Assessing CaDPA levels, metabolic activity, and spore detection through deuterium labeling.

Many strains among spore-forming bacteria species are associated with food spoilage, foodborne disease, and hospital-acquired infections. Understanding the impact of environmental conditions and decontamination techniques on the metabolic activity, viability, and biomarkers of these spores is crucial for combatting them. To distinguish and track spores and to understand metabolic mechanisms, spores must be labeled. Staining or genetic modification are current methods for this, however, these methods can be time-consuming, and affect the viability and function of spore samples. In this work, we investigate the use of heavy water for permanent isotope labeling of spores and Raman spectroscopy for tracking sporulation/germination mechanisms. We also discuss the potential of this method in observing decontamination. We find that steady-state deuterium levels in the spore are achieved after only ∼48 h of incubation with 30% D2O-infused broth and sporulation, generating Raman peaks at cell silent region of 2200 and 2300 cm-1. These deuterium levels then decrease rapidly upon spore germination in non-deuterated media. We further find that unlike live spores, spores inactivated using various methods do not lose these Raman peaks upon incubation in growth media, suggesting these peaks may be used to indicate the viability of a spore sample. We further observe several Raman peaks exclusive to deuterated DPA, a spore-specific chemical biomarker, at e.g. 988 and 2300 cm-1, which can be used to track underlying changes in spores involving DPA. In conclusion, permanent spore labeling using deuterium offers a robust and non-invasive way of labeling bacterial spores for marking, viability determination, and characterising spore activity.

Hsp70 Inhibits Aggregation of IAPP by Binding to the Heterogeneous Prenucleation Oligomers.

Molecular chaperone Hsp70 plays important roles in the pathology of amyloid diseases by inhibiting aberrant aggregation of proteins. However, the biophysical mechanism of the interaction of Hsp70 with the intrinsically disordered proteins (IDPs) is unclear. Here, we report that Hsp70 inhibits aggregation of islet amyloid polypeptide (IAPP) at substoichiometric concentrations under diverse solution conditions, including in the absence of ATP. The inhibitory effect is strongest if Hsp70 is added in the beginning of aggregation but progressively less if added later, indicating a role for Hsp70 in preventing nucleation of IAPP. However, ensemble measurement of the binding affinity suggests poor interactions between Hsp70 and IAPP. Therefore, we hypothesize that the interaction must involve a rare species (e.g., the oligomeric intermediates of IAPP). Size exclusion chromatography and field flow fractionation are then used to fractionate the constituent species. Multiangle light scattering and fluorescence correlation spectroscopy measurements indicate that the dominant fraction in size exclusion chromatography contains a few nanomolar Hsp70-IAPP complexes amid several µmoles of free Hsp70. Using single-particle two-color coincidence detection measurements, we detected a minor fraction that exhibits fluorescence bursts arising from heterogeneous oligomeric complexes of IAPP and Hsp70. Taken together, our results indicate that Hsp70 interacts poorly with the monomers but strongly with oligomers of IAPP. This is likely a generic feature of the interactions of Hsp70 chaperones with the amyloidogenic IDPs. Whereas high-affinity interactions with the oligomers prevent aberrant aggregation, poor interaction with the monomers averts interference with the physiological functions of the IDPs.

High-affinity multivalent interactions between apolipoprotein E and the oligomers of amyloid-ß.

Although the interaction of apoE isoforms with amyloid-ß (Aß) peptides plays a critical role in the progression of Alzheimer's disease, how they interact with each other remains poorly understood. Here, we investigate the molecular mechanism of apoE-Aß interactions by comparing the effects of the different domains of apoE on Aß. The kinetics of aggregation of Aß1-42 are delayed dramatically in the presence of substoichiometric, nanomolar concentrations of N-terminal fragment (NTF), C-terminal fragment (CTF) and full-length apoE both in lipid-free and in lipidated forms. However, interactions between apoE and Aß as measured by intermolecular Förster resonance energy transfer (FRET) analysis were found to be minimal at t = 0 but to increase in a time-dependent manner. Thus, apoE must interact with one or more 'intermediates' rather than the monomers of Aß. Kinetics of FRET between full-length apoE4 labelled with EDANS at position 62 or 139 or 210 or 247 or 276, and tetramethylrhodamine-labelled Aß (TMR-Aß), further support an involvement of all the three domains of apoE in the interactions. However, the above-mentioned residues do not appear to form a single pocket in the 3-dimensional structure of apoE. A competitive binding assay examining the effects of unlabelled fragments or full-length apoE on the FRET between EDANS-apoE and TMR-Aß show that binding affinity of the full-length apoE to Aß is much higher than that of the fragments. Furthermore, apoE4 is found to interact more strongly than apoE3. We hypothesize that high affinity of the apoE-Aß interaction is attained due to multivalent binding mediated by multiple interactions between oligomeric Aß and full-length apoE.

Probing transient non-native states in amyloid beta fiber elongation by NMR.

Using NMR to probe transient binding of Aß1-40 monomers to fibers, we find partially bound conformations with the highest degree of interaction near F19-K28 and a lesser degree of interaction near the C-terminus (L34-G37). This represents a shift away from the KLVFFA recognition sequence (residues 16-21) currently used for inhibitor design.

