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Automatic detection of falls is important for enabling people who are older to safely live independently longer within their homes. Current automated fall detection systems are typically designed using inertial sensors positioned on the body that generate an alert if there is an abrupt change in motion. These inertial sensors provide no information about the context of the person being monitored and are prone to false positives that can limit their ongoing usage. We describe a fall-detection system consisting of a wearable inertial measurement unit (IMU) and an RF time-of-flight (ToF) transceiver that ranges with other ToF beacons positioned throughout a home. The ToF ranging enables the system to track the position of the person as they move around a home. We describe and show results from three machine learning algorithms that integrate context-related position information with IMU based fall detection to enable a deeper understanding of where falls are occurring and also to improve the specificity of fall detection. The beacons used to localize the falls were able to accurately track to within 0.39 meters of specific waypoints in a simulated home environment. Each of the three algorithms was evaluated with and without the context-based false alarm detection on simulated falls done by 3 volunteer subjects in a simulated home. False positive rates were reduced by 50% when including context.

Heme-linked proteins, such as cytochromes, are popular subjects for protein folding studies. There is the underlying question of whether the heme affects the structure of the denatured state by cross-linking it and forming other interactions, which would perturb the folding pathway. We have studied wild-type and mutant cytochrome b562 from Escherichia coli, a 106 residue four-alpha-helical bundle. The holo protein apparently refolds with a half-life of 4 micros in its ferrous state. We have analysed the folding of the apo protein using continuous-flow fluorescence as well as stopped-flow fluorescence and CD. The apo protein folded much more slowly with a half-life of 270 micros that was unaffected by the presence of exogenous heme. We examined the nature of the denatured states of both holo and apo proteins by NMR methods over a range of concentrations of guanidine hydrochloride. The starting point for folding of the holo protein in concentrations of denaturant around the denaturation transition was a highly ordered native-like species with heme bound. Fully denatured holo protein at higher concentrations of denaturant consisted of denatured apo protein and free heme. Our results suggest that the very fast folding species of denatured holo protein is in a compact state, whereas the normal folding pathway from fully denatured holo protein consists of the slower folding of the apo protein followed by the binding of heme. These data should be considered in the analysis of folding of heme proteins.

The earliest folding events in single-tryptophan mutants of RNase A were investigated by fluorescence measurements by using a combination of stopped-flow and continuous-flow mixing experiments covering the time range from 70 micros to 10 s. An ultrarapid double-jump mixing protocol was used to study refolding from an unfolded ensemble containing only native proline isomers. The continuous-flow measurements revealed a series of kinetic events on the submillisecond time scale that account for the burst-phase signal observed in previous stopped-flow experiments. An initial increase in fluorescence within the 70-micros dead time of the continuous-flow experiment is consistent with a relatively nonspecific collapse of the polypeptide chain whereas a subsequent decrease in fluorescence with a time constant of approximately 80 micros is indicative of a more specific structural event. These rapid conformational changes are not observed if RNase A is allowed to equilibrate under denaturing conditions, resulting in formation of nonnative proline isomers. Thus, contrary to previous expectations, the isomerization state of proline peptide bonds can have a major impact on the structural events during early stages of folding.

Information on the time-dependence of molecular species is critical for elucidating reaction mechanisms in chemistry and biology. Rapid flow experiments involving turbulent mixing of two or more solutions continue to be the main source of kinetic information on protein folding and other biochemical processes, such as ligand binding and enzymatic reactions. Recent advances in mixer design and detection methods have opened a new window for exploring conformational changes in proteins on the microsecond time scale. These developments have been especially important for exploring early stages of protein folding.

A continuous-flow mixing device with a dead time of 100 micros coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal beta-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT(*) SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step ( approximately 100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 micros. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the beta-barrel domain while the specific interactions in the alpha-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal beta-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT(*) SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.

The folding of cytochrome c(551) from Pseudomonas aeruginosa was previously thought to follow a simple sequential mechanism, consistent with the lack of histidine residues, other than the native His16 heme ligand, that can give rise to mis-coordinated species. However, further kinetic analysis reveals complexities indicative of a folding mechanism involving parallel pathways. Double-jump interrupted refolding experiments at low pH indicate that approximately 50% of the unfolded cytochrome c(551) population can reach the native state via a fast (10 ms) folding track, while the rest follows a slower folding path with populated intermediates. Stopped-flow experiments using absorbance at 695 nm to monitor refolding confirm the presence of a rapidly folding species containing the native methionine-iron bond while measurements on carboxymethylated cytochrome c(551) (which lacks the Met-Fe coordination bond) indicate that methionine ligation occurs late during folding along the fast folding track, which appears to be dominant at physiological pH. Continuous-flow measurements of tryptophan-heme energy transfer, using a capillary mixer with a dead time of about 60 micros, show evidence for a rapid chain collapse within 100 micros preceding the rate-limiting folding phase on the milliseconds time scale. A third process with a time constant in the 10-50 ms time range is consistent with a minor population of molecules folding along a parallel channel, as confirmed by quantitative kinetic modeling. These findings indicate the presence of two or more slowly inter-converting ensembles of denatured states that give rise to pH-dependent partitioning among fast and slow-folding pathways.

Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2-5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of approximately 150 micros, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.

For many proteins, compact conformations are known to accumulate in advance of the rate-limiting step in folding. To understand the nature and significance of these early conformational events, we employed ultrarapid mixing methods to fully characterize the kinetics of folding of the 57-residue B1 domain of protein G. Continuous-flow fluorescence measurements exhibit a major exponential phase on the submillisecond time scale (600-700 micros), which is followed by a slower phase with a denaturant-dependent time constant (2-30 ms) observable by conventional stopped-flow measurements. The combined kinetic traces quantitatively account for the total change in Trp 43 fluorescence upon folding, including the previously unresolved 'burst phase' signal. The denaturant dependence of the two rate constants and their relative amplitudes are fully consistent with a three-state mechanism, U right harpoon over left harpoon I right harpoon over left harpoon N, where I is a productive intermediate with native-like fluorescence properties. The relatively slow rate and exponential time course of the initial folding phase indicates that a substantial free energy barrier is encountered during chain condensation, resulting in a partially organized ensemble of states distinct from the initial unfolded conformations.

Although important structural events in protein folding are known to occur on the submillisecond time scale, the limited time resolution of conventional kinetic methods has precluded direct observation of the initial collapse of the polypeptide chain. A continuous-flow capillary mixing method recently developed by us made it possible to account for the entire fluorescence change associated with refolding of cytochrome c from approximately 5-10(-5)-10(2) s, including the previously unresolved quenching of Trp 59 fluorescence (burst phase) indicative of the formation of compact states. The kinetics of folding exhibits a major exponential process with a time constant of approximately 50 micros, independent of initial conditions and heme ligation state, indicating that a common free energy barrier is encountered during the initial collapse of the polypeptide chain. The resulting loosely packed intermediate accumulates prior to the rate-limiting formation of specific tertiary interactions, confirming previous indications that folding involves at least two distinct stages.

A continuous-flow capillary mixing apparatus, based on the original design of Regenfuss et al. (Regenfuss, P., R. M. Clegg, M. J. Fulwyler, F. J. Barrantes, and T. M. Jovin. 1985. Rev. Sci. Instrum. 56:283-290), has been developed with significant advances in mixer design, detection method and data analysis. To overcome the problems associated with the free-flowing jet used for observation in the original design (instability, optical artifacts due to scattering, poor definition of the geometry), the solution emerging from the capillary is injected directly into a flow-cell joined to the tip of the outer capillary via a ground-glass joint. The reaction kinetics are followed by measuring fluorescence versus distance downstream from the mixer, using an Hg(Xe) arc lamp for excitation and a digital camera with a UV-sensitized CCD detector for detection. Test reactions involving fluorescent dyes indicate that mixing is completed within 15 micros of its initiation and that the dead time of the measurement is 45 +/- 5 micros, which represents a >30-fold improvement in time resolution over conventional stopped-flow instruments. The high sensitivity and linearity of the CCD camera have been instrumental in obtaining artifact-free kinetic data over the time window from approximately 45 micros to a few milliseconds with signal-to-noise levels comparable to those of conventional methods. The scope of the method is discussed and illustrated with an example of a protein folding reaction.

The reverse micellar system formed by the negatively charged surfactant AOT and the organic solvent isooctane is used to solubilize the protein RNase T1. The physicochemical properties of the entrapped protein have been studied using intrinsic tryptophan fluorescence and far-and near-UV CD. These studies indicate a similar structure for the protein in reverse micelles and in pH 7.0 buffer. Thermal unfolding has been studied as a function of W0, the molar ratio of water to AOT, in the solution. Measuring the change in fluorescence intensity as a function of temperature, we observe a reversible transition for W0 in the range 5-12. Heating rate dependencies carried out on these transitions (0.6-3.0 degrees C/min) indicate that the transition temperature and the apparent van't Hoff enthalpy change depend on the scanning rate as well as on W0. The values of the transition temperature, T(m) and the enthalpy change, delta H degrees(un), extrapolated to an infinitely slow scanning rate, are analyzed considering the electrostatic interaction of the charged residues of the protein with the charges of the surfactant molecules forming reverse micelles, the variation of the size of the reverse micelles, and the relative rates of unfolding, refolding, and irreversible denaturation.

