Catalytic diversity in self-propagating peptide assemblies.

The protein-only infectious agents known as prions exist within cellular matrices as populations of assembled polypeptide phases ranging from particles to amyloid fibres. These phases appear to undergo Darwinian-like selection and propagation, yet remarkably little is known about their accessible chemical and biological functions. Here we construct simple peptides that assemble into well-defined amyloid phases and define paracrystalline surfaces able to catalyse specific enantioselective chemical reactions. Structural adjustments of individual amino acid residues predictably control both the assembled crystalline order and their accessible catalytic repertoire. Notably, the density and proximity of the extended arrays of enantioselective catalytic sites achieve template-directed polymerization of new polymers. These diverse amyloid templates can now be extended as dynamic self-propagating templates for the construction of even more complex functional materials.

Neurofibrillar tangle surrogates: histone H1 binding to patterned phosphotyrosine peptide nanotubes.

Living cells contain a range of densely phosphorylated surfaces, including phospholipid membranes, ribonucleoproteins, and nucleic acid polymers. Hyperphosphorylated surfaces also accumulate in neurodegenerative diseases as neurofibrillar tangles. We have synthesized and structurally characterized a precisely patterned phosphotyrosine surface and establish this assembly as a surrogate of the neuronal tangles by demonstrating its high-affinity binding to histone H1. This association with nucleic acid binding proteins underscores the role such hyperphosphorylated surfaces may play in disease and opens functional exploration into protein-phosphorylated surface interactions in a wide range of other complex assemblies.

Mapping amyloid-ß(16-22) nucleation pathways using fluorescence lifetime imaging microscopy.

The cross-ß peptide architecture is associated with numerous functional biomaterials and deleterious disease related aggregates. While these diverse and ubiquitous paracrystalline assemblies have been widely studied, a fundamental understanding of the nucleation and aggregation pathways to these structures remains elusive. Here we highlight a novel application of fluorescence lifetime imaging microscopy in characterising the critical stages of peptide aggregation. Using the central nucleating core of the amyloid-ß (Aß), Aß(16-22), as a model cross-ß system, and utilising a small fraction of rhodamine labelled peptide (Rh110-Aß(17-22)), we map out a folding pathway from monomer to paracrystalline nanotube. Using this intrinsic fluorescence reporter, we demonstrate the effects of interfaces and evaporation on the nucleation of sub-critical concentration solutions, providing access to previously uncharacterised intermediate morphologies. Using fluorescence lifetime we follow the local peptide environment through the stages of nucleation and hydrophobic collapse, ending in a stable final structure. This work provides a metric for future implementations of measuring fluorescence lifetimes of intrinsic fluorescence reporters during the very dynamic processes relating to peptide nucleation and maturation.

τFCS: multi-method global analysis enhances resolution and sensitivity in fluorescence fluctuation measurements.

Fluorescence fluctuation methods have become invaluable research tools for characterizing the molecular-level physical and chemical properties of complex systems, such as molecular concentrations, dynamics, and the stoichiometry of molecular interactions. However, information recovery via curve fitting analysis of fluctuation data is complicated by limited resolution and challenges associated with identifying accurate fit models. We introduce a new approach to fluorescence fluctuation spectroscopy that couples multi-modal fluorescence measurements with multi-modal global curve fitting analysis. This approach yields dramatically enhanced resolution and fitting model discrimination capabilities in fluctuation measurements. The resolution enhancement allows the concentration of a secondary species to be accurately measured even when it constitutes only a few percent of the molecules within a sample mixture, an important new capability that will allow accurate measurements of molecular concentrations and interaction stoichiometry of minor sample species that can be functionally important but difficult to measure experimentally. We demonstrate this capability using τFCS, a new fluctuation method which uses simultaneous global analysis of fluorescence correlation spectroscopy and fluorescence lifetime data, and show that τFCS can accurately recover the concentrations, diffusion coefficients, lifetimes, and molecular brightness values for a two component mixture over a wide range of relative concentrations.

Electronic cameras for low-light microscopy.

This chapter introduces to electronic cameras, discusses the various parameters considered for evaluating their performance, and describes some of the key features of different camera formats. The chapter also presents the basic understanding of functioning of the electronic cameras and how these properties can be exploited to optimize image quality under low-light conditions. Although there are many types of cameras available for microscopy, the most reliable type is the charge-coupled device (CCD) camera, which remains preferred for high-performance systems. If time resolution and frame rate are of no concern, slow-scan CCDs certainly offer the best available performance, both in terms of the signal-to-noise ratio and their spatial resolution. Slow-scan cameras are thus the first choice for experiments using fixed specimens such as measurements using immune fluorescence and fluorescence in situ hybridization. However, if video rate imaging is required, one need not evaluate slow-scan CCD cameras. A very basic video CCD may suffice if samples are heavily labeled or are not perturbed by high intensity illumination. When video rate imaging is required for very dim specimens, the electron multiplying CCD camera is probably the most appropriate at this technological stage. Intensified CCDs provide a unique tool for applications in which high-speed gating is required. The variable integration time video cameras are very attractive options if one needs to acquire images at video rate acquisition, as well as with longer integration times for less bright samples. This flexibility can facilitate many diverse applications with highly varied light levels.

Global analysis in fluorescence correlation spectroscopy and fluorescence lifetime microscopy.

Fluorescence correlation spectroscopy (FCS) and related fluctuation spectroscopy and microscopy methods have become important research tools that enable detailed investigations of the chemical and physical properties of molecules and molecular systems in a variety of complex environments. Information recovery via curve fitting of fluctuation data can present complicating challenges due to limited resolution and/or problems with fitting model verification. We discuss a new approach to data analysis called τFCS that couples multiple modes of signal acquisition, here specifically FCS and fluorescence lifetimes, with global analysis. We demonstrate enhanced resolution using τFCS, including the capability to recover the concentration of both molecular species in a two-component mixture even when the species have identical diffusion coefficients and molecular brightness values, provided their fluorescent lifetimes are distinct. We also demonstrate how τFCS provides useful tools for model discrimination in FCS curve fitting.

Phase networks of cross-ß peptide assemblies.

Recent evidence suggests that simple peptides can access diverse amphiphilic phases, and that these structures underlie the robust and widely distributed assemblies implicated in nearly 40 protein misfolding diseases. Here we exploit a minimal nucleating core of the Aß peptide of Alzheimer's disease to map its morphologically accessible phases that include stable intermolecular molten particles, fibers, twisted and helical ribbons, and nanotubes. Analyses with both fluorescence lifetime imaging microscopy (FLIM) and transmission electron microscopy provide evidence for liquid-liquid phase separations, similar to the coexisting dilute and dense protein-rich liquid phases so critical for the liquid-solid transition in protein crystallization. We show that the observed particles are critical for transitions to the more ordered cross-ß peptide phases, which are prevalent in all amyloid assemblies, and identify specific conditions that arrest assembly at the phase boundaries. We have identified a size dependence of the particles in order to transition to the para-crystalline phase and a width of the cross-ß assemblies that defines the transition between twisted fibers and helically coiled ribbons. These experimental results reveal an interconnected network of increasing molecularly ordered cross-ß transitions, greatly extending the initial computational models for cross-ß assemblies.

Direct observation of nucleation and growth in amyloid self-assembly.

Access to native protein structure depends on precise polypeptide folding and assembly pathways. Identifying folding missteps that may lead to the nearly 40 protein misfolding diseases could feature prominently in the development of intervention strategies. Accordingly, we have investigated the earliest steps of assembly by the folding nucleus of the Alzheimer's disease Abeta peptide with real-time imaging and fluorescence correlation spectroscopy. These analyses reveal the immediate formation of large micrometer size clusters maintaining properties of intermolecular molten globules. These dynamic unstructured aggregates serve as the nucleating sites for amyloid growth and, as with native protein folding, appear important for backbone desolvation. The resulting amyloid nucleus however is able to template monomer addition from solution at rates from 2K peptides/s at millimolar peptide concentrations. This direct observation of amyloid assembly unifies several divergent models that currently exist for protein misfolding.

Fibrillogenesis in continuously spun synthetic collagen fiber.

The universal structural role of collagen fiber networks has motivated the development of collagen gels, films, coatings, injectables, and other formulations. However, reported synthetic collagen fiber fabrication schemes have either culminated in short, discontinuous fiber segments at unsuitably low production rates, or have incompletely replicated the internal fibrillar structure that dictates fiber mechanical and biological properties. We report a continuous extrusion system with an off-line phosphate buffer incubation step for the manufacture of synthetic collagen fiber. Fiber with a cross-section of 53+ or - 14 by 21 + or - 3 microm and an ultimate tensile strength of 94 + or - 19 MPa was continuously produced at 60 m/hr from an ultrafiltered monomeric collagen solution. The effect of collagen solution concentration, flow rate, and spinneret size on fiber size was investigated. The fiber was further characterized by microdifferential scanning calorimetry, transmission electron microscopy (TEM), second harmonic generation (SHG) analysis, and in a subcutaneous murine implant model. Calorimetry demonstrated stabilization of the collagen triple helical structure, while TEM and SHG revealed a dense, axially aligned D-periodic fibril structure throughout the fiber cross-section. Implantation of glutaraldehyde crosslinked and noncrosslinked fiber in the subcutaneous tissue of mice demonstrated limited inflammatory response and biodegradation after a 6-week implant period.

The intracellular mobility of nuclear import receptors and NLS cargoes.

