Α-synuclein and ß-synuclein enhance secretion protein production in baculovirus expression vector system.

The baculovirus expression vector system (BEVS) is widely used as a tool for expressing of recombinant proteins in insect cells or larvae. However, the expression level of secretion pathway proteins is often lower than that of cytosolic and nucleus proteins. Thus, we attempted to improve production of secreted proteins by using a secretory alkaline phosphatase-EGFP fusion protein (SEFP)-based bi-cistronic baculovirus vector to identify chaperones that have potential on boosting secreted protein production. As co-expressed SEFP with a chaperone, calreticulin (CALR), it was found that the secreted SEFP enzyme activity can be boosted up to twofold. This result demonstrated the SEFP-based bi-cistronic approach can be used to identify the genes that can enhance secretion protein production in BEVS. Thus, the chaperone activity of α-synuclein (α-syn) and ß-synuclein (ß-syn) was evaluated in cells co-expressed with SEFP and compared that with CALR by analyzing localization, alkaline phosphatase enzyme activity, and mRNA expression levels of SEFP. Our results showed that SEFP enzyme activity from cells co-expressed with both synuclein proteins can be enhanced up to 2.3-fold and this increment was better than that caused by CALR. Moreover, this enhancement might arise from the transcription enhancement or higher RNA stability. By this novel approach, we provided evidences that α- and ß-syn can enhance secretion proteins production in BEVS.

Crystal structures of rice (Oryza sativa) glyceraldehyde-3-phosphate dehydrogenase complexes with NAD and sulfate suggest involvement of Phe37 in NAD binding for catalysis.

Cytosolic Oryza sativa glyceraldehyde-3-phosphate dehydrogenase (OsGAPDH), the enzyme involved in the ubiquitous glycolysis, catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate (BPG) using nicotinamide adenine dinucleotide (NAD) as an electron acceptor. We report crystal structures of OsGAPDH in three conditions of NAD-free, NAD-bound and sulfate-soaked forms to discuss the molecular determinants for coenzyme specificity. The structure of OsGAPDH showed a homotetramer form with each monomer comprising three domains-NAD-binding, catalytic and S-loop domains. NAD binds to each OsGAPDH subunits with some residues forming positively charged grooves that attract sulfate anions, as a simulation of phosphate groups in the product BPG. Phe37 not only forms a bottleneck to improve NAD-binding but also combines with Pro193 and Asp35 as key conserved residues for NAD-specificity in OsGAPDH. The binding of NAD alters the side-chain conformation of Phe37 with a 90° rotation related to the adenine moiety of NAD, concomitant with clamping the active site about 0.6 Å from the "open" to "closed" form, producing an increased affinity specific for NAD. Phe37 exists only in higher organisms, whereas it is replaced by other residues (Thr or Leu) with smaller side chains in lower organisms, which makes a greater distance between Leu34 and NAD of E. coli GAPDH than that between Phe37 and NAD of OsGAPDH. We demonstrated that Phe37 plays a crucial role in stabilizing NAD binding or intermediating of apo-holo transition, resulting in a greater NAD-dependent catalytic efficiency using site-directed mutagenesis. Phe37 might be introduced by evolution generating a catalytic advantage in cytosolic GAPDH.

Functional expression of rat neuroligin-1 extracellular fragment by a bi-cistronic baculovirus expression vector.

The interaction between the synaptic adhesion molecules neuroligins and neurexins is essential for connecting the pre- and post-synaptic neurons, modulating neuronal signal transmission, and facilitating neuronal axogenesis. Here, we describe the simultaneous expression of the extracellular domain of rat neuroligin-1 (NL1) proteins along with the enhanced green fluorescent protein (EGFP) using the bi-cistronic baculovirus expression vector system (bi-BEVS). Recombinant rat NL1 protein, fused with signal sequence derived from human Azurocidin gene (AzSP), was secreted into the culture medium and the optimum harvest time for NL1 protein before the lysis of infected cells was determined through the release of cytosolic EGFP. The NL1 protein (0.129±0.013 mg/8×10(7) High Five cells; ~96% purity by metal affinity chromatography) was obtained from the supernatant of the recombinant virus-infected insect cells. A novel chip was employed to address whether the recombinant NL1 is functional in axogenesis. The purified rat NL1 promoted and enhanced the growth rate (137.07±9.74 µm/day) of the axon on NL1/PLL (poly-L-lysine)-coated fine lines on the chip compared to those lines that were coated with PLL alone (105.53±4.53 µm/day). These results were confirmed by fluorescence immunocytochemistry and demonstrated that the recombinant protein can be purified by a one-step process using IMAC combined with monitoring of cell lysis by bi-BEVS. This technique along with our novel chip offers a simple, cost-effective and useful platform for understanding the roles of NL1 protein in neuronal regeneration and synaptic formation studies.

Purification, crystallization and preliminary X-ray crystallographic analysis of the receiver and stalk domains (PA3346RS) of the response regulator PA3346 from Pseudomonas aeruginosa PAO1.

The regulatory domain (PA3346RS), comprising the receiver and stalk domains, of the response regulator PA3346 requires phosphorylation for activation with magnesium ions as cofactors in order to modulate the downstream protein phosphatase activity for the regulation of swarming motility in Pseudomonas aeruginosa PAO1. Fusion-tagged recombinant PA3346RS of total molecular mass 25.3 kDa has been overexpressed in Escherichia coli, purified using Ni(2+)-NTA and Q-Sepharose ion-exchange columns and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from PA3346RS crystals to 2.0 Šresolution. The crystal belonged to space group P4(1) or P4(3), with unit-cell parameters a = 82.38, c = 73.34 Å. Preliminary analysis indicated the presence of a dimer of PA3346RS in the asymmetric unit, with a solvent content of 48.6%.

