Homeocurvature adaptation of phospholipids to pressure in deep-sea invertebrates.

Hydrostatic pressure increases with depth in the ocean, but little is known about the molecular bases of biological pressure tolerance. We describe a mode of pressure adaptation in comb jellies (ctenophores) that also constrains these animals' depth range. Structural analysis of deep-sea ctenophore lipids shows that they form a nonbilayer phase at pressures under which the phase is not typically stable. Lipidomics and all-atom simulations identified phospholipids with strong negative spontaneous curvature, including plasmalogens, as a hallmark of deep-adapted membranes that causes this phase behavior. Synthesis of plasmalogens enhanced pressure tolerance in Escherichia coli, whereas low-curvature lipids had the opposite effect. Imaging of ctenophore tissues indicated that the disintegration of deep-sea animals when decompressed could be driven by a phase transition in their phospholipid membranes.

Reversible disassembly-reassembly of C-phycocyanin in pressurization-depressurization cycles of high hydrostatic pressure.

Hydrostatic pressure can reversibly modulate protein-protein and protein-chromophore interactions of C-phycocyanin (C-PC) from Spirulina platensis. Small-angle X-ray scattering combined with UV-Vis spectrophotometry and protein modeling was used to explore the color and structural changes of C-PC under high pressure conditions at different pH levels. It was revealed that pressures up to 350 MPa were enough to fully disassemble C-PC from trimers to monomers at pH 7.0, or from monomers to detached subunits at pH 9.0. These disassemblies were accompanied by protein unfolding that caused these high-pressure induced structures to be more extended. These changes were reversible following depressurization. The trimer-to-monomer transition proceeded through a collection of previously unrecognized, L-shaped intermediates resembling C-PC dimers. Additionally, pressurized C-PC showed decayed Q-band absorption and fortified Soret-band absorption. This was evidence that the folded tetrapyrroles, which had folded at ambient pressure, formed semicyclic unfolded conformations at a high pressure. Upon depressurization, the peak intensity and shift all recovered stepwise, showing pressure can precisely manipulate C-PC's structure as well as its color. Overall, a protein-chromophore regulatory theory of C-PC was unveiled. The pressure-tunability could be harnessed to modify and stabilize C-PC's structure and photochemical properties for designing new delivery and optical materials.

Understanding the Impacts of Molecular and Macromolecular Crowding Agents on Protein-Polymer Complex Coacervates.

Complex coacervation refers to the liquid-liquid phase separation (LLPS) process occurring between charged macromolecules. The study of complex coacervation is of great interest due to its implications in the formation of membraneless organelles (MLOs) in living cells. However, the impacts of the crowded intracellular environment on the behavior and interactions of biomolecules involved in MLO formation are not fully understood. To address this knowledge gap, we investigated the effects of crowding on a model protein-polymer complex coacervate system. Specifically, we examined the influence of sucrose as a molecular crowder and polyethylene glycol (PEG) as a macromolecular crowder. Our results reveal that the presence of crowders led to the formation of larger coacervate droplets that remained stable over a 25-day period. While sucrose had a minimal effect on the physical properties of the coacervates, PEG led to the formation of coacervates with distinct characteristics, including higher density, increased protein and polymer content, and a more compact internal structure. These differences in coacervate properties can be attributed to the effects of crowders on individual macromolecules, such as the conformation of model polymers, and nonspecific interactions among model protein molecules. Moreover, our results show that sucrose and PEG have different partition behaviors: sucrose was present in both the coacervate and dilute phases, while PEG was observed to be excluded from the coacervate phase. Collectively, our findings provide insights into the understanding of crowding effects on complex coacervation, shedding light on the formation and properties of coacervates in the context of MLOs.

Characterizing Protein-Protein Interactions and Viscosity of a Monoclonal Antibody from Low to High Concentration Using Small-Angle X-ray Scattering and Molecular Dynamics Simulations.