Building, Characterization, and Applications of Cuvette-FCS in Denaturant-Induced Expansion of Globular and Disordered Proteins.

Fluorescence correlation spectroscopy (FCS) is a single-molecule sensitive technique with widespread applications in biophysics. However, conventional microscope-based FCS setups have limitations in performing certain experiments such as those requiring agitations such as stirring or heating, and those involving measurements in solvents with the mismatch of refractive indices. We have recently developed an FCS setup that is suitable for performing measurements inside regular cuvettes. The cuvette-FCS is suitable for performing single-molecule measurements in experiments that are regularly performed in spectrofluorometers but are generally avoided in microscope-based FCS. Here we describe building and characterization of the performance of the cuvette-FCS setup in detail. Finally, we have used a natively folded protein and an intrinsically disordered protein to demonstrate and describe how cuvette-FCS can be applied conveniently to measure urea-dependent expansion of hydrodynamic size of proteins.

A Fluorescence Correlation Spectrometer for Measurements in Cuvettes.

We have developed a fluorescence correlation spectroscopy (FCS) setup for performing single-molecule measurements on samples inside regular cuvettes. The cuvette FCS uses a horizontally mounted extra-long working distance, 0.7 NA, air objective with a working distance of >1.8 mm instead of a high NA water or oil immersion objective. The performance of the cuvette FCS is found to be highly sensitive to the quality and alignment of the cuvette. The radial resolution and effective observation volume obtained using the optimized setup are ∼340 nm and 1.8 fL, respectively. The highest molecular brightness and the signal/noise ratio in the autocorrelation data achieved using an aqueous solution of rhodamine B are greater than 44 kHz and 110, respectively. Here, we demonstrate two major advantages of cuvette FCS. For example, the cuvette FCS can be used for measurements over a wide range of temperatures that is beyond the range permitted in the microscope-based FCS. Furthermore, cuvette FCS can be coupled to automatic titrators to study urea-dependent unfolding of proteins with unprecedented accuracy. The ease of use and compatibility with various accessories will enable applications of cuvette FCS in the experiments that are regularly performed in spectrofluorometers but are generally avoided in microscope-based FCS.

Quantitative Characterization of Metastability and Heterogeneity of Amyloid Aggregates.

Amyloids are heterogeneous assemblies of extremely stable fibrillar aggregates of proteins. Although biological activities of the amyloids are dependent on its conformation, quantitative evaluation of heterogeneity of amyloids has been difficult. Here we use disaggregation of the amyloids of tetramethylrhodamine-labeled Aß (TMR-Aß) to characterize its stability and heterogeneity. Disaggregation of TMR-Aß amyloids, monitored by fluorescence recovery of TMR, was negligible in native buffer even at low nanomolar concentrations but the kinetics increased exponentially with addition of denaturants such as urea or GdnCl. However, dissolution of TMR-Aß amyloids is different from what is expected in the case of thermodynamic solubility. For example, the fraction of soluble amyloids is found to be independent of total concentration of the peptide at all concentrations of the denaturants. Additionally, soluble fraction is dependent on growth conditions such as temperature, pH, and aging of the amyloids. Furthermore, amyloids undissolved in a certain concentration of the denaturant do not show any further dissolution after dilution in the same solvent; instead, these require higher concentrations of the denaturant. Taken together, our results indicate that amyloids are a heterogeneous ensemble of metastable states. Furthermore, dissolution of each structurally homogeneous member requires a unique threshold concentration of denaturant. Fraction of soluble amyloids as a function of concentration of denaturants is found to be sigmoidal. The sigmoidal curve becomes progressively steeper with progressive seeding of the amyloids, although the midpoint remains unchanged. Therefore, heterogeneity of the amyloids is a major determinant of the steepness of the sigmoidal curve. The sigmoidal curve can be fit assuming a normal distribution for the population of the amyloids of various kinetic stabilities. We propose that the mean and the standard deviation of the normal distribution provide quantitative estimates of mean kinetic stability and heterogeneity, respectively, of the amyloids in a certain preparation.

A single fluorophore to address multiple logic gates.

Logic gates with different radixes have been constructed using a biologically active molecule, 2-(4'-N,N-dimethylaminophenyl)imidazo[4,5-b]pyridine (DMAPIP-b). Taking advantage of the multiple binding sites of the fluorophore, a series of different molecular logic gates are developed using fluorescence intensities at different wavelengths. The high emission of the molecule is drastically quenched in the presence of Fe(3+). It is regained by the addition of an equivalent amount of F(-). The fluorescence On-Off nature has been used to construct molecular full subtractor and molecular keypad lock system with Boolean logic. A ternary system is generated by considering three defined fluorescence intensities at particular wavelengths. The smooth dependency of emission intensities with analyte concentration is utilized to construct an infinite-valued fuzzy logic system. The fuzzy logic system is further coupled with a neuro-adaptation method to predict more accurately the dependency of molecular intensity on external inputs.