Modulation of frustration in folding by sequence permutation.

Folding of globular proteins can be envisioned as the contraction of a random coil unfolded state toward the native state on an energy surface rough with local minima trapping frustrated species. These substructures impede productive folding and can serve as nucleation sites for aggregation reactions. However, little is known about the relationship between frustration and its underlying sequence determinants. Chemotaxis response regulator Y (CheY), a 129-amino acid bacterial protein, has been shown previously to populate an off-pathway kinetic trap in the microsecond time range. The frustration has been ascribed to premature docking of the N- and C-terminal subdomains or, alternatively, to the formation of an unproductive local-in-sequence cluster of branched aliphatic side chains, isoleucine, leucine, and valine (ILV). The roles of the subdomains and ILV clusters in frustration were tested by altering the sequence connectivity using circular permutations. Surprisingly, the stability and buried surface area of the intermediate could be increased or decreased depending on the location of the termini. Comparison with the results of small-angle X-ray-scattering experiments and simulations points to the accelerated formation of a more compact, on-pathway species for the more stable intermediate. The effect of chain connectivity in modulating the structures and stabilities of the early kinetic traps in CheY is better understood in terms of the ILV cluster model. However, the subdomain model captures the requirement for an intact N-terminal domain to access the native conformation. Chain entropy and aliphatic-rich sequences play crucial roles in biasing the early events leading to frustration in the folding of CheY.

Sub-millisecond time-resolved SAXS using a continuous-flow mixer and X-ray microbeam.

Small-angle X-ray scattering (SAXS) is a well established technique to probe the nanoscale structure and interactions in soft matter. It allows one to study the structure of native particles in near physiological environments and to analyze structural changes in response to variations in external conditions. The combination of microfluidics and SAXS provides a powerful tool to investigate dynamic processes on a molecular level with sub-millisecond time resolution. Reaction kinetics in the sub-millisecond time range has been achieved using continuous-flow mixers manufactured using micromachining techniques. The time resolution of these devices has previously been limited, in part, by the X-ray beam sizes delivered by typical SAXS beamlines. These limitations can be overcome using optics to focus X-rays to the micrometer size range providing that beam divergence and photon flux suitable for performing SAXS experiments can be maintained. Such micro-SAXS in combination with microfluidic devices would be an attractive probe for time-resolved studies. Here, the development of a high-duty-cycle scanning microsecond-time-resolution SAXS capability, built around the Kirkpatrick-Baez mirror-based microbeam system at the Biophysics Collaborative Access Team (BioCAT) beamline 18ID at the Advanced Photon Source, Argonne National Laboratory, is reported. A detailed description of the microbeam small-angle-scattering instrument, the turbulent flow mixer, as well as the data acquisition and control and analysis software is provided. Results are presented where this apparatus was used to study the folding of cytochrome c. Future prospects for this technique are discussed.

Phase-space analysis and experimental results for secondary focusing at X-ray beamlines.

Micro-focusing optical devices at synchrotron beamlines usually have a limited acceptance, but more flux can be intercepted if such optics are used to focus secondary sources created by the primary optics. Flux throughput can be maximized by placing the secondary focusing optics close to or exactly at the secondary source position. However, standard methods of beamline optics analysis, such as the lens equation or matching the mirror surface to an ellipse, work poorly when the source-to-optics distance is very short. In this paper the general characteristics of the focusing of beams with Gaussian profiles by a ;thin lens' are analysed under the paraxial approximation in phase space, concluding that the focusing of a beam with a short source-to-optics distance is distinct from imaging the source; slope errors are successfully included in all the formulas so that they can be used to calculate beamline focusing with good accuracy. A method is also introduced to use the thin-lens result to analyse the micro-focusing produced by an elliptically bent trapezoid-shaped Kirkpatrick-Baez mirror. The results of this analysis are in good agreement with ray-tracing simulations and are confirmed by the experimental results of the secondary focusing at the 18-ID Bio-CAT beamline (at the APS). The result of secondary focusing carried out at 18-ID using a single-bounce capillary can also be explained using this phase-space analysis. A discussion of the secondary focusing results is presented at the end of this paper.

Synchrotron X-ray imaging reveals a correlation of tumor copper speciation with Clioquinol's anticancer activity.

Tumor development and metastasis depend on angiogenesis that requires certain growth factors, proteases, and the trace element copper (Cu). Recent studies suggest that Cu could be used as a novel target for cancer therapies. Clioquinol (CQ), an antibiotic that is able to form stable complexes with Cu or zinc (Zn), has shown proteasome-inhibitory, androgen receptor-suppressing, apoptosis-inducing, and antitumor activities in human cancer cells and xenografts. The mechanisms underlying the interaction of CQ with cellular Cu, the alteration of the Cu/Zn ratio and the antitumor role of CQ in vivo have not been fully elucidated. We report here that Cu accumulates in tumor tissue and that the Cu/Zn balances in tumor, but not normal, tissue change significantly after the treatment with CQ. Cu speciation analysis showed that the Cu(I) species is predominant in both normal and tumor tissues and that Cu(II) content was significantly increased in tumor, but not normal tissue after CQ treatment. Our findings indicate that CQ can interact with cellular Cu in vivo, dysregulates the Cu/Zn balance and is able to convert Cu(I) to Cu(II) in tumor tissue. This conversion of Cu(I) to Cu(II) may be associated with CQ-induced proteasome inhibition and growth suppression in the human prostate tumor xenografts.