Understanding protein-protein interactions and formation of reversible oligomers (clusters) in concentrated monoclonal antibody (mAb) solutions is necessary for designing stable, low viscosity (η) concentrated formulations for processing and subcutaneous injection. Here we characterize the strength (K) of short-range anisotropic attractions (SRA) for 75-200 mg/mL mAb2 solutions at different pH and cosolute conditions by analyzing structure factors (Seff(q)) from small-angle X-ray scattering (SAXS) using coarse-grained molecular dynamics simulations. Best fit simulations additionally provide cluster size distributions, fractal dimensions, cluster occluded volume, and mAb coordination numbers. These equilibrium properties are utilized in a model to account for increases in viscosity caused by occluded volume in the clusters (packing effects) and dissipation of stress across lubricated fractal clusters. Seff(q) is highly sensitive to K at 75 mg/mL where mAbs can mutually align to form SRA contacts but becomes less sensitive at 200 mg/mL as steric repulsion due to packing becomes dominant. In contrast, η at 200 mg/mL is highly sensitive to SRA and the average cluster size from SAXS/simulation, which is observed to track the cluster relaxation time from shear thinning. By analyzing the distribution of sub-bead hot spots on the 3D mAb surface, we identify a strongly attractive hydrophobic patch in the complementarity determining region (CDR) at pH 4.5 that contributes to the high K and consequently large cluster sizes and high η. Adding NaCl screens electrostatic interactions and increases the impact of hydrophobic attraction on cluster size and raises η, whereas nonspecific binding of Arg attenuates all SRA, reducing η. The hydrophobic patch is absent at higher pH values, leading to smaller K, smaller clusters, and lower η. This work constitutes a first attempt to use SAXS and CG modeling to link both structural and rheological properties of concentrated mAb solutions to the energetics of specific hydrophobic patches on mAb surfaces. As such, our work opens an avenue for future research, including the possibility of designing coarse-grained models with physically meaningful interacting hot spots.

pH-Dependent Solution Micellar Structure of Amphoteric Polypeptoid Block Copolymers with Positionally Controlled Ionizable Sites.

While solution micellization of ionic block copolymers (BCP) with randomly distributed ionization sites along the hydrophilic segments has been extensively studied, the roles of positionally controlled ionization sites along the BCP chains in their micellization and resulting micellar structure remain comparatively less understood. Herein, three amphoteric polypeptoid block copolymers carrying two oppositely charged ionizable sites, with one fixed at the hydrophobic terminus and the other varyingly positioned along the hydrophilic segment, have been synthesized by sequential ring-opening polymerization method. The presence of the ionizable site at the hydrophobic segment terminus is expected to promote polymer association toward equilibrium micellar structures in an aqueous solution. The concurrent presence of oppositely charged ionizable sites on the polymer chains allows the polymer association to be electrostatically modulated in a broad pH range (ca. 2-12). Micellization of the amphoteric polypeptoid BCP in dilute aqueous solution and the resulting micellar structure at different solution pHs was investigated by a combination of scattering and microscopic methods. Negative-stain transmission-electron microscopy (TEM), small-angle neutron scattering (SANS), and small-angle X-ray scattering (SAXS) analyses revealed the dominant presence of core-shell-type spherical micelles and occasional rod-like micelles with liquid crystalline (LC) domains in the micellar core. The micellar structures (e.g., aggregation number, radius of gyration, chain packing in the micelle) were found to be dependent on the solution pH and the position of the ionizable site along the chain. This study has highlighted the potential of controlling the position of ionizable sites along the BCP polymer to modulate the electrostatic and LC interactions, thus tailoring the micellar structure at different solution pH values in water.

Development of in-line anoxic small-angle X-ray scattering and structural characterization of an oxygen-sensing transcriptional regulator.

