Sequence Determinants of the Folding Free-Energy Landscape of beta alpha-Repeat Proteins: A Dissertation

The most common structural platform in biology, the βα-repeat classes of proteins, are represented by the (βα)8TIM barrel topology and the α/β/α sandwich, CheY-like topology. Previous studies on the folding mechanisms of several members of these proteins have suggested that the initial event during refolding involves the formation of a kinetically trapped species that at least partially unfolds before the native conformation can be accessed. The simple topologies of these proteins are thought to permit access to locally folded regions that may coalesce in non-native ways to form stable interactions leading to misfolded intermediates. In a pair of TIM barrel proteins, αTS and sIGPS, it has been shown that the core of the off-pathway folding intermediates is comprised of locally connected clusters of isoleucine, leucine and valine (ILV) residues. These clusters of Branched Aliphatic Side Chains (BASiC) have the unique ability to very effectively prevent the penetration of water to the underlying hydrogen bond networks. This property retards hydrogen exchange with solvent, strengthening main chain hydrogen bonds and linking tertiary and secondary structure in a cooperative network of interactions. This property would also promote the rapid formation of collapsed species during refolding. From this viewpoint, the locally connected topology and the appropriate distribution of ILV residues in the sequence can modulate the energy landscapes of TIM barrel proteins. Another sequence determinant of protein stability that can significantly alter the structure and stability of TIM barrels is the long-range main chain-side chain hydrogen bond. Three of these interactions have been shown to form the molecular underpinnings for the cooperative access to the native state in αTS. Global analysis results presented in Chapter II and Chapter III, suggest that the off-pathway mechanism is common to three proteins of the CheY-like topology, namely CheY, NT-NtrC and Spo0F. These results are corroborated by Gō-simulations that are able to identify the minimal structure of kinetically trapped species during the refolding of CheY and Spo0F. The extent of transient, premature structure appears to correlate with the number of ILV side chains involved in a large sequence-local cluster that is formed between the central β-sheet and helices α2, α3 and α4. The failure of Gō-simulations to detect off-pathway species during the refolding of NT-NtrC may reflect the smaller number of ILV side chains in its corresponding hydrophobic cluster. In Chapter IV, comparison of the location of large ILV clusters with the hydrogen exchange protected regions in 19 proteins, suggest that clusters of BASiC residues are the primarily determinants of the stability cores of globular proteins. Although the location of the ILV clusters is sufficient to determine a majority of the protected amides in a protein structure, the extent of protection is over predicted by the ILV cluster method. The survey of 71 TIM barrel proteins presented in Chapter V, suggests that a specific type of long-range main chain-side chain hydrogen bond, termed “βα hairpin clamp” is a common feature in the βα-repeat proteins. The location and sequence patterns observed demonstrate an evolutionary signature of the βαβ modules that are the building blocks of several βα-repeat protein families. In summary, the work presented in this thesis recognizes the role of sequence in modulating the folding free energy landscapes of proteins. The formation of off-pathway folding intermediates in three CheY-like proteins and the differences in the proposed extent of structure formed in off-pathway intermediates of these three proteins, suggest that both topology and sequence play important and concerted roles in the folding of proteins. Locally connected ILV can clusters lead to off-pathway traps, whereas the formation of the productive folding path requires the development of long-range nativelike topological features to form the native state. The ability of ILV clusters to link secondary and tertiary structure formation enables them to be at the core of this cooperative folding process. Very good correlations between the locations of ILV clusters and both strong protection against exchange and the positions of folding nuclei for a variety of proteins reported in the literature support the generality of the BASiC hypothesis. Finally, the discovery of a novel pattern of H-bond interactions in the TIM barrel architecture, between the amide hydrogen of a core ILV residue with a polar side chain, bracketing βαβ modules, suggests a means for establishing cooperativity between different types of side chain interactions towards formation of the native structure. See Additional Files for copies of the source code for the global analysis program and the cluster analysis program

MARKET ACCESS FOR HIGH-VALUE FOODS

Market access remains a major impediment for expansion of global trade in high-value foods, particularly processed foods. Countries use tariffs and other measures that effectively stimulate imports of relatively unprocessed agricultural commodities at the expense of processed products. Tariff escalation, in which tariffs rise with the level of processing, discourages trade in high-value foods, and trade remedy measures, such as antidumping duties, are concentrated among high-value products. Globalization has provided countries with easier access to capital and technology needed to produce processed food, further affecting trade patterns and markets for high-value foods. A uniform cut in tariffs increases trade in high-value foods more than trade in raw agricultural commodities and improves real wages in developing and developed countries.Food trade, processed food, high-value foods, tariff, tariff escalation, trade remedy measures, sanitary and phytosanitary measures, safeguard measures, revealed comparative advantage, trade complementarities, International Relations/Trade,

Computer design of microfluidic mixers for protein/RNA folding studies

Kinetic studies of biological macromolecules increasingly use microfluidic mixers to initiate and monitor reaction progress. A motivation for using microfluidic mixers is to reduce sample consumption and decrease mixing time to microseconds. Some applications, such as small-angle x-ray scattering, also require large ( \u3e 10 micron) sampling areas to ensure high signal-to-noise ratios and to minimize parasitic scattering. Chaotic to marginally turbulent mixers are well suited for these applications because this class of mixers provides a good middle ground between existing laminar and turbulent mixers. In this study, we model various chaotic to marginally turbulent mixing concepts such as flow turning, flow splitting, and vortex generation using computational fluid dynamics for optimization of mixing efficiency and observation volume. Design iterations show flow turning to be the best candidate for chaotic/marginally turbulent mixing. A qualitative experimental test is performed on the finalized design with mixing of 10 M urea and water to validate the flow turning unsteady mixing concept as a viable option for RNA and protein folding studies. A comparison of direct numerical simulations (DNS) and turbulence models suggests that the applicability of turbulence models to these flow regimes may be limited

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

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-timeresolution 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

Atomistic structural ensemble refinement reveals non-native structure stabilizes a sub-millisecond folding intermediate of CheY

The dynamics of globular proteins can be described in terms of transitions between a folded native state and less-populated intermediates, or excited states, which can play critical roles in both protein folding and function. Excited states are by definition transient species, and therefore are difficult to characterize using current experimental techniques. Here, we report an atomistic model of the excited state ensemble of a stabilized mutant of an extensively studied flavodoxin fold protein CheY. We employed a hybrid simulation and experimental approach in which an aggregate 42 milliseconds of all-atom molecular dynamics were used as an informative prior for the structure of the excited state ensemble. This prior was then refined against small-angle X-ray scattering (SAXS) data employing an established method (EROS). The most striking feature of the resulting excited state ensemble was an unstructured N-terminus stabilized by non-native contacts in a conformation that is topologically simpler than the native state. Using these results, we then predict incisive single molecule FRET experiments as a means of model validation. This study demonstrates the paradigm of uniting simulation and experiment in a statistical model to study the structure of protein excited states and rationally design validating experiments

Microstructural characterization of AISI 431 martensitic stainless steel laser-deposited coatings

High cooling rates during laser cladding of stainless steels may alter the microstructure and phase constitution of the claddings and consequently change their functional properties. In this research, solidification structures and solid state phase transformation products in single and multi layer AISI 431 martensitic stainless steel coatings deposited by laser cladding at different processing speeds are investigated by optical microscopy, Scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), orientation imaging microscopy (OIM), ternary phase diagram, Schaeffler and TTT diagrams. The results of this study show how partitionless solidification and higher solidification rates alter the microstructure and phase constitution of martensitic stainless steel laser deposited coatings. In addition, it is shown that while different cladding speeds have no effect on austenite–martensite orientation relationship in the coatings, increasing the cladding speed has resulted in a reduction of hardness in deposited coatings which is in contrast to the common idea about obtaining higher hardness values at higher cladding speeds.

Do Multinational enterprises push up wages of domestic firms in the Italian Manufacturing sector?

This paper analyzes the effects of foreign direct investment on wages paid by domestic firms in the Italian manufacturing sector over the period 2002–2007. In particular, the authors investigate the im-pact of multinational enterprises on wages paid by local firms which operate in the same industry, known and horizontal wage spillovers, or have linkages with multinational enterprises in both downstream and upstream industries, known as vertical wage spillovers. By using a large panel dataset, consisting of 551,000 observations, the authors find evidence of wage spillovers only at inter-industry level and, more specifically, for those firms who supply their goods to multinational enterprises, described as backward wage spillovers. Moreover, findings suggest that the wage spillover effect is strongly affected by the technological gap between local and foreign firms: only workers employed in domestic firms with a low-medium technological absorptive capacity seem to benefit from the presence of multinational enterprises in terms of higher wages

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

Predicting Gains With Visuospatial Training After Stroke Using an EEG Measure of Frontoparietal Circuit Function

The heterogeneity of stroke prompts the need for predictors of individual treatment response to rehabilitation therapies. We previously studied healthy subjects with EEG and identified a frontoparietal circuit in which activity predicted training-related gains in visuomotor tracking. Here we asked whether activity in this same frontoparietal circuit also predicts training-related gains in visuomotor tracking in patients with chronic hemiparetic stroke. Subjects (n = 12) underwent dense-array EEG recording at rest, then received 8 sessions of visuomotor tracking training delivered via home-based telehealth methods. Subjects showed significant training-related gains in the primary behavioral endpoint, Success Rate score on a standardized test of visuomotor tracking, increasing an average of 24.2 ± 21.9% (p = 0.003). Activity in the circuit of interest, measured as coherence (20–30Hz) between leads overlying ipsilesional frontal (motor cortex) and parietal lobe, significantly predicted training-related gains in visuomotor tracking change, measured as change in Success Rate score (r = 0.61, p = 0.037), supporting the main study hypothesis. Results were specific to the hypothesized ipsilesional motor-parietal circuit, as coherence within other circuits did not predict training-related gains. Analyses were repeated after removing the four subjects with injury to motor or parietal areas; this increased the strength of the association between activity in the circuit of interest and training-related gains. The current study found that (1) Eight sessions of training can significantly improve performance on a visuomotor task in patients with chronic stroke, (2) this improvement can be realized using home-based telehealth methods, (3) an EEG-based measure of frontoparietal circuit function predicts training-related behavioral gains arising from that circuit, as hypothesized and with specificity, and (4) incorporating measures of both neural function and neural injury improves prediction of stroke rehabilitation therapy effects