Rita Graceffa

Minireview: structural insights into early folding events using continuous-flow time-resolved small-angle X-ray scattering.

Small-angle X-ray scattering (SAXS) is a powerful method for obtaining quantitative structural information on the size and shape of proteins, and it is increasingly used in kinetic studies of folding and association reactions. In this minireview, we discuss recent developments in using SAXS to obtain structural information on the unfolded ensemble and early folding intermediates of proteins using continuous-flow mixing devices. Interfacing of these micromachined devices to SAXS beamlines has allowed access to the microsecond time regime. The experimental constraints in implementation of turbulence and laminar flow-based mixers with SAXS detection and a comparison of the two approaches are presented. Current improvements and future prospects of microsecond time-resolved SAXS and the synergy with ab initio structure prediction and molecular dynamics simulations are discussed.

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

The development of a high-duty-cycle microsecond time-resolution SAXS capability at the Biophysics Collaborative Access Team beamline (BioCAT) 18ID at the Advanced Photon Source, Argonne National Laboratory, USA, is reported.

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59-heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~27 μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency>90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.

Advances in turbulent mixing techniques to study microsecond protein folding reactions

Recent experimental and computational advances in the protein folding arena have shown that the readout of the one-dimensional sequence information into three-dimensional structure begins within the first few microseconds of folding. The initiation of refolding reactions has been achieved by several means, including temperature jumps, flash photolysis, pressure jumps, and rapid mixing methods. One of the most commonly used means of initiating refolding of chemically denatured proteins is by turbulent flow mixing with refolding dilution buffer, where greater than 99% mixing efficiency has been achieved within 10s of microseconds. Successful interfacing of turbulent flow mixers with complementary detection methods, including time-resolved Fluorescence Spectroscopy (trFL), Förster Resonance Energy Transfer, Circular Dichroism, Small-Angle X-ray Scattering, Hydrogen Exchange followed by Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy, Infrared Spectroscopy (IR), and Fourier Transform IR Spectroscopy, has made this technique very attractive for monitoring various aspects of structure formation during folding. Although continuous-flow (CF) mixing devices interfaced with trFL detection have a dead time of only 30 µs, burst phases have been detected in this time scale during folding of peptides and of large proteins (e.g., CheY and TIM barrels). Furthermore, a major limitation of the CF mixing technique has been the requirement of large quantities of sample. In this brief communication, we will discuss the recent flurry of activity in micromachining and microfluidics, guided by computational simulations, which are likely to lead to dramatic improvements in time resolution and sample consumption for CF mixers over the next few years.

X-rays Reveal the Internal Structure of Keratin Bundles in Whole Cells.

In recent years, X-ray imaging of biological cells has emerged as a complementary alternative to fluorescence and electron microscopy. Different techniques were established and successfully applied to macromolecular assemblies and structures in cells. However, while the resolution is reaching the nanometer scale, the dose is increasing. It is essential to develop strategies to overcome or reduce radiation damage. Here we approach this intrinsic problem by combing two different X-ray techniques, namely ptychography and nanodiffraction, in one experiment and on the same sample. We acquire low dose ptychography overview images of whole cells at a resolution of 65 nm. We subsequently record high-resolution nanodiffraction data from regions of interest. By comparing images from the two modalities, we can exclude strong effects of radiation damage on the specimen. From the diffraction data we retrieve quantitative structural information from intracellular bundles of keratin intermediate filaments such as a filament radius of 5 nm, hexagonal geometric arrangement with an interfilament distance of 14 nm and bundle diameters on the order of 70 nm. Thus, we present an appealing combined approach to answer a broad range of questions in soft-matter physics, biophysics and biology.

Modulation of frustration in folding by sequence permutation

Significance Folding mechanisms of large proteins are often complicated by the existence of kinetic traps that impede progress toward the native conformation. We have tested the role of chain connectivity in creating such traps by permuting the sequence of a small α/β/α sandwich protein, the chemotaxis response regulator Y. An approach combining experimental and native-centric simulations reveals that chain entropy and aliphatic-rich sequences conspire to create frustrated species whose structures and stabilities vary with connectivity. The initial events in folding reflect not a random collapse driven by the hydrophobic effect but rather the accumulation of substructures favored by low-contact-order nonpolar interactions in the polypeptide. The conserved global free-energy minimum of the native conformation ultimately resolves these early frustrations in folding. 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.

Following DNA Compaction During the Cell Cycle by X-ray Nanodiffraction

X-ray imaging of intact biological cells is emerging as a complementary method to visible light or electron microscopy. Owing to the high penetration depth and small wavelength of X-rays, it is possible to resolve subcellular structures at a resolution of a few nanometers. Here, we apply scanning X-ray nanodiffraction in combination with time-lapse bright-field microscopy to nuclei of 3T3 fibroblasts and thus relate the observed structures to specific phases in the cell division cycle. We scan the sample at a step size of 250 nm and analyze the individual diffraction patterns according to a generalized Porods law. Thus, we obtain information on the aggregation state of the nuclear DNA at a real space resolution on the order of the step size and in parallel structural information on the order of few nanometers. We are able to distinguish nucleoli, heterochromatin, and euchromatin in the nuclei and follow the compaction and decompaction during the cell division cycle.

High-speed detector for time-resolved diffraction studies

There are a growing number of high brightness synchrotron sources that require high-frame-rate detectors to provide the time-scales required for performing time-resolved diffraction experiments. We report on the development of a very high frame rate CMOS X-ray detector for time-resolved muscle diffraction and time-resolved solution scattering experiments. The detector is based on a low-afterglow scintillator, provides a megapixel resolution with frame rates of up to 120,000 frames per second, an effective pixel size of 64 µm, and can be adapted for various X-ray energies. The paper describes the detector design and initial results of time-resolved diffraction experiments on a synchrotron beamline.

High-speed CMOS detector for time-resolved synchrotron applications

We report on the development of a high-speed CMOS X-ray detector for time-resolved muscle diffraction and time-resolved solution scattering experiments. The detector is based on a 1024×1024 pixel resolution CMOS sensor with 17 μm × 17 μm pixels, and a low-afterglow microcolumnar scintillator film developed by RMD, fiber optically coupled to the sensor through an image intensifier. The detector can operate at up to 2,000 fps with full 1024×1024 pixel resolution and up to 120,000 fps with a reduced imaging area without any binning. The detector has an effective pixel size of 64 μm, an imaging area of 7 cm×7 cm, and the sensitivity to measure a single 12 keY photon with a signal-to-noise ratio of better than 2:1. The detector design and time-resolved diffraction experiments performed at a synchrotron using this detector are described here.