Biomolecular interaction of purified recombinant Arabidopsis thaliana's alternative oxidase 1A with TCA cycle metabolites: Biophysical and molecular docking studies.

In higher plants, the mitochondrial alternative oxidase (AOX) pathway plays an essential role in maintaining the TCA cycle/cellular carbon and energy balance under various physiological and stress conditions. Though the activation of AOX pathway upon exogenous addition of α-ketoacids/TCA cycle metabolites [pyruvate, α-ketoglutarate (α-KG), oxaloacetic acid (OAA), succinate and malic acid] to isolated mitochondria is known, the molecular mechanism of interaction of these metabolites with AOX protein is limited. The present study is designed to understand the biomolecular interaction of pure recombinant Arabidopsis thaliana AOX1A with TCA cycle metabolites under in vitro conditions using various biophysical and molecular docking studies. The binding of α-KG, fumaric acid and OAA to rAtAOX1A caused conformational change in the microenvironment of tryptophan residues as evidenced by red shift in the synchronous fluorescence spectra (∆λ = 60 nm). Besides, a decrease in conventional fluorescence emission spectra, tyrosine specific synchronous fluorescence spectra (∆λ = 15 nm) and α-helical content of CD spectra revealed the conformation changes in rAtAOX1A structure associated with binding of various TCA cycle metabolites. Further, surface plasmon resonance (SPR) and microscale thermophoresis (MST) studies revealed the binding affinity, while docking studies identified binding pocket residues, respectively, for these metabolites on rAtAOX1A.

Advancing large-scale production of TEV protease through an innovative NT* tag-based fusion construct.

Tobacco etch virus Protease (TEVp), a cysteine protease, is renowned for its remarkable specific proteolysis, making it an invaluable tool for removing fusion tags from recombinant proteins. However, TEV protease's inherent insolubility limits its broad application. Fusion constructs like an N-terminal MBP fusion, known for its improved solubility, have been employed for TEVp production to address this issue. In this study, we fused the TEVp with the N-terminal domain of the spider silk protein, specifically utilizing a charge-reversed mutant (D40K/K65D) of the N-terminal domain of major ampullate spidroin-1 protein from Euprosthenops australis, referred to as NT*. This fusion construct contains a TEVp cleavage site, enabling intracellular self-processing and the release of a His7-tagged protease. The significant increase in soluble protein expression allowed us to purify approximately 90-100 mg of TEVp from a 1-L E. coli culture, surpassing previous findings by a considerable margin. The enzyme remained stable and catalytically active even after several months of storage in a deep freezer (-80 °C).

Measuring Dipolar Order Parameters in Nondeuterated Proteins Using Solid-State NMR at the Magic-Angle-Spinning Frequency of 100 kHz.

Proteins are dynamic molecules, relying on conformational changes to carry out function. Measurement of these conformational changes can provide insight into how function is achieved. For proteins in the solid state, this can be done by measuring the decrease in the strength of anisotropic interactions due to motion-induced fluctuations. The measurement of one-bond heteronuclear dipole-dipole coupling at magic-angle-spinning (MAS) frequencies >60 kHz is ideal for this purpose. However, rotational-echo double resonance (REDOR), an otherwise gold-standard technique for the quantitative measurement of these couplings, is difficult to implement under these conditions, especially in nondeuterated samples. We present here a combination of strategies based on REDOR variants ϵ-REDOR and DEDOR (deferred REDOR) and simultaneously measure residue-specific 15N-1H and 13Cα-1Hα dipole-dipole couplings in nondeuterated systems at the MAS frequency of 100 kHz. These strategies open up avenues to access dipolar order parameters in a variety of systems at the increasingly fast MAS frequencies that are now available.

Assignment of aromatic side-chain spins and characterization of their distance restraints at fast MAS.