Oxygen-sensitive metalloenzymes are responsible for many of the most fundamental biochemical processes in nature, from the reduction of dinitrogen in nitrogenase to the biosynthesis of photosynthetic pigments. However, biophysical characterization of such proteins under anoxic conditions can be challenging, especially at noncryogenic temperatures. In this study, we introduce the first in-line anoxic small-angle X-ray scattering (anSAXS) system at a major national synchrotron source, featuring both batch-mode and chromatography-mode capabilities. To demonstrate chromatography-coupled anSAXS, we investigated the oligomeric interconversions of the fumarate and nitrate reduction (FNR) transcription factor, which is responsible for the transcriptional response to changing oxygen conditions in the facultative anaerobe Escherichia coli. Previous work has shown that FNR contains a labile [4Fe-4S] cluster that is degraded when oxygen is present and that this change in cluster composition leads to the dissociation of the DNA-binding dimeric form. Using anSAXS, we provide the first direct structural evidence for the oxygen-induced dissociation of the E. coli FNR dimer and its correlation with cluster composition. We further demonstrate how complex FNR-DNA interactions can be studied by investigating the promoter region of the anaerobic ribonucleotide reductase genes, nrdDG, which contains tandem FNR-binding sites. By coupling size-exclusion chromatography-anSAXS with full-spectrum UV-Vis analysis, we show that the [4Fe-4S] cluster-containing dimeric form of FNR can bind to both sites in the nrdDG promoter region. The development of in-line anSAXS greatly expands the toolbox available for the study of complex metalloproteins and provides a foundation for future expansions.

Pressure pushes tRNALys3 into excited conformational states.

Conformational dynamics play essential roles in RNA function. However, detailed structural characterization of excited states of RNA remains challenging. Here, we apply high hydrostatic pressure (HP) to populate excited conformational states of tRNALys3, and structurally characterize them using a combination of HP 2D-NMR, HP-SAXS (HP-small-angle X-ray scattering), and computational modeling. HP-NMR revealed that pressure disrupts the interactions of the imino protons of the uridine and guanosine U-A and G-C base pairs of tRNALys3. HP-SAXS profiles showed a change in shape, but no change in overall extension of the transfer RNA (tRNA) at HP. Configurations extracted from computational ensemble modeling of HP-SAXS profiles were consistent with the NMR results, exhibiting significant disruptions to the acceptor stem, the anticodon stem, and the D-stem regions at HP. We propose that initiation of reverse transcription of HIV RNA could make use of one or more of these excited states.

Development of in-line anoxic small-angle X-ray scattering and structural characterization of an oxygen-sensing transcriptional regulator.

Oxygen-sensitive metalloenzymes are responsible for many of the most fundamental biochemical processes in nature, from the reduction of di-nitrogen in nitrogenase to the biosynthesis of photosynthetic pigments. However, biophysical characterization of such proteins under anoxic conditions can be challenging, especially at non-cryogenic temperatures. In this study, we introduce the first in-line anoxic small-angle X-ray scattering (anSAXS) system at a major national synchrotron source, featuring both batch-mode and chromatography-mode capabilities. To demonstrate chromatography-coupled anSAXS, we investigated the oligomeric interconversions of the Fumarate and Nitrate Reduction (FNR) transcription factor, which is responsible for the transcriptional response to changing oxygen conditions in the facultative anaerobe Escherichia coli . Previous work has shown that FNR contains a labile [4Fe-4S] cluster that is degraded when oxygen is present, and that this change in cluster composition leads to the dissociation of the DNA-binding dimeric form. Using anSAXS, we provide the first direct structural evidence for the oxygen-induced dissociation of the E. coli FNR dimer and its correlation with cluster composition. We further demonstrate how complex FNR-DNA interactions can be studied by investigating the promoter region of the anaerobic ribonucleotide reductase genes, nrdDG , which contains tandem FNR binding sites. By coupling SEC-anSAXS with full spectrum UV-Vis analysis, we show that the [4Fe-4S] clustercontaining dimeric form of FNR can bind to both sites in the nrdDG promoter region. The development of in-line anSAXS greatly expands the toolbox available for the study of complex metalloproteins and provides a foundation for future expansions.

