An apparatus suitable for pressure-jump experiments with variable pressure amplitude and a fast response time to facilitate time-resolved small-angle x-ray scattering at synchrotron facilities is described. The high pressure-jump apparatus is capable of performing bidirectional pressure jumps at a time resolution as high as 5 ms. The high pressure sample cell presented has flat diamond windows and is suited for pressures up to 0.7 GPa operating in the temperature range from −40 to 120 °C. The cell is designed for investigating biological and other soft condensed matter materials. Modifications on the window supports allow also simultaneous wide-angle x-ray scattering data to be taken. We have used the equipment to study the kinetics of protein folding reactions. The performance of the apparatus is demonstrated by presenting data on the pressure-induced un/refolding reaction of the water-soluble protein SNase WT.
The kinetics of chain disruption and collapse of staphylococcal nuclease after positive or negative pressure jumps was monitored by real-time small-angle x-ray scattering under pressure. We used this method to probe the overall conformation of the protein by measuring its radius of gyration and pair-distance-distribution function p(r) which are sensitive to the spatial extent and shape of the particle. At all pressures and temperatures tested, the relaxation profiles were well described by a single exponential function. No fast collapse was observed, indicating that the rate limiting step for chain collapse is the same as that for secondary and tertiary structure formation. Whereas refolding at low pressures occurred in a few seconds, at high pressures the relaxation was quite slow, approximately 1 h, due to a large positive activation volume for the rate-limiting step for chain collapse. A large increase in the system volume upon folding implies significant dehydration of the transition state and a high degree of similarity in terms of the packing density between the native and transition states in this system. This study of the time-dependence of the tertiary structure in pressure-induced folding/unfolding reactions demonstrates that novel information about the nature of protein folding transitions and transition states can be obtained from a combination of small-angle x-ray scattering using high intensity synchrotron radiation with the high pressure perturbation technique.
In this work we discuss the use of small-angle x-ray scattering methods for investigating the temperature and pressure dependent structure and phase behaviour of soft condensed matter and in particular of biomolecular systems, such as lipid mesophases, model biomembrane systems as well as proteins in solution. In addition to temperature, pressure has also been used as a physical parameter in these studies, in particular for studying the energetics and phase behaviour of these systems, but also because high pressure is a feature of certain natural environments and because the high pressure phase behaviour of biomolecules is also of importance for biotechnological applications. By using the pressure jump relaxation technique in combination with time-resolved synchrotron small-angle x-ray scattering, the kinetics of biomolecular phase transitions can be investigated. We applied the technique for studying lipid phase transitions and protein unfolding/refolding reactions. After the discussion of the underlying theoretical concepts, several characteristic examples are presented and discussed.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a fast analysis tool employed for the detection of a broad range of analytes. However, MALDI-MS has a reputation of not being suitable for quantitative analysis. Inhomogeneous analyte/matrix co-crystallization, spot-to-spot inhomogeneity, as well as a typically low number of replicates are the main contributing factors. Here, we present a novel MALDI sample target for quantitative MALDI-MS applications, which addresses the limitations mentioned above. The platform is based on the recently developed microarray for mass spectrometry (MAMS) technology and contains parallel lanes of hydrophilic reservoirs. Samples are not pipetted manually but deposited by dragging one or several sample droplets with a metal sliding device along these lanes. Sample is rapidly and automatically aliquoted into the sample spots due to the interplay of hydrophilic/hydrophobic interactions. With a few microliters of sample, it is possible to aliquot up to 40 replicates within seconds, each aliquot containing just 10 nL. The analyte droplet dries immediately and homogeneously, and consumption of the whole spot during MALDI-MS analysis is typically accomplished within few seconds. We evaluated these sample targets with respect to their suitability for use with different samples and matrices. Furthermore, we tested their application for generating calibration curves of standard peptides with α-cyano-4-hdydroxycinnamic acid as a matrix. For angiotensin II and [Glu(1)]-fibrinopeptide B we achieved coefficients of determination (r(2)) greater than 0.99 without the use of internal standards.
It has been demonstrated that the application of hydrostatic pressure to enzymes placed in surfactant "nanocontainers", such as reversed micelles, can bring additional advantages for both increasing the enzyme stability and modulating the enzyme activity. The effects of pressure on the structural properties of the surfactant matrices are essentially unknown, however. In this paper, small-angle neutron scattering experiments were carried out to study the effect of pressure on the structure of AOT-n-octane-water mesophases. The applied pressure range was 0.1-3000 bar. The hydration degree w0 ) cH 2 O/cAOT (mol/mol) was varied from 5 to 46, and the AOT concentration was varied from 0.1 to 1 M. Depending on the AOT concentration and hydration degree, a pressure-induced elongation of the micellar structure or a pressureinduced phase transition from a dense water-in-oil droplet structure to a bicontinuous L 3 phase or lamellar phase is observed. Incorporation of R-chymotrypsin, a water-soluble enzyme, has a significant influence on the topology of the surfactant mesophase, in particular if the radius of the water droplet inside the reversed micelle reaches the size of the protein, i.e., at hydration degree of w0 ≈ 10. Under these conditions, lipid-protein interactions lead to significant changes of the structure and phase behavior of the system, and the changes observed are enhanced with increasing pressure.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.