Dynamics of ANS Binding to Tuna Apomyoglobin Measured with Fluorescence Correlation Spectroscopy

AbstractThe dynamics of the binding reaction of ANS to native and partly folded (molten globule) tuna and horse apomyoglobins has been investigated by fluorescence correlation spectroscopy and frequency domain fluorometry. The reaction rate has been measured as a function of apomyoglobin and ANS concentrations, pH, and temperature. Examination of the autocorrelation functions shows that the reaction rate is fast enough to be observed in tuna apomyoglobin, whereas the reaction rate in horse apomyoglobin is on the same time scale as diffusion through the volume or longer. Specifically, for tuna apomyoglobin at pH 7 and room temperature the on rate is 2200μM−1 s−1 and the off rate is 5900s−1, in comparison with kon=640μM−1 s−1 and koff=560s−1 for horse myoglobin as measured previously. The independence of the reaction rate from the ANS concentration indicates that the reaction rate is dominated by the off rate. The temperature dependence of the on-rate shows that this rate is diffusion limited. The temperature dependence of the off rates analyzed by Arrhenius and Ferry models indicates that the off rate depends on the dynamics of the protein. The differences between horse and tuna apomyoglobins in the ANS binding rate can be explained in terms of the three-dimensional apoprotein structures obtained by energy minimization after heme removal starting from crystallographic coordinates. The comparison of the calculated apomyoglobin surfaces shows a 15% smaller cavity for tuna apomyoglobin. Furthermore, a negative charge (D44) is present in the heme cavity of tuna apomyoglobin that could decrease the strength of ANS binding. At pH 5 the fluorescence lifetime distribution of ANS-apomyoglobin is bimodal, suggesting the presence of an additional binding site in the protein. The binding rates determined by FCS under these conditions show that the protein is either in the open configuration or is more flexible, making it much easier to bind. At pH 3, the protein is in a partially denatured state with multiple potential binding sites for ANS molecule, and the interpretation of the autocorrelation function is not possible by simple models. This conclusion is consistent with the broad distribution of ANS fluorescence lifetimes observed in frequency domain measurements

Dynamics of HIV-1 Gag Processing as Revealed by Fluorescence Lifetime Imaging Microscopy and Single Virus Tracking

The viral polyprotein Gag plays a central role for HIV-1 assembly, release and maturation. Proteolytic processing of Gag by the viral protease is essential for the structural rearrangements that mark the transition from immature to mature, infectious viruses. The timing and kinetics of Gag processing are not fully understood. Here, fluorescence lifetime imaging microscopy and single virus tracking are used to follow Gag processing in nascent HIV-1 particles in situ. Using a Gag polyprotein labelled internally with eCFP, we show that proteolytic release of the fluorophore from Gag is accompanied by an increase in its fluorescence lifetime. By tracking nascent virus particles in situ and analyzing the intensity and fluorescence lifetime of individual traces, we detect proteolytic cleavage of eCFP from Gag in a subset (6.5%) of viral particles. This suggests that for the majority of VLPs, Gag processing occurs with a delay after particle assembly

Range or Meadow Regrowth Grazing and Weaning Effects on Two Year-Old Cows

Body condition of cows at calving affects pregnancy rate and breeding date. Body condition at calving of spring calving cows wintered on range is influenced by fall body condition. A Montana study showed that lactating cows grazing range lost body condition during August and September. The loss of body condition was attributed to an inadequate consumption of crude protein. Diet samples of cattle grazing Sandhills range during August to October contain 6% to 8% crude protein. Loss of body condition of spring calving, primiparous cows grazing Nebraska Sandhills range during the fall is a concern. Subirrigated meadow regrowth is a higher quality forage than upland range in the fall. Diet samples collected from cattle grazing regrowth from subirrigated meadow during October contained approximately 11% crude protein. Two potential ways of maintaining or increasing cow body condition during the fall is to wean the calf, thus reducing the cow\u27s nutrient requirements, or increase the potential to meet crude protein requirements with higher quality forage. Our objectives were to determine if September weaning or grazing subirrigated meadows would improve body condition score of spring calving primiparous beef cows during September and October. and to determine nutrient intakes by dry and lactating cows grazing native range or subirrigated meadow regrowth

A multiscale wavelet algorithm for atom tracking in STM movies

High-speed scanning tunneling microscopy (STM) data have become available that provide movies of time-dependent surface processes. To track adsorbed atoms and molecules in such data automatic routines are required. We introduce a multiresolution wavelet particle detection algorithm for this purpose. To identify the particles, the images are decomposed by means of a discrete wavelet transform into wavelet planes of different resolutions. An 'à trous' low-pass filter is applied. The coefficients from the wavelet planes are filtered to remove noise. Wavelet planes with significant coefficients from the particles are multiplied, and the product is transformed into a binary particle mask. The precision of the method is tested with data sets of adsorbed CO molecules and O atoms on a Ru(0001) surface. The algorithm can safely detect and localize these particles with high precision, even in the presence of the enhanced noise characteristic for high-speed, constant-height STM data. By linking the particle positions, we obtain extended trajectories with a resolution of ∼0.5 Å or better allowing us to investigate the detailed motion of single atoms on a surface

Nanoresolution real-time 3D orbital tracking for studying mitochondrial trafficking in vertebrate axons in vivo

We present the development and in vivo application of a feedback-based tracking microscope to follow individual mitochondria in sensory neurons of zebrafish larvae with nanometer precision and millisecond temporal resolution. By combining various technical improvements, we tracked individual mitochondria with unprecedented spatiotemporal resolution over distances of >100 mu m. Using these nanoscopic trajectory data, we discriminated five motional states: a fast and a slow directional motion state in both the anterograde and retrograde directions and a stationary state. The transition pattern revealed that, after a pause, mitochondria predominantly persist in the original direction of travel, while transient changes of direction often exhibited longer pauses. Moreover, mitochondria in the vicinity of a second, stationary mitochondria displayed an increased probability to pause. The capability of following and optically manipulating a single organelle with high spatiotemporal resolution in a living organism offers a new approach to elucidating their function in its complete physiological context

Multicolor 3D Orbital Tracking

Feedback-based single-particle tracking (SPT) is a powerful technique for investigating particle behavior with very high spatiotemporal resolution. The ability to follow different species and their interactions independently adds a new dimension to the information available from SPT. However, only a few approaches have been expanded to multiple colors and no method is currently available that can follow two differently labeled biomolecules in 4 dimensions independently. In this proof-of-concept paper, the new modalities available when performing 3D orbital tracking with a second detection channel are demonstrated. First, dual-color tracking experiments are described studying independently diffusing particles of different types. For interacting particles where their motion is correlated, a second modality is implemented where a particle is tracked in one channel and the position of the second fluorescence species is monitored in the other channel. As a third modality, 3D orbital tracking is performed in one channel while monitoring its spectral signature in a second channel. This last modality is used to successfully readout accurate Forster Resonance Energy Transfer (FRET) values over time while tracking a mobile particle

Seasonal Changes in Protein Degradabilities of Sandhills Native Range and Subirrigated Meadow Diets and Application of a Metabolizable Protein System

Meadow and range diets increased in digestibility, crude protein. and escape protein during periods of active growth