Biological applications of X-ray microprobes.

PURPOSE: To present an overview of the workshop on X-ray fluorescence microscopy (XFM). RESULTS: Talks presented at the workshop and the associated works are highlighted. CONCLUSIONS: Use of XFM in biomedical sciences is growing and may be advanced even further by adding (i) high resolution microprobes, and (ii) high throughput approaches to the XFM toolbox.

Clioquinol, a therapeutic agent for Alzheimer's disease, has proteasome-inhibitory, androgen receptor-suppressing, apoptosis-inducing, and antitumor activities in human prostate cancer cells and xenografts.

Tumor growth and metastasis depend on angiogenesis that requires the cofactor copper. Consistently, high levels of copper have been found in many types of human cancers, including prostate, breast, colon, and lung. Recent studies suggest that copper could be used as a novel selective target for cancer therapies. Clioquinol is capable of forming stable complexes with copper and currently used in clinics for treatment of Alzheimer's disease. Most recently, it has been reported that clioquinol possesses antitumor effects. However, the underlying molecular mechanism is unclear. We report here that after binding to copper, clioquinol can inhibit the proteasomal chymotrypsin-like activity, repress androgen receptor (AR) protein expression, and induce apoptotic cell death in human prostate cancer LNCaP and C4-2B cells. In addition, clioquinol alone exhibits similar effects in prostate cancer cell lines with elevated copper at concentrations similar to those found in patients. Addition of dihydrotestosterone did not affect clioquinol-mediated proteasome inhibition in both prostate cancer cell lines. However, dihydrotestosterone partially inhibited clioquinol-induced AR suppression and apoptosis only in androgen-dependent LNCaP cells. Animal studies show that clioquinol treatment significantly inhibits the growth of human prostate tumor C4-2B xenografts (by 66%), associated with in vivo proteasome inhibition, AR protein repression, angiogenesis suppression, and apoptosis induction. Our study provides strong evidence that clioquinol is able to target tumor proteasome in vivo in a copper-dependent manner, resulting in formation of an active AR inhibitor and apoptosis inducer that is responsible for its observed antiprostate tumor effect.

1s2p resonant inelastic X-ray scattering of iron oxides.

1s2p resonant inelastic X-ray scattering (RIXS) spectroscopy has been measured for a series of iron oxides, including octahedral and tetrahedral Fe(II) and Fe(III) systems. Their spectral shapes have been analyzed and explained using crystal-field multiplet simulations. The RIXS planes and the K-edge and L-edge X-ray absorption spectra related to these RIXS planes will be discussed with respect to their analytical opportunities. It is concluded that the full power and possibilities of 1s2p RIXS needs an overall resolution of 0.3 eV. This will yield a technique with more detailed information than K-edge and L-edge X-ray absorption combined, obtained in a single experiment. Another major advantage is that 1s2p RIXS involves only hard X-rays, and experiments under essentially any condition and on any system are feasible.

L X-ray fluorescence cross sections experimentally determined for elements with 45 < Z < 50 at 9 keV.

Experimental determination of L fluorescence cross-sections for elements with 45

High-flux hard X-ray microbeam using a single-bounce capillary with doubly focused undulator beam.

A pre-focused X-ray beam at 12 keV and 9 keV has been used to illuminate a single-bounce capillary in order to generate a high-flux X-ray microbeam. The BioCAT undulator X-ray beamline 18ID at the Advanced Photon Source was used to generate the pre-focused beam containing 1.2 x 10(13) photons s(-1) using a sagittal-focusing double-crystal monochromator and a bimorph mirror. The capillary entrance was aligned with the focal point of the pre-focused beam in order to accept the full flux of the undulator beam. Two alignment configurations were tested: (i) where the center of the capillary was aligned with the pre-focused beam (;in-line') and (ii) where one side of the capillary was aligned with the beam (;off-line'). The latter arrangement delivered more flux (3.3 x 10(12) photons s(-1)) and smaller spot sizes (< or =10 microm FWHM in both directions) for a photon flux density of 4.2 x 10(10) photons s(-1) microm(-2). The combination of the beamline main optics with a large-working-distance (approximately 24 mm) capillary used in this experiment makes it suitable for many microprobe fluorescence applications that require a micrometer-size X-ray beam and high flux density. These features are advantageous for biological samples, where typical metal concentrations are in the range of a few ng cm(-2). Micro-XANES experiments are also feasible using this combined optical arrangement.