Chaperone-mediated native folding of a ß-scorpion toxin in the periplasm of Escherichia coli.

BACKGROUND: Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria. METHODS: The ß-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the Escherichia coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined. RESULTS: Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold. CONCLUSIONS: The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method. GENERAL SIGNIFICANCE: We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors.

Increased CRMP2 phosphorylation is observed in Alzheimer's disease; does this tell us anything about disease development?

Collapsin response mediator protein-2 (CRMP2) was recently identified as a physiological substrate for GSK3 and Cdk5, two protein kinases suggested to exhibit greater activity in Alzheimer's disease (AD). Indeed, phosphorylation of CRMP2, at the residues targeted by GSK3 and Cdk5, is relatively high in cortex isolated from human AD brain, as well as in the brains of animal models of AD, while phospho-CRMP2 is found in neurofibrillary tangles. In mouse models of AD, increased phosphorylation occurs prior to pathology. Although CRMP2 has no known enzymatic activity, a great deal of information is appearing on its importance in neuronal development and polarity, as well as in axon growth and guidance. In this mini-review, we examine what is known about CRMP2 function, how that is controlled by phosphorylation, what alterations in molecular mechanisms could lead to the abnormally high CRMP2 phosphorylation in AD, and whether this is likely to be specific to AD or occur in other forms of neurodegeneration. This will include discussion of the evidence for increased GSK3 or Cdk5 activity, for decreased phosphatase activity, or the upregulation of other CRMP2 protein kinases in AD. Importantly, we will compare the processes that may contribute to increased CRMP2 phosphorylation with those known to increase tau hyperphosphorylation in AD, and whether these are likely to be part of disease development or a useful early marker for AD.

Proteomic analysis of colonic crypts from normal, multiple intestinal neoplasia and p53-null mice: a comparison with colonic polyps.

In order to observe cellular changes caused by mutation of the tumor suppressors, APC and p53, we have generated protein expression profiles of mouse colon epithelial cells using two-dimensional electrophoresis (2-DE). Crypts, polyps and stroma were isolated from normal, multiple intestinal neoplasia (MIN) and p53-null mice, each with a C57Black/6J background, and subjected to 2-DE in two separate pH ranges (pH 3-10 and pH 6-11). No significant differences in protein expression patterns were observed between the normal, MIN and p53-null colon epithelial crypts. However, 64 proteins from the MIN polyps showed a 2-fold or greater difference in intensity that was statistically significant as assessed by the Wilcoxon rank-sum test (p < or = 0.05). Of these, calreticulin, carbonic anhydrase I and a new member of the glutathione-S-transferase theta family of proteins have so far been identified using an in-gel digestion protocol coupled with reversed-phase high performance liquid chromatography (RP-HPLC) ion-trap mass spectrometry. In addition, 38 marker proteins have been identified in a continuing effort to generate a comprehensive 2-DE database of proteins expressed by mouse colon epithelial cells (these databases are available at http://www.ludwig.edu.au/jpsl/jpslhome. html).

Disulfide bond structure and N-glycosylation sites of the extracellular domain of the human interleukin-6 receptor.

The high affinity interleukin-6 (IL-6) receptor is a hexameric complex consisting of two molecules each of IL-6, IL-6 receptor (IL-6R), and the high affinity converter and signaling molecule, gp130. The extracellular "soluble" part of the IL-6R (sIL-6R) consists of three domains: an amino-terminal Ig-like domain and two fibronectin-type III (FN III) domains. The two FN III domains comprise the cytokine-binding domain defined by a set of 4 conserved cysteine residues and a WSXWS sequence motif. Here, we have determined the disulfide structure of the human sIL-6R by peptide mapping in the absence and presence of reducing agent. Mass spectrometric analysis of these peptides revealed four disulfide bonds and two free cysteines. The disulfides Cys102-Cys113 and Cys146-Cys157 are consistent with known cytokine-binding domain motifs, and Cys28-Cys77 with known Ig superfamily domains. An unusual cysteine connectivity between Cys6-Cys174, which links the Ig-like and NH2-terminal FN III domains causing them to fold back onto each other, has not previously been observed among cytokine receptors. The two free cysteines (Cys192 and Cys258) were detected as cysteinyl-cysteines, although a small proportion of Cys258 was reactive with the alkylating agent 4-vinylpyridine. Of the four potential N-glycosylation sites, carbohydrate moieties were identified on Asn36, Asn74, and Asn202, but not on Asn226.

Visual manifestations of occipital lobe infarction in three patients on a geriatric psychiatry unit.

The authors present three cases of hospitalized patients on a geriatric psychiatry floor who were found to have previously undiagnosed occipital lobe infarctions associated with visual manifestations. The manifestations discussed are visual field defects, visual hallucinations, and color anomia. The incidence of undiagnosed occipital lobe infarctions and the contribution of these infarctions to visual perception changes in this patient population are unknown. The authors suggest that for patients who present with visual perception changes, a high index of suspicion for occipital lobe infarction should be maintained. Careful visual field testing is an essential part of the admitting work-up for hospitalized geriatric patients.

Precise measurements with a compact vacuum infrared spectrometer.

A 3-m vacuum grating IR spectrometer with digital recording is described. Resolution better than 0.025 cm(-1) at 3000 cm(-1) is demonstrated, and details are given of a fast deconvolution procedure by which the resolution is enhanced to near 0.010 cm(-1) . The grating drive incorporates a Merton nut, which facilitates the measurement of highly precise wave numbers. Lines in the 12 degrees 1 band of N(2)O near 4630 cm(-1) have been measured and agree within an average of +/-0.0004 cm(-1) with interferometric values.

Calibration of far-infrared spectrometers in the 100-200-cm(-1) region with the nu(5)-nu(4) band of acetylene.

The R branch of the nu(5)-nu(4) difference band of acetylene, 12C(2)H(2), is recommended as a calibration standard for the 100-200-cm(-1) region. The lines are split by l-type doubling into pairs with a 3:1 intensity ratio, but this can only be observed under high resolution. The position of the calculated maximum of each blended line contour is displaced by the l-type doubling from the accurately calculated wavenumber of the more intense component of each J pair, but not by more than one-fifteenth of the spectral slit width. It is recommended that values calculated from constants derived from high resolution mid-ir measurements be used for calibration.

Rotation lines of hydrogen chloride for wavenumber calibration of far infrared spectrometers.

Contours are calculated for the pairs of H(35)Cl and H(37)Cl rotation lines for a range of spectral slitwidths between 0.10 cm(-1) and 3.0 cm(-1). The peak positions are compared with the frequencies of the H(35)Cl lines with a view to predicting the displacement of each peak due to overlap of the H(37)Cl lines. Experimental measurements of the J = 12 and 13 lines show that the predictions are correct. It is suggested that the calculated peaks be used as calibration data for instruments, although it is pointed out that if the wavenumber error of the instrument exceeds one-twentieth of the spectral slitwidth, the displacements can be ignored.

Vacuum grating spectrometer for the mid and far infrared.

The construction and performance of a single beam large aperture spectrometer totally enclosed in a vacuum chamber are described. The focal length of the Czerny-Turner monochromator is 90 cm and plane gratings up to 23 cm x 18 cm are used. The grating drive incorporates a Merton nut. With a Golay detector, the resolution achieved is about 0.12 cm(-1) at 1000 cm(-1), 0.20-0.30 cm(-1) in the range 300-27 cm(-1) Illustrative spectra of ammonia, water vapor, and glyoxal are given.