Osman Bilsel

Dye-Sensitized Core/Active Shell Upconversion Nanoparticles for Optogenetics and Bioimaging Applications.

Near-infrared (NIR) dye-sensitized upconversion nanoparticles (UCNPs) can broaden the absorption range and boost upconversion efficiency of UCNPs. Here, we achieved significantly enhanced upconversion luminescence in dye-sensitized core/active shell UCNPs via the doping of ytterbium ions (Yb(3+)) in the UCNP shell, which bridged the energy transfer from the dye to the UCNP core. As a result, we synergized the two most practical upconversion booster effectors (dye-sensitizing and core/shell enhancement) to amplify upconversion efficiency. We demonstrated two biomedical applications using these UCNPs. By using dye-sensitized core/active shell UCNP embedded poly(methyl methacrylate) polymer implantable systems, we successfully shifted the optogenetic neuron excitation window to a biocompatible and deep tissue penetrable 800 nm wavelength. Furthermore, UCNPs were water-solubilized with Pluronic F127 with high upconversion efficiency and can be imaged in a mouse model.

Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein

The earliest kinetic folding events for (βα)8 barrels reflect the appearance of off-pathway intermediates. Continuous-flow microchannel mixing methods interfaced to small-angle x-ray scattering (SAXS), circular dichroism (CD), time-resolved Förster resonant energy transfer (trFRET), and time-resolved fluorescence anisotropy (trFLAN) have been used to directly monitor global and specific dimensional properties of the partially folded state in the microsecond time range for a representative (βα)8 barrel protein. Within 150 μs, the α-subunit of Trp synthase (αTS) experiences a global collapse and the partial formation of secondary structure. The time resolution of the folding reaction was enhanced with trFRET and trFLAN to show that, within 30 μs, a distinct and autonomous partially collapsed structure has already formed in the N-terminal and central regions but not in the C-terminal region. A distance distribution analysis of the trFRET data confirmed the presence of a heterogeneous ensemble that persists for several hundreds of microseconds. Ready access to locally folded, stable substructures may be a hallmark of repeat-module proteins and the source of early kinetic traps in these very common motifs. Their folding free-energy landscapes should be elaborated to capture this source of frustration.

Barriers in protein folding reactions.

Publisher Summary This chapter focuses on the experimental thermodynamic and structural characterization of the barriers in the folding of single-domain globular proteins. The chapter also focuses on the structural aspects of the transition state of folding reactions, primarily, on mutational analyses and other protein engineering studies in which folding and/or unfolding kinetics have been investigated. The thermodynamic properties and the dynamics of the barrier crossings are also discussed. Studies of the elementary events in protein folding through simple model systems and a comparative overview of insights obtained from experimental and computational results are presented in the chapter. Many single-domain proteins, typically composed of 300 or fewer amino acids, exhibit intermediates in their folding reactions. Studies in which the properties of the intermediates have been the focus, as opposed to the transition states, are discussed. The chapter concludes that the merging of computational and experimental studies offers the prospect of unprecedented insight into the structure and dynamics of barriers in protein folding reactions.

A microchannel solution mixer for studying microsecond protein folding reactions

Many proteins fold through intermediates that are populated in the submillisecond time regime. To monitor directly the formation of these kinetic intermediates, we have developed a simple, robust, easy to assemble continuous flow mixer for studying folding reactions in the 35–1000μs time regime. The mixer is constructed by laser-machining 75-μm channels in a 127-μm-thick polyimide or polyetheretherketone polymer wafer. Mixing times of ∼25to∼50μs can be achieved for a 1∕10 dilution reaction of 8M urea with flow rates of 10–20mL∕min. CCD-based steady-state and time-correlated single-photon-counting-based fluorescence detection strategies are described. Preliminary results on the early events in the refolding of cytochrome c are presented.

Metal Deficiency Increases Aberrant Hydrophobicity of Mutant Superoxide Dismutases That Cause Amyotrophic Lateral Sclerosis

The mechanisms by which mutant variants of Cu/Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis are not clearly understood. Evidence to date suggests that altered conformations of amyotrophic lateral sclerosis mutant SOD1s trigger perturbations of cellular homeostasis that ultimately cause motor neuron degeneration. In this study we correlated the metal contents and disulfide bond status of purified wild-type (WT) and mutant SOD1 proteins to changes in electrophoretic mobility and surface hydrophobicity as detected by 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. As-isolated WT and mutant SOD1s were copper-deficient and exhibited mobilities that correlated with their expected negative charge. However, upon disulfide reduction and demetallation at physiological pH, both WT and mutant SOD1s underwent a conformational change that produced a slower mobility indicative of partial unfolding. Furthermore, although ANS did not bind appreciably to the WT holoenzyme, incubation of metal-deficient WT or mutant SOD1s with ANS increased the ANS fluorescence and shifted its peak toward shorter wavelengths. This increased interaction with ANS was greater for the mutant SOD1s and could be reversed by the addition of metal ions, especially Cu2+, even for SOD1 variants incapable of forming the disulfide bond. Overall, our findings support the notion that misfolding associated with metal deficiency may facilitate aberrant interactions of SOD1 with itself or with other cellular constituents and may thereby contribute to neuronal toxicity.

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.

Metal-free ALS variants of dimeric human Cu,Zn-superoxide dismutase have enhanced populations of monomeric species

Amino acid replacements at dozens of positions in the dimeric protein human, Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS). Although it has long been hypothesized that these mutations might enhance the populations of marginally-stable aggregation-prone species responsible for cellular toxicity, there has been little quantitative evidence to support this notion. Perturbations of the folding free energy landscapes of metal-free versions of five ALS-inducing variants, A4V, L38V, G93A, L106V and S134N SOD1, were determined with a global analysis of kinetic and thermodynamic folding data for dimeric and stable monomeric versions of these variants. Utilizing this global analysis approach, the perturbations on the global stability in response to mutation can be partitioned between the monomer folding and association steps, and the effects of mutation on the populations of the folded and unfolded monomeric states can be determined. The 2- to 10-fold increase in the population of the folded monomeric state for A4V, L38V and L106V and the 80- to 480-fold increase in the population of the unfolded monomeric states for all but S134N would dramatically increase their propensity for aggregation through high-order nucleation reactions. The wild-type-like populations of these states for the metal-binding region S134N variant suggest that even wild-type SOD1 may also be prone to aggregation in the absence of metals.

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.

Microsecond subdomain folding in dihydrofolate reductase.

The characterization of microsecond dynamics in the folding of multisubdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain α/β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼550 μs phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR--in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse--emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed.