A method for determination of transient (on the millisecond timescale) intramolecular distance distributions (IDDs) by time-resolved dynamic non-radiative excitation energy transfer measurements was developed. The time-course of the development of the IDD between residues 73 and 203 in the CORE domain of Escherichia coli adenylate kinase throughout refolding from the GuHCl-induced denatured state was determined. The mean of the apparent IDD reduced to a value close to its magnitude in the native protein, within 2 ms (the dead-time of the instrument). At that time the width of that distribution was rather large (16+/-2 A). The large width implies that the intramolecular diffusion coefficient of the labeled segment does not exceed 10(-7) cm(2)/second. In a second slower phase of the refolding transition, the width was reduced to its native value (6+/-4 A).
"trFRET experiments are ideal for detecting the formation of each closed long loop since it is possible to follow selected distances between two sites that are separated by large number of residues, their distributions, and fast fluctuations (Beechem and Haas 1989; Haas 2005). This led to our efforts towards the development of the trFRET-based " double kinetics " method for the detection of transient intramolecular distance distribution in the rapid collapsed state of globular proteins and during the full folding transition (Ratner et al. 2000; Jacob et al. 2005; Ben Ishay et al. 2012a). The NLI-based model for initiation of folding raises several questions that must be addressed. "
[Show abstract][Hide abstract] ABSTRACT: The extremely fast and efficient folding transition (in seconds) of globular proteins led to the search for some unifying principles embedded in the physics of the folding polypeptides. Most of the proposed mechanisms highlight the role of local interactions that stabilize secondary structure elements or a folding nucleus as the starting point of the folding pathways, i.e., a “bottom–up” mechanism. Non-local interactions were assumed either to stabilize the nucleus or lead to the later steps of coalescence of the secondary structure elements. An alternative mechanism was proposed, an “up–down” mechanism in which it was assumed that folding starts with the formation of very few non-local interactions which form closed long loops at the initiation of folding. The possible biological advantage of this mechanism, the “loop hypothesis”, is that the hydrophobic collapse is associated with ordered compactization which reduces the chance for degradation and misfolding. In the present review the experiments, simulations and theoretical consideration that either directly or indirectly support this mechanism are summarized. It is argued that experiments monitoring the time-dependent development of the formation of specifically targeted early-formed sub-domain structural elements, either long loops or secondary structure elements, are necessary. This can be achieved by the time-resolved FRET-based “double kinetics” method in combination with mutational studies. Yet, attempts to improve the time resolution of the folding initiation should be extended down to the sub-microsecond time regime in order to design experiments that would resolve the classes of proteins which first fold by local or non-local interactions.
[Show abstract][Hide abstract] ABSTRACT: Single-molecule spectroscopy has developed into a powerful tool for the study of biological systems. The ability to observe single protein changes has revealed a great deal of information about the heterogeneity of these systems. In this dissertation, single-molecule techniques have been used to investigate the effect of Ca2+ on millisecond and microsecond fluctuations of the protein calmodulin (CaM). The first part of this dissertation discusses the development and testing of a home-built, two-channel, confocal microscope system used for fluorescence correlation spectroscopy (FCS) and scanning single-molecule measurements. Secondly, the newly built system was tested by performing two-channel FCS measurements using a FRET-pair labeled synthetic polyproline peptide. Polyproline has been shown to approximate a "rigid-rod" and therefore was not expected to show any FRET fluctuations on the FCS timescale. The results from the polyproline correlations led to an investigation to develop expressions to describe the differences in the initial amplitudes of the correlations. These expressions were dependent on the presence of multiple FRET states in the solution and fits were demonstrated using both simulations and real data. Next, the dynamics of several FRET-pair labeled mutants of CaM were measured using FCS techniques. The resulting correlations were globally fit to reveal inter-lobe dynamics on the 100 microsecond timescale that were diminished upon the removal of Ca2+. Intra-lobe dynamics of the N-terminus were also investigated demonstrating an increase in the dynamics in the apo state when compared to the Ca2+ bound state. Finally, CaM was immobilized in unilamellar vesicles to probe millisecond dynamics of the CaM 34-110 mutant in the presence and absence of Ca2+. Rates of interchange between conformational substates of CaM were measured demonstrating an increase in the rates of interchange between conformations in the presence of Ca2+. This supports the view that when bound to Ca2+, CaM is in a more dynamic state leading to its ability to bind a wide variety of targets.
[Show abstract][Hide abstract] ABSTRACT: The maximum likelihood method (MLM) provides high resolution direction-of-arrival (DOA) performance for an array of sensor elements. Its performance, however, is significantly degraded under conditions of individual element phase errors. Such phase perturbations can be caused by channel phase errors, array element placement errors, and frequency errors. By introducing robust constraints into the MLM structure, a phase-robust DOA algorithm is developed. The algorithm protects output signal-to-noise performance of signals impinging on the array against phase errors. Simulations of the algorithm are presented in the presence of random element placement errors
Signals, Systems and Computers, 1993. 1993 Conference Record of The Twenty-Seventh Asilomar Conference on; 12/1993
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