Temperature dependence of protein backbone motion from carbonyl 13C and amide 15N NMR relaxation.

The NMR spin-lattice relaxation rate (R1) and the rotating-frame spin-lattice relaxation rate (R1rho) of amide 15N and carbonyl 13C (13C') of the uniformly 13C- and 15N-labeled ubiquitin were measured at different temperatures and field strengths to investigate the temperature dependence of overall rotational diffusion and local backbone motion. Correlation between the order parameter of the N-H vector, SNH2, and that of the carbonyl carbon, S2C', was investigated. The effective S2C' was estimated from the direct fit of the experimental relaxation rates and from the slope of 2R2-R1 vs. B2 using Lipari-Szabo formalism. The average SNH2 decreased by 5.9%, while the average S2C' decreased by 4.6% from 15 to 47 degrees C. At the extreme low and high temperatures the difference in the temperature dependence of the order parameters vanishes. At the intermediate temperatures they do not change by the same amount but they follow the same trend. On the same peptide plane along the protein sequence, S2C' and SNH2 are highly correlated. The results suggest that fast local motion experienced at the site of the N-H vector and carbonyl nucleus is more complicated than previously thought and it cannot be easily described by one single type of motion in a broad range of temperature.

Enhanced protein secretion from insect cells by co-expression of the chaperone calreticulin and translation initiation factor eIF4E.

Host protein synthesis is shut down in the lytic baculovirus expression vector system (BEVS). This also affects host proteins involved in routing secretory proteins through the endoplasmic reticulum (ER)-Golgi system. It has been demonstrated that a secretory alkaline phosphatase-EGFP fusion protein (SEFP) can act as a traceable and sensitive secretory reporter protein in BEVS. In this study, a chaperone, calreticulin (CALR), and the translation initiation factor eIF4E were co-expressed with SEFP using a bicistronic baculovirus expression vector. We observed that the intracellular distribution of SEFP in cells co-expressing CALR was different from co-expressing eIF4E. The increased green fluorescence emitted by cells co-expressing CALR had a good correlation with the abundance of intracellular SEFP protein and an unconventional ER expansion. Cells co-expressing eIF4E, on the other hand, showed an increase in extracellular SEAP activity compared to the control. Utilization of these baculovirus expression constructs containing either eIF4E or CALR offers a significant advantage for producing secreted proteins for various biotechnological and therapeutic applications.

Refinement of protein structure against non-redundant carbonyl 13C NMR relaxation.

Carbonyl (13)C' relaxation is dominated by the contribution from the (13)C' chemical shift anisotropy (CSA). The relaxation rates provide useful and non-redundant structural information in addition to dynamic parameters. It is straightforward to acquire, and offers complimentary structural information to the (15)N relaxation data. Furthermore, the non-axial nature of the (13)C' CSA tensor results in a T(1)/T(2) value that depends on an additional angular variable even when the diffusion tensor of the protein molecule is axially symmetric. This dependence on an extra degree of freedom provides new geometrical information that is not available from the NH dipolar relaxation. A protocol that incorporates such structural restraints into NMR structure calculation was developed within the program Xplor-NIH. Its application was illustrated with the yeast Fis1 NMR structure. Refinement against the (13)C' T(1)/T(2) improved the overall quality of the structure, as evaluated by cross-validation against the residual dipolar coupling as well as the (15)N relaxation data. In addition, possible variations of the CSA tensor were addressed.

Model-free analysis for large proteins at high magnetic field strengths.

Protein backbone dynamics is often characterized using model-free analysis of three sets of (15)N relaxation data: longitudinal relaxation rate (R1), transverse relaxation rate (R2), and (15)N-{H} NOE values. Since the experimental data is limited, a simplified model-free spectral density function is often used that contains one Lorentzian describing overall rotational correlation but not one describing internal motion. The simplified spectral density function may be also used in estimating the overall rotational correlation time, by making the R2/R1 largely insensitive to internal motions, as well as used as one of the choices in the model selection protocol. However, such approximation may not be valid for analysis of relaxation data of large proteins recorded at high magnetic field strengths since the contribution to longitudinal relaxation from the Lorentzian describing the overall rotational diffusion of the molecule is comparably small relative to that describing internal motion. Here, we quantitatively estimate the errors introduced by the use of the simplified spectral density in model-free analysis for large proteins at high magnetic field strength.

Temperature dependence of domain motions of calmodulin probed by NMR relaxation at multiple fields.

Interdomain motions of Ca(2+)-ligated calmodulin were characterized by analyzing the nuclear magnetic resonance (15)N longitudinal relaxation rate R(1), transverse relaxation rate R(2), and steady-state {(1)H}-(15)N NOE of the backbone amide group at three different magnetic field strengths (18.8, 14.1, and 8.5 T) and four different temperatures (21, 27, 35, and 43 degrees C). Between 35 and 43 degrees C, a larger than expected change in the amplitude and the time scale of the interdomain motion for both N- and C-domains was observed. We attribute this to the shift in population of four residues (74-77) in the central linker from predominantly helical to random coil in this temperature range. This is consistent with the conformation of these residues in the calmodulin-peptide complex, where they are nonhelical. The doubling of the disordered region of the central helix (residues 78-81 at room temperature) when temperature is raised from 35 to 43 degrees C results in larger amplitude interdomain motion. Our analysis of the NMR relaxation data quantifies subtle changes in the interdomain dynamics and provides an additional tool to monitor conformational changes in multidomain proteins.