Two models are commonly used to describe the poorly understood earliest steps of protein folding. The framework model stresses very early formation of nascent secondary structures, which coalesce into a compact, molten, globule-like form from which tertiary structure slowly develops. The hydrophobic collapse model gives overriding precedence to a nonspecific collapse of the polypeptide chain which facilitates subsequent formation of specific secondary and tertiary structure. Here we report our analysis of the earliest observable events of the major folding pathway of barstar, a small protein. We compare the kinetics of folding using circular dichroism at 222 nm and 270 nm, intrinsic tryptophan fluorescence, fluorescence of the hydrophobic dye 8-anilino-1-naphthalene-sulphonic acid on binding, and restoration of tryptophan-dansyl fluorescence energy transfer as structure-monitoring probes. We show that the polypeptide chain rapidly collapses (within 4 ms) to a compact globule with a solvent-accessible hydrophobic core, but with no optically active secondary or tertiary structure. Thus the earliest event of the major folding pathway of barstar is a nonspecific hydrophobic collapse that does not involve concomitant secondary structure formation.

The mechanism of folding of the small protein barstar in the pre-transition zone at pH 7, 25 degrees C has been characterized using rapid-mixing techniques. Earlier studies had established the validity of the three-state US <--> UF <--> N mechanism for folding and unfolding in the presence of guanidine hydrochloride (GdnHCl) at concentrations greater than 2.0 M, where US and UF are the slow-refolding and fast-refolding unfolded forms, respectively, and N is the fully folded form. It is now shown that early intermediates, IS1 and IS2 as well as a late native-like intermediate, IN, are present on the folding pathways of US, and an early intermediate IF1 on the folding pathway of UF, when barstar is refolded in concentrations of GdnHCl below 2.0 M. The rates of formation and disappearance of IN, and the rates of formation of N at three different concentrations of GdnHCl in the pre-transition zone have been measured. The data indicate that in 1.5 M GdnHCl, IN is not fully populated on the US-->IS1-->IN-->N pathway because the rate of its formation is so slow that the US <--> UF <--> N pathway can effectively compete with that pathway. In 1.0 M GdnHCl, the US-->IS1-->IN transition is so fast that IN is fully populated. In 0.6 M GdnHCl, IN appears not to be fully populated because an alternative folding pathway, US-->IS2-->N, becomes available for the folding of US, in addition to the US-->IS1-->IN-->N pathway. Measurement of the binding of the hydrophobic dye 1-anilino-8-naphthalenesulphonate (ANS) during folding indicates that ANS binds to two distinct intermediates, IM1 and IM2, that form within 2 ms on the US-->IM1-->IS1-->IN-->N and US-->IM2-->IS2-->N pathways. There is no evidence for the accumulation of intermediates that can bind ANS on the folding pathway of UF.

The fluorescence-monitored kinetics of folding and unfolding of barstar by guanidine hydrochloride (GdnHCl) in the folding transition zone, at pH 7, 25 degrees C, have been quantitatively analyzed using a 3-state mechanism: U(S)<-->UF<-->N. U(S) and UF are slow-refolding and fast-refolding unfolded forms of barstar, and N is the native protein. U(S) and UF probably differ in possessing trans and cis conformations, respectively, of the Tyr 47-Pro 48 bond. The 3-state model could be used because the kinetics of folding and unfolding of barstar show 2 phases, a fast phase and a slow phase, and because the relative amplitudes of the 2 phases depend only on the final refolding conditions and not on the initial conditions. Analysis of the observed kinetics according to the 3-state model yields the values of the 4 microscopic rate constants that describe the transitions between the 3 states at different concentrations of GdnHCl. The value of the equilibrium unfolded ratio U(S):UF (K21) and the values of the rate constants of the U(S)-->UF and UF-->U(S) reactions, k12 and k21, respectively, are shown to be independent of the concentration of GdnHCl. K21 has a value of 2.1 +/- 0.1, and k12 and k21 have values of 5.3 x 10(-3) s-1 and 11.2 x 10(-3) s-1, respectively. Double-jump experiments that monitor reactions that are silent to fluorescence monitoring were used to confirm the values of K21, k12, and k21 obtained from the 3-state analysis and thereby the validity of the 3-state model.(ABSTRACT TRUNCATED AT 250 WORDS)