Micelle Formation and Phase Separation of Poloxamer 188 and Preservative Molecules in Aqueous Solutions Studied by Small Angle X-ray Scattering.

Multi-injection pharmaceutical products such as insulin must be formulated to prevent aggregation and microbial contamination. Small-molecule preservatives and nonionic surfactants such as poloxamer 188 (P188) are thus often employed in protein drug formulations. However, mixtures of preservatives and surfactants can induce aggregation and even phase separation over time, despite the fact that all components are well dissolvable when used alone in aqueous solution. A systematic study is conducted here to understand the phase behavior and morphological causes of aggregation of P188 in the presence of the preservatives phenol and benzyl alcohol, primarily using small-angle x-ray scattering (SAXS). Based on SAXS results, P188 remains as unimers in solution when below a certain phenol concentration. Upon increasing the phenol concentration, a regime of micelle formation is observed due to the interaction between P188 and phenol. Further increasing the phenol concentration causes mixtures to become turbid and phase-separate over time. The effect of benzyl alcohol on the phase behavior is also investigated.

High Pressure CPMG and CEST Reveal That Cavity Position Dictates Distinct Dynamic Disorder in the PP32 Repeat Protein.

Given the central role of conformational dynamics in protein function, it is essential to characterize the time scales and structures associated with these transitions. High pressure (HP) perturbation favors transitions to excited states because they typically occupy a smaller molar volume, thus facilitating characterization of conformational dynamics. Repeat proteins, with their straightforward architecture, provide good models for probing the sequence dependence of protein conformational dynamics. Investigations of chemical exchange by 15N CPMG relaxation dispersion analysis revealed that introduction of a cavity via substitution of isoleucine 7 by alanine in the N-terminal capping motif of the pp32 leucine-rich repeat protein leads to pressure-dependent conformational exchange detected on the 500 µs-2 ms CPMG time scale. Exchange amplitude decreased from the N- to C-terminus, revealing a gradient of conformational exchange across the protein. In contrast, introduction of a cavity in the central core of pp32 via the L60A mutation led to pressure-induced exchange on a slower (>2 ms) time scale detected by 15N-CEST analysis. Excited state 15N chemical shifts indicated that in the excited state detected by HP CEST, the N-terminal region is mostly unfolded, while the core retains native-like structure. These HP chemical exchange measurements reveal that cavity position dictates exchange on distinct time scales, highlighting the subtle, yet central role of sequence in determining protein conformational dynamics.

Inline small-angle X-ray scattering-coupled chromatography under extreme hydrostatic pressure.

As continuing discoveries highlight the surprising abundance and resilience of deep ocean and subsurface microbial life, the effects of extreme hydrostatic pressure on biological structure and function have attracted renewed interest. Biological small-angle X-ray scattering (BioSAXS) is a widely used method of obtaining structural information from biomolecules in solution under a wide range of solution conditions. Due to its ability to reduce radiation damage, remove aggregates, and separate monodisperse components from complex mixtures, size-exclusion chromatography-coupled SAXS (SEC-SAXS) is now the dominant form of BioSAXS at many synchrotron beamlines. While BioSAXS can currently be performed with some difficulty under pressure with non-flowing samples, it has not been clear how, or even if, continuously flowing SEC-SAXS, with its fragile media-packed columns, might work in an extreme high-pressure environment. Here we show, for the first time, that reproducible chromatographic separations coupled directly to high-pressure BioSAXS can be achieved at pressures up to at least 100 MPa and that pressure-induced changes in folding and oligomeric state and other properties can be observed. The apparatus described here functions at a range of temperatures (0°C-50°C), expanding opportunities for understanding biomolecular rules of life in deep ocean and subsurface environments.