We have investigated classical nuclear localization sequence (NLS) mediated protein trafficking by measuring biomolecular dynamics within living cells using two-photon fluorescence correlation spectroscopy. By directly observing the behavior of specific molecules in their native cellular environment, it is possible to uncover functional details that are not apparent from traditional biochemical investigations or functional assays. We show that the intracellular mobility of NLS cargoes and their import receptor proteins, karyopherin-alpha and karyopherin-beta, can be robustly measured and that quantitative comparison of intracellular diffusion coefficients provides new insights into nuclear transport mechanisms. Import cargo complexes are assembled throughout the cytoplasm, and their diffusion is slower than predicted by molecular weight due to specific interactions. Analysis of NLS cargo diffusion in the cytoplasm indicates that these interactions are likely disrupted by NLS cargo binding. Our results suggest that delivery of import receptors and NLS cargoes to nuclear pores may complement selective translocation through the pores as a functional mechanism for regulating transport of proteins into the nucleus.

Light harvesting antenna on an amyloid scaffold.

A pigment array has been constructed within a paracrystalline amyloid nanotube and Förster energy transfer along the nanotube surface has been demonstrated to self-assembled acceptor dyes.

Comparing the intracellular mobility of fluorescent proteins following in vitro expression or cell loading with streptolysin-O.

The application of fluorescent proteins in live cells has greatly improved our ability to study molecular mobility, which both reflects molecular function in live cells and reveals the properties of the local environment. Although measuring molecular mobility with fluorescent fusion proteins is powerful and convenient, certain experiments still require exogenous macromolecules to be loaded into cells. Cell viability provides a rough gauge of cellular damage following membrane permeabilization, but it is unknown how permeabilization will affect intracellular mobility. We have used fluorescence correlation spectroscopy to measure the intracellular dynamics of the enhanced green fluorescent protein (EGFP) in living human embryonic kidney (HEK) cells under conditions where the EGFP is either expressed or loaded using streptolysin O (SLO) permeabilization to determine how permeabilization effects mobility. We found that purified EGFP loaded with SLO has the same mobility as the expressed EGFP, while the mobility of the expressed EGFP after SLO permeabilization treatment becomes slightly slower. Our results indicate that SLO permeabilization is often accompanied by the loss of cellular soluble proteins to the surrounding medium, which explains the apparent decrease in diffusion rates following treatment. These measurements are also relevant to the role of molecular crowding in the intracellular mobility of proteins.

Propagators and time-dependent diffusion coefficients for anomalous diffusion.

Complex diffusive dynamics are often observed when one is investigating the mobility of macromolecules in living cells and other complex environments, yet the underlying physical or chemical causes of anomalous diffusion are often not fully understood and are thus a topic of ongoing research interest. Theoretical models capturing anomalous dynamics are widely used to analyze mobility data from fluorescence correlation spectroscopy and other experimental measurements, yet there is significant confusion regarding these models because published versions are not entirely consistent and in some cases do not appear to satisfy the diffusion equation. Further confusion is introduced through variations in how fitting parameters are reported. A clear definition of fitting parameters and their physical significance is essential for accurate interpretation of experimental data and comparison of results from different studies acquired under varied experimental conditions. This article aims to clarify the physical meaning of the time-dependent diffusion coefficients associated with commonly used fitting models to facilitate their use for investigating the underlying causes of anomalous diffusion. We discuss a propagator for anomalous diffusion that captures the power law dependence of the mean-square displacement and can be shown to rigorously satisfy the extended diffusion equation provided one correctly defines the time-dependent diffusion coefficient. We also clarify explicitly the relation between the time-dependent diffusion coefficient and fitting parameters in fluorescence correlation spectroscopy.

Recognition of polyadenosine RNA by zinc finger proteins.

Messenger RNA transcripts are coated from cap to tail with a dynamic combination of RNA binding proteins that process, package, and ultimately regulate the fate of mature transcripts. One class of RNA binding proteins essential for multiple aspects of mRNA metabolism consists of the poly(A) binding proteins. Previous studies have concentrated on the canonical RNA recognition motif-containing poly(A) binding proteins as the sole family of poly(A)-specific RNA binding proteins. In this study, we present evidence for a previously uncharacterized poly(A) recognition motif consisting of tandem CCCH zinc fingers. We have probed the nucleic acid binding properties of a yeast protein, Nab2, that contains this zinc finger motif. Results of this study reveal that the seven tandem CCCH zinc fingers of Nab2 specifically bind to polyadenosine RNA with high affinity. Furthermore, we demonstrate that a human protein, ZC3H14, which contains CCCH zinc fingers homologous to those found in Nab2, also specifically binds polyadenosine RNA. Thus, we propose that these proteins are members of an evolutionarily conserved family of poly(A) RNA binding proteins that recognize poly(A) RNA through a fundamentally different mechanism than previously characterized RNA recognition motif-containing poly(A) binding proteins.