The assignment of aromatic side-chain spins has always been more challenging than assigning backbone and aliphatic spins. Selective labeling combined with mutagenesis has been the approach for assigning aromatic spins. This manuscript reports a method for assigning aromatic spins in a fully protonated protein by connecting them to the backbone atoms using a low-power TOBSY sequence. The pulse sequence employs residual polarization and sequential acquisitions techniques to record HN- and HC-detected spectra in a single experiment. The unambiguous assignment of aromatic spins also enables the characterization of 1H-1H distance restraints involving aromatic spins. Broadband (RFDR) and selective (BASS-SD) recoupling sequences were used to generate HN-ΗC, HC-HN and HC-HC restraints involving the side-chain proton spins of aromatic residues. This approach has been demonstrated on a fully protonated U-[13C,15N] labeled GB1 sample at 95-100 kHz MAS.

Mechanism of selective polarization exchange amongst chemically similar and distinct protons during weak rf irradiation at fast magic angle spinning.

Band Selective Spectral Spin-Diffusion (BASS-SD) is a method to obtain selective 1H-1H contacts between chemically similar protons within a distance range of 5-6 Å in fully protonated proteins. BASS-SD combines low-amplitude proton spinlock radio frequency (rf) pulses with fast MAS frequency to enable selective polarization exchange in fully protonated molecules. The selectivity of transfer is dictated by the bandwidth of the spinlock pulse and has been used to observe selective HN-HN, Hα-Ηα and Hmethyl-Hmethyl correlations. These proton-proton spatial contacts are similar to those observed in perdeuterated samples and serve as useful structural restraints towards de novo protein structure determination. This study employs bimodal Floquet theory to derive the first- and second-order effective Hamiltonians necessary to understand the spin dynamics during BASS-SD. Analytical calculations combined with numerical simulations delineate two different mechanisms for polarization transfer amongst the proton spins. The BASS-SD recoupling condition has been reoptimized to observe selective correlations between chemically different protons (e.g., HN-Hα) while retaining the spatial contacts between chemically similar protons (e.g., HN-HN). The new BASS-SD condition is integrated with simultaneous and sequential acquisition approaches to generate four different types of structural restraints (HN-HN, Hα-Ηα, HN-Hα, Hα-HN) in one experiment. The approach has been demonstrated on microcrystalline U-[13C,15N] labeled GB1 protein at âˆ¼ 95-100 kHz MAS.

CURD: a Single-Shot Strategy to Obtain Assignments and Distance Restraints for Proteins Using Solid-State MAS NMR Spectroscopy.

We present a strategy dubbed CURD (correlations using recycle delays) to acquire chemical-shift assignments and distance restraints for proteins in a single experimental block under slow-moderate magic-angle spinning conditions. This is done by concatenating the 3D-CCC and 3D-NNC experiments, both of which individually require long experimental times for sufficient resolution and sensitivity to be realized. Unlike previous approaches, the CURD strategy does not increase the amount of radio-frequency deposition on the sample and does not require lengthy procedures to optimize any of the pulse sequence elements. Instead, time savings is obtained by using the hitherto unused recycle delay of one of the experiments (2D-CC/3D-CCC) to establish inter-residue correlations for the second experiment (2D-NN/3D-NNC). Experiments are demonstrated on a model protein at the MAS frequency of 12.5 kHz and are shown to result in time savings of the order of days for most of the routine cases.

A comparison between MBP- and NT* as N-terminal fusion partner for recombinant protein production in E. coli.

Advances in structural biology have been fueled in part by developing techniques for large-scale heterologous expression and purification of proteins. Nevertheless, this step is still a bottleneck in biophysical studies of many proteins. Often, fusion proteins are used to increase expression levels, solubility, or both. Here, we compare a recently reported fusion tag, NT*, with Maltose Binding Protein (MBP), a well-known fusion tag and solubility enhancer. NT* shows high expression and solubility when used as an N-terminal fusion partner for several aggregation-prone peptides. Its efficacy in enhancing the solubility of aggregation-prone globular proteins has, however, not been tested. We find here that although the overall expression levels for NT* fusions are much higher than those for the MBP fusion, MBP was far superior for enhancing the solubility of the passenger protein. Nevertheless, the effective yield after purification from the soluble fraction of both MBP-fusion and NT*-fusion was comparable, mainly due to higher expression levels in NT*-fusion and a smaller fraction of the passenger protein net weight being locked in the fusion protein. We conclude that NT* is an excellent fusion tag to improve the overall expression of globular proteins but does not increase the passenger protein's solubility compared to MBP. Proteins that are partially soluble or can be refolded in-vitro will significantly benefit from N-terminal NT* fusions. MBP, however, still remains one of the very few options for an N-terminal fusion if the solubility of the protein after expression is critical for preserving its proper fold or activity.