A round-robin approach provides a detailed assessment of biomolecular small-angle scattering data reproducibility and yields consensus curves for benchmarking.

Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0-1 Å-1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å-1, but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H2O and in D2O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å-1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set.

High-pressure SAXS, deep life, and extreme biophysics.

The biological relevance of hydrostatic pressure is becoming much more widely understood and appreciated as discoveries of new niches for extreme life continue to emerge. The unusual chemistry and physiological adaptations of organisms under extreme pressure promises to be a rich source of new insights in the years ahead if structural information can be obtained at the molecular level. Fortunately, recent advances in instrumentation are making structural biology techniques easier to perform at extreme pressures and more widely available. In addition to biological applications, hydrostatic pressure is a useful biophysical tool that can perturb systems in ways directly connected to the presence of atomic-level voids, cavities, and other volumetric properties. Under pressure, individual molecular complexes can dissociate, and monomers can unfold; transitions can occur in lipid mesophases, and liquid phases can dissolve and re-form. Small angle X-ray solution scattering (SAXS) can detect and characterize pressure-induced changes in all these situations. This chapter reviews what is known about pressure effects in a wide variety of biomolecular systems and how those effects display in X-ray scattering data. The influence of hydrostatic pressure on solution scattering is discussed, and the most widely used data processing methods are re-examined considering pressure effects. The chapter concludes with an overview of the high-pressure SAXS instrument design followed by recommended data collection protocol.

Antimicrobial Peptide Mechanism Studied by Scattering-Guided Molecular Dynamics Simulation.

In an effort to combat rising antimicrobial resistance, our labs have rationally designed cationic, helical, amphipathic antimicrobial peptides (AMPs) as alternatives to traditional antibiotics since AMPs incur bacterial resistance in weeks, rather than days. One highly positively charged AMP, WLBU2 (+13e), (RRWV RRVR RWVR RVVR VVRR WVRR), has been shown to be effective in killing both Gram-negative (G(-)) and Gram-positive (G(+)) bacteria by directly perturbing the bacterial membrane nonspecifically. Previously, we used two equilibrium experimental methods: synchrotron X-ray diffuse scattering (XDS) providing lipid membrane thickness and neutron reflectometry (NR) providing WLBU2 depth of penetration into three lipid model membranes (LMMs). The purpose of the present study is to use the results from the scattering experiments to guide molecular dynamics (MD) simulations to investigate the detailed biophysics of the interactions of WLBU2 with LMMs of Gram-negative outer and inner membranes, and Gram-positive cell membranes, to elucidate the mechanisms of bacterial killing. Instead of coarse-graining, backmapping, or simulating without bias for several microseconds, all-atom (AA) simulations were guided by the experimental results and then equilibrated for ∼0.5 µs. Multiple replicas of the inserted peptide were run to probe stability and reach a combined time of at least 1.2 µs for G(-) and also 2.0 µs for G(+). The simulations with experimental comparisons help rule out certain structures and orientations and propose the most likely set of structures, orientations, and effects on the membrane. The simulations revealed that water, phosphates, and ions enter the hydrocarbon core when WLBU2 is positioned there. For an inserted peptide, the three types of amino acids, arginine, tryptophan, and valine (R, W, V), are arranged with the 13 Rs extending from the hydrocarbon core to the phosphate group, Ws are located at the interface, and Vs are more centrally located. For a surface state, R, W, and V are positioned relative to the bilayer interface as expected from their hydrophobicities, with Rs closest to the phosphate group, Ws close to the interface, and Vs in between. G(-) and G(+) LMMs are thinned ∼1 Å by the addition of WLBU2. Our results suggest a dual anchoring mechanism for WLBU2 both in the headgroup and in the hydrocarbon region that promotes a defect region where water and ions can flow across the slightly thinned bacterial cell membrane.

Terminase Subunits from the Pseudomonas-Phage E217.

Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae E217, a Pseudomonas-phage used in an experimental cocktail to eradicate P. aeruginosa in vitro and in animal models. We identified the large (TerL) and small (TerS) terminase subunits in two genes ∼58 kbs away from each other in the E217 genome. TerL presents a classical two-domain architecture, consisting of an N-terminal ATPase and C-terminal nuclease domain arranged into a bean-shaped tertiary structure. A 2.05 Å crystal structure of the C-terminal domain revealed an RNase H-like fold with two magnesium ions in the nuclease active site. Mutations in TerL residues involved in magnesium coordination had a dominant-negative effect on phage growth. However, the two ions identified in the active site were too far from each other to promote two-metal-ion catalysis, suggesting a conformational change is required for nuclease activity. We also determined a 3.38 Å cryo-EM reconstruction of E217 TerS that revealed a ring-like decamer, departing from the most common nonameric quaternary structure observed thus far. E217 TerS contains both N-terminal helix-turn-helix motifs enriched in basic residues and a central channel lined with basic residues large enough to accommodate double-stranded DNA. Overexpression of TerS caused a more than a 4-fold reduction of E217 burst size, suggesting a catalytic amount of the protein is required for packaging. Together, these data expand the molecular repertoire of viral terminase subunits to Pseudomonas-phages used for phage therapy.

Recognition of the TDP-43 nuclear localization signal by importin α1/ß.

Cytoplasmic mislocalization of the TAR-DNA binding protein of 43 kDa (TDP-43) leads to large, insoluble aggregates that are a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. Here, we study how importin α1/ß recognizes TDP-43 bipartite nuclear localization signal (NLS). We find that the NLS makes extensive contacts with importin α1, especially at the minor NLS-binding site. NLS binding results in steric clashes with the C terminus of importin α1 that disrupts the TDP-43 N-terminal domain (NTD) dimerization interface. A putative phosphorylation site in the proximity of TDP-43 R83 at the minor NLS site destabilizes binding to importins by reducing the NLS backbone dynamics. Based on these data, we explain the pathogenic role of several post-translational modifications and mutations in the proximity of TDP-43 minor NLS site that are linked to disease and shed light on the chaperone activity of importin α1/ß.

Differential recognition of canonical NF-κB dimers by Importin α3.

Nuclear translocation of the p50/p65 heterodimer is essential for NF-κB signaling. In unstimulated cells, p50/p65 is retained by the inhibitor IκBα in the cytoplasm that masks the p65-nuclear localization sequence (NLS). Upon activation, p50/p65 is translocated into the nucleus by the adapter importin α3 and the receptor importin ß. Here, we describe a bipartite NLS in p50/p65, analogous to nucleoplasmin NLS but exposed in trans. Importin α3 accommodates the p50- and p65-NLSs at the major and minor NLS-binding pockets, respectively. The p50-NLS is the predominant binding determinant, while the p65-NLS induces a conformational change in the Armadillo 7 of importin α3 that stabilizes a helical conformation of the p65-NLS. Neither conformational change was observed for importin α1, which makes fewer bonds with the p50/p65 NLSs, explaining the preference for α3. We propose that importin α3 discriminates between the transcriptionally active p50/p65 heterodimer and p50/p50 and p65/65 homodimers, ensuring fidelity in NF-κB signaling.

Substitution of the Methionine Axial Ligand of the T1 Copper for the Fungal-like Phenylalanine Ligand (M298F) Causes Local Structural Perturbations that Lead to Thermal Instability and Reduced Catalytic Efficiency of the Small Laccase from Streptomyces coelicolor A3(2).

