We report here on the genome sequence of Pasteurella multocida Razi 0002 of avian origin, isolated in Iran. The genome has a size of 2,289,036 bp, a GC content of 40.3%, and is predicted to contain 2,079 coding sequences.

Single-molecule fluorescence spectroscopy addresses biological mechanisms and enables ultrasensitive diagnostics. We describe a new family of single-molecule fluorescence methods that uses alternating-laser excitation (ALEX) of diffusing or immobilized biomolecules to study their structure, interactions, and dynamics. This is accomplished using ratios that report on the distance between and the stoichiometry of fluorophores attached to the molecules of interest. The principle of alternation is compatible with several time scales, allowing monitoring of fast dynamics or simultaneous monitoring of a large number of individual molecules.

We study protein and nucleic acid structure and dynamics using single-molecule FRET and alternating-laser excitation. Freely diffusing molecules are sorted into subpopulations based on singlemolecule signals detected within 100 s to 1 ms. Distance distributions caused by fluctuations faster than 100 s are studied within these subpopulations by using time-correlated singlephoton counting. Measured distance distributions for dsDNA can be accounted for by considering fluorophore linkers and fluorophore rotational diffusion, except that we find smaller fluctuations for internally labeled dsDNA than DNA with one of the fluorophores positioned at a terminal site. We find that the electrostatic portion of the persistence length of short single-stranded poly(dT) varies approximately as the ionic strength (I) to the ؊1͞2 power (I ؊1/2 ), and that the average contribution to the contour length per base is 0.40 -0.45 nm. We study unfolded chymotrypsin inhibitor 2 (CI2) and unfolded acyl-CoA binding protein (ACBP) even under conditions where folded and unfolded subpopulations coexist (contributions from folded proteins are excluded by using alternating-laser excitation). At lower denaturant concentrations, unfolded CI2 and ACBP are more compact and display larger fluctuations than at higher denaturant concentrations where only unfolded proteins are present. The experimentally measured fluctuations are larger than the fluctuations predicted from a Gaussian chain model or a wormlike chain model. We propose that the larger fluctuations may indicate transient residual structure in the unfolded state.conformational dynamics ͉ protein folding ͉ single-molecule fluorescence spectroscopy ͉ nucleic acid structure ͉ fluorescence resonance energy transfer I n contrast to neutral homopolymers (1), biopolymers such as proteins and nucleic acids have long-range Coulomb interactions (2) and specific, intrachain interactions that strongly affect their structure and dynamics. Protein folding is the most spectacular manifestation of these interactions (3). Understanding biopolymer energy landscapes requires measurements of fluctuating distance distributions occurring over short distances (0.1-100 nm) and many time scales (picoseconds to minutes) (4). The structure and dynamics of charged polymer chains (polyelectrolytes), including dsDNA and ssDNA, are strongly affected by long-range, electrostatic repulsion (2). Because simulations and theory have focused on dilute, single-chain properties, a regime difficult to access by conventional methods, experimental validation of many predictions has been lacking (5). In protein folding studies, folded, unfolded, and partially folded species may be simultaneously present and rapidly interconverting, obscuring the properties of individual species. An experimental method that unravels distance distributions and fast conformational fluctuations is therefore much-needed.Previous protein folding studies with single-molecule FRET (6-8) measured distance distributions with time resolutions of Ϸ100 s. Using ...

We have developed a microfluidic mixer for studying protein folding and other reactions with a mixing time of