Fluorescence intensity is a poor predictor of saturation effects in two-photon microscopy: artifacts in fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) has become an increasingly important measurement tool for biological and biomedical investigations, with the capability to assay molecular dynamics and interactions both in vitro and within living cells. Information recovery in FCS requires an accurate characterization and calibration of the observation volume. A number of recent reports have demonstrated that the calibration of the observation volume is excitation power dependent, a complication that arises due to excitation saturation. While quantitative models are now available to account for these volume variations, many researchers attempt to avoid saturation issues by working with low nonsaturating excitation intensities. For two-photon excited fluorescence, this is typically thought to be achievable by working with excitation powers for which the total measured fluorescence signal maintains its quadratic dependence on excitation intensity. We demonstrate that observing only the power dependence of the fluorescence intensity will tend to underestimate the importance of saturation, and explain these findings in terms of basic physical models.

Nuclear localization signal receptor affinity correlates with in vivo localization in Saccharomyces cerevisiae.

Nuclear localization signals (NLSs) target proteins into the nucleus through mediating interactions with nuclear import receptors. Here, we perform a quantitative analysis of the correlation between NLS receptor affinity and the steady-state distribution of NLS-bearing cargo proteins between the cytoplasm and the nucleus of live yeast, which reflects the relative import rates of various NLS sequences. We find that there is a complicated, but monotonic quantitative relationship between the affinity of an NLS for the import receptor, importin alpha, and the steady-state accumulation of the cargo in the nucleus. This analysis takes into consideration the impact of protein size. In addition, the hypothetical upper limit to an NLS affinity for the receptors is explored through genetic approaches. Overall, our results indicate that there is a correlation between the binding affinity of an NLS cargo for the NLS receptor, importin alpha, and the import rate for this cargo. This correlation, however, is not maintained for cargoes that bind to the NLS receptor with very weak or very strong affinity.

Characterizing observation volumes and the role of excitation saturation in one-photon fluorescence fluctuation spectroscopy.

Fluorescence correlation spectroscopy (FCS) and related distribution analysis techniques have become extremely important and widely used research tools for analyzing the dynamics, kinetics, interactions, and mobility of biomolecules. However, it is not widely recognized that photophysical dynamics can dramatically influence the calibration of fluctuation spectroscopy instrumentation. While the basic theories for fluctuation spectroscopy methods are well established, there have not been quantitative models to characterize the photophysical-induced variations observed in measured fluctuation spectroscopy data under varied excitation conditions. We introduce quantitative models to characterize how the fluorescence observation volumes in one-photon confocal microscopy are modified by excitation saturation as well as corresponding models for the effect of the volume changes in FCS. We introduce a simple curve fitting procedure to model the role of saturation in FCS measurements and demonstrate its accuracy in fitting measured correlation curves over a wide range of excitation conditions.

Observation volumes and {gamma}-factors in two-photon fluorescence fluctuation spectroscopy.

Fluorescence fluctuation spectroscopy has become an important measurement tool for investigating molecular dynamics, molecular interactions, and chemical kinetics in biological systems. Although the basic theory of fluctuation spectroscopy is well established, it is not widely recognized that saturation of the fluorescence excitation can dramatically alter the size and profile of the fluorescence observation volume from which fluorescence fluctuations are measured, even at relatively modest excitation levels. A precise model for these changes is needed for accurate analysis and interpretation of fluctuation spectroscopy data. We here introduce a combined analytical and computational approach to characterize the observation volume under saturating conditions and demonstrate how the variation in the volume is important in two-photon fluorescence correlation spectroscopy. We introduce a simple approach for analysis of fluorescence correlation spectroscopy data that can fully account for the effects of saturation, and demonstrate its success for characterizing the observed changes in both the amplitude and relaxation timescale of measured correlation curves. We also discuss how a quantitative model for the observed phenomena may be of broader importance in fluorescence fluctuation spectroscopy.

Saturation modified point spread functions in two-photon microscopy.

Excitation saturation can dramatically alter the effective imaging point spread function (PSF) in two-photon fluorescence microscopy. The saturation-modified PSF can have important implications for resolution in fluorescence imaging as saturation leads to both an increased fluorescence observation volume and an altered spatial profile for the PSF. We introduce here a computational approach to accurately quantify molecular excitation profiles that represent the modified imaging PSF in two-photon microscopy under the influence of excitation saturation. An analytical model that accounts for pulsed laser excitation is developed to calculate the influence of saturation at any location within the excitation laser profile. The overall saturation modified molecular excitation profiles are then evaluated numerically. Our results demonstrate that saturation can play an important role in two-photon fluorescence microscopy even with relatively modest excitation levels.