1 structure and obtained a backbone root-mean-square deviation of 2.0 Å from the high-resolution structure of the protein. Therefore, this rf-efficient PERSPIRATIONCP method is useful for obtaining many long-range distance restraints for protein structure determination.