Selective 1H-1H recoupling via symmetry sequences in fully protonated samples at fast magic angle spinning.

Proton-detected solid-state NMR at fast Magic Angle Spinning (MAS) is becoming the norm to characterize molecules. Routinely 1H-1H and 1H-X dipolar couplings are used to characterize the structure and dynamics of molecules. Selective proton recoupling techniques are emerging as a method for structural characterization via estimation of qualitative and quantitative distances. In the present study, we demonstrate through numerical simulations and experiments that the well-characterized CNvn sequences can also be tailored for selective recoupling of proton spins by employing C elements of the type (ß)Φ(4ß)Φ+π(3ß)Φ. Herein, several CNvn sequences were examined through numerical simulations and experiments. C614 recoupling sequence with a modified POST-element ((ß)Φ(4ß)Φ+π(3ß)Φ) shows selective polarization transfer efficiencies on the order of 40-50% between various proton spin pairs in fully protonated samples at rf amplitudes ranging from 0.3 to 0.8 times the MAS frequency. These selective recoupling sequences have been labeled as frequency-selective-CNvn sequences. The extent of selectivity, polarization transfer efficiency and the feasibility of experimentally measuring proton-proton distances in fully protonated samples are explored here. The development of efficient and robust selective 1H-1H recoupling experiments is required to structurally characterize molecules without artificial isotope enrichment or the need for diffracting crystals.

Efficient refolding and functional characterization of PfAMA1(DI+DII) expressed in E. coli.

Apical membrane antigen 1 (AMA1) is a surface protein of Plasmodium sp. that plays a crucial role in forming moving junction (MJ) during the invasion of human red blood cells. The obligatory presence of AMA1 in the parasite lifecycle designates this protein as a potential vaccine candidate and an essential target for the development of novel peptide or protein therapeutics. However, due to multiple cysteine residues in the protein sequence, attaining the native fold with correct disulfide linkages during the refolding process after expression in bacteria has remained challenging for years. Although several approaches to obtain the refolded protein from bacterial expression have been reported previously, achieving high yield during refolding and proper functional validation of the expressed protein was lacking. We report here an improved method of refolding to obtain higher quantity of refolded protein. We have also validated the refolded protein's functional activity by evaluating the expressed AMA1 protein binding with a known inhibitory peptide, rhoptry neck protein 2 (RON2), using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC).

Accuracy of 1H-1H distances measured using frequency selective recoupling and fast magic-angle spinning.

Selective recoupling of protons (SERP) is a method to selectively and quantitatively measure magnetic dipole-dipole interaction between protons and, in turn, the proton-proton distance in solid-state samples at fast magic-angle spinning. We present a bimodal operator-based Floquet approach to describe the numerically optimized SERP recoupling sequence. The description calculates the allowed terms in the first-order effective Hamiltonian, explains the origin of selectivity during recoupling, and shows how different terms are modulated as a function of the radio frequency amplitude and the phase of the sequence. Analytical and numerical simulations have been used to evaluate the effect of higher-order terms and offsets on the polarization transfer efficiency and quantitative distance measurement. The experimentally measured 1H-1H distances on a fully protonated thymol sample are ∼10%-15% shorter than those reported from diffraction studies. A semi-quantitative model combined with extensive numerical simulations is used to rationalize the effect of the third-spin and the role of different parameters in the experimentally observed shorter distances. Measurements at high magnetic fields improve the match between experimental and diffraction distances. The measurement of 1H-1H couplings at offsets different from the SERP-offset has also been explored. Experiments were also performed on a perdeuterated ubiquitin sample to demonstrate the feasibility of simultaneously measuring multiple quantitative distances and to evaluate the accuracy of the measured distance in the absence of multispin effects. The estimation of proton-proton distances provides a boost to structural characterization of small pharmaceuticals and biomolecules, given that the positions of protons are generally not well defined in x-ray structures.

Can proton-proton recoupling in fully protonated solids provide quantitative, selective and efficient polarization transfer?

Dipolar recoupling sequences have been used to probe spatial proximity of nuclear spins and were traditionally designed to probe rare spins such as 13C and/or 15N nuclei. The multi-spin dipolar-coupling network of the rare spins is weak due to smaller couplings and large chemical shift dispersion. Therefore, the recoupling approaches were tailored to design offset compensated or broadband sequences. In contrast, protons have a substantially stronger dipolar-coupling network and much narrower chemical shift range. Broadband recoupling sequences such as radio-frequency driven recoupling (RFDR), back-to-back (BABA), and lab frame proton-proton spin diffusion have been routinely used to characterize the structures of protein/macromolecules and small molecules. Recently selective 1H-1H recoupling sequences have been proposed that combine chemical shift offset of the resolved proton spectrum (at fast MAS) with first- and second-order dipolar recoupling Hamiltonians to obtain quantitative and qualitative proton distances, respectively. Herein, we evaluate the performances of broadband and selective proton recoupling sequences such as finite pulse RFDR (fp-RFDR), band-selective spectral spin diffusion (BASS-SD), second-order cross-polarization (SOCP), and selective recoupling of proton (SERP) in terms of the selectivity and efficiency of 1H-1H polarization transfers in a dense network of proton spins and explore the possibility of measuring 1H-1H distances. We use theoretical considerations, numerical simulations, and experiments to support the distinct advantages and disadvantages of each recoupling sequence. Experiments were performed on L-histidine.HCl.H2O at a MAS frequency of 71.43 kHz. This study rationalizes the proper selection of 1H-1H recoupling sequences when working with fully protonated solids.

High-resolution crystal structures of the D1 and D2 domains of protein tyrosine phosphatase epsilon for structure-based drug design.

Here, new crystal structures are presented of the isolated membrane-proximal D1 and distal D2 domains of protein tyrosine phosphatase epsilon (PTPℇ), a protein tyrosine phosphatase that has been shown to play a positive role in the survival of human breast cancer cells. A triple mutant of the PTPℇ D2 domain (A455N/V457Y/E597D) was also constructed to reconstitute the residues of the PTPℇ D1 catalytic domain that are important for phosphatase activity, resulting in only a slight increase in the phosphatase activity compared with the native D2 protein. The structures reported here are of sufficient resolution for structure-based drug design, and a microarray-based assay for high-throughput screening to identify small-molecule inhibitors of the PTPℇ D1 domain is also described.

Quantitative 1H-1H Distances in Protonated Solids by Frequency-Selective Recoupling at Fast Magic Angle Spinning NMR.

Nuclear magnetic resonance (NMR) spectroscopy of protons in protonated solids is challenging. Fast magic angle spinning (MAS) and homonuclear decoupling schemes, in conjunction, with high magnetic fields have improved the proton resolution. However, experiments to quantitatively measure 1H-1H distances still remain elusive due to the dense proton-proton dipolar coupling network. A novel MAS solid-state NMR pulse sequence is proposed to selectively recouple and measure interproton distances in protonated samples. The phase-modulated sequence combined with a judicious choice of transmitter frequency is used to measure quantitative 1H-1H distances on the order of 3 Å in l-histidine·HCl·H2O, despite the presence of other strongly coupled protons. This method provides a major boost to NMR crystallography approaches for structural determination of pharmaceutical molecules by directly measuring 1H-1H distances. The band-selective nature of the sequence also enables observation of selective 1H-1H correlations (e.g., HN-HN/HN-Hα/ΗΝ-ΗMethyl) in peptides and proteins, which should serve as useful restraints in structure determination.

Crystal structure of the human dual specificity phosphatase 1 catalytic domain.

The dual specificity phosphatase DUSP1 was the first mitogen activated protein kinase phosphatase (MKP) to be identified. It dephosphorylates conserved tyrosine and threonine residues in the activation loops of mitogen activated protein kinases ERK2, JNK1 and p38-alpha. Here, we report the crystal structure of the human DUSP1 catalytic domain at 2.49 Å resolution. Uniquely, the protein was crystallized as an MBP fusion protein in complex with a monobody that binds to MBP. Sulfate ions occupy the phosphotyrosine and putative phosphothreonine binding sites in the DUSP1 catalytic domain.

Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag.

Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His6- or a dual His6-MBP tagged fusion protein by Gateway® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His6 tag or a His6-MBP tag can be made on the basis of this solubility test.

Removal of Affinity Tags with TEV Protease.

Although affinity tags are highly effective tools for the expression and purification of recombinant proteins, they generally need to be removed prior to structural and functional studies. This chapter describes a simple method for overproducing a soluble form of a stable variant of tobacco etch virus (TEV) protease in Escherichia coli and a protocol for purifying it to homogeneity so that it can be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. Further, we cleave a model substrate protein (MBP-NusG) in vitro using the purified TEV protease to illustrate a protease cleavage protocol that can be employed for simple pilot experiments and large-scale protein preparations.

A dual protease approach for expression and affinity purification of recombinant proteins.

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification.

Phosphotyrosine Substrate Sequence Motifs for Dual Specificity Phosphatases.

Protein tyrosine phosphatases dephosphorylate tyrosine residues of proteins, whereas, dual specificity phosphatases (DUSPs) are a subgroup of protein tyrosine phosphatases that dephosphorylate not only Tyr(P) residue, but also the Ser(P) and Thr(P) residues of proteins. The DUSPs are linked to the regulation of many cellular functions and signaling pathways. Though many cellular targets of DUSPs are known, the relationship between catalytic activity and substrate specificity is poorly defined. We investigated the interactions of peptide substrates with select DUSPs of four types: MAP kinases (DUSP1 and DUSP7), atypical (DUSP3, DUSP14, DUSP22 and DUSP27), viral (variola VH1), and Cdc25 (A-C). Phosphatase recognition sites were experimentally determined by measuring dephosphorylation of 6,218 microarrayed Tyr(P) peptides representing confirmed and theoretical phosphorylation motifs from the cellular proteome. A broad continuum of dephosphorylation was observed across the microarrayed peptide substrates for all phosphatases, suggesting a complex relationship between substrate sequence recognition and optimal activity. Further analysis of peptide dephosphorylation by hierarchical clustering indicated that DUSPs could be organized by substrate sequence motifs, and peptide-specificities by phylogenetic relationships among the catalytic domains. The most highly dephosphorylated peptides represented proteins from 29 cell-signaling pathways, greatly expanding the list of potential targets of DUSPs. These newly identified DUSP substrates will be important for examining structure-activity relationships with physiologically relevant targets.

Positional effects of fusion partners on the yield and solubility of MBP fusion proteins.

Escherichia coli maltose-binding protein (MBP) is exceptionally effective at promoting the solubility of its fusion partners. However, there are conflicting reports in the literature claiming that (1) MBP is an effective solubility enhancer only when it is joined to the N-terminus of an aggregation-prone passenger protein, and (2) MBP is equally effective when fused to either end of the passenger. Here, we endeavor to resolve this controversy by comparing the solubility of a diverse set of MBP fusion proteins that, unlike those analyzed in previous studies, are identical in every way except for the order of the two domains. The results indicate that fusion proteins with an N-terminal MBP provide an excellent solubility advantage along with more robust expression when compared to analogous fusions in which MBP is the C-terminal fusion partner. We find that only intrinsically soluble passenger proteins (i.e., those not requiring a solubility enhancer) are produced as soluble fusions when they precede MBP. We also report that even subtle differences in inter-domain linker sequences can influence the solubility of fusion proteins.

Unrelated solubility-enhancing fusion partners MBP and NusA utilize a similar mode of action.

The tendency of recombinant proteins to accumulate in the form of insoluble aggregates in Escherichia coli is a major hindrance to their overproduction. One of the more effective approaches to circumvent this problem is to use translation fusion partners {solubility-enhancers (SEs)}. E. coli maltose-binding protein (MBP) and N-utilization substance A (NusA) are arguably the most effective solubilizing agents that have been discovered so far. Here, we show that although these two proteins are structurally, functionally, and physicochemically distinct, they influence the solubility and folding of their fusion partners in a very similar manner. These SEs act as "holdases" that prevent the aggregation of their fusion partners. Subsequent folding of the passenger proteins, when it occurs, is either spontaneous or chaperone-mediated.