Many industrial processes operate at elevated temperatures or within broad pH and salinity ranges. However, the utilization of enzymes to carry out biocatalysis in such processes is often impractical or even impossible. Laccases (EC 1.10.3.2), which constitute a large family of multicopper oxidases, have long been used in the industrial setting. Although fungal laccases are in many respects considered superior to their bacterial counterparts, the bacterial laccases have been receiving greater attention recently. Albeit lower in redox potential than fungal laccases, bacterial laccases are commonly thermally more stable, act within broader pH ranges, do not contain posttranslational modifications, and could therefore serve as a high potential scaffold for directed evolution for the production of enzymes with enhanced properties. Several examples focusing on the axial ligand mutations of the T1 copper site have been published in the past. However, structural evidence on the local and global changes induced by those mutations have thus far been of computational nature only. In this study, we set out to structurally and kinetically characterize a few of the most commonly reported axial ligand mutations of a bacterial small laccase (SLAC) from Streptomyces coelicolor. While one of the mutations (Met to Leu) equips the enzyme with better thermal stability, the other (Met to Phe) induces an opposite effect. These mutations cause local structural rearrangement of the T1 site as demonstrated by X-ray crystallography. Our analysis confirms past findings that for SLACs, single point mutations that change the identity of the axial ligand of the T1 copper are not enough to provide a substantial increase in the catalytic efficiency but can in some cases have a detrimental effect on the enzyme's thermal stability parameters instead.

Tuning C-Phycocyanin Photoactivity via pH-Mediated Assembly-Disassembly.

Environment-triggered protein conformational changes have garnered wide interest in both fundamental research, for deciphering in vivo acclimatory responses, and practical applications, for designing stimuli-responsive probes. Here, we propose a protein-chromophore regulatory mechanism that allows for manipulation of C-phycocyanin (C-PC) from Spirulina platensis by environmental pH and UV irradiation. Using small-angle X-ray scattering, a pH-mediated C-PC assembly-disassembly pathway, from monomers to nonamers, was unraveled. Such flexible protein matrices impart tunability to the embedded tetrapyrroles, whose photochemical behaviors were found to be modulated by protein assembly states. UV irradiation on C-PC triggers pH-dependent singlet oxygen (1O2) generation and conformational changes. Intermolecular photo-crosslinking occurs at pH 5.0 via dityrosine species, which bridges solution-based C-PC oligomers into unprecedented dodecamers and 24-mers. These supramolecular assemblies impart C-PC at pH 5.0, which significantly enhanced 1O2 yield, fluorescence, and photostability relative to those at other pH values, a finding that makes C-PC appealing for tumor-targeted photodynamic therapy.

Protein unfolded states populated at high and ambient pressure are similarly compact.

The relationship between the dimensions of pressure-unfolded states of proteins compared with those at ambient pressure is controversial; resolving this issue is related directly to the mechanisms of pressure denaturation. Moreover, a significant pressure dependence of the compactness of unfolded states would complicate the interpretation of folding parameters from pressure perturbation and make comparison to those obtained using alternative perturbation approaches difficult. Here, we determined the compactness of the pressure-unfolded state of a small, cooperatively folding model protein, CTL9-I98A, as a function of temperature. This protein undergoes both thermal unfolding and cold denaturation, and the temperature dependence of the compactness at atmospheric pressure is known. High-pressure small angle x-ray scattering studies, yielding the radius of gyration and high-pressure diffusion ordered spectroscopy NMR experiments, yielding the hydrodynamic radius were carried out as a function of temperature at 250 MPa, a pressure at which the protein is unfolded. The radius of gyration values obtained at any given temperature at 250 MPa were similar to those reported previously at ambient pressure, and the trends with temperature are similar as well, although the pressure-unfolded state appears to undergo more pronounced expansion at high temperature than the unfolded state at atmospheric pressure. At 250 MPa, the compaction of the unfolded chain was maximal between 25 and 30°C, and the chain expanded upon both cooling and heating. These results reveal that the pressure-unfolded state of this protein is very similar to that observed at ambient pressure, demonstrating that pressure perturbation represents a powerful approach for observing the unfolded states of proteins under otherwise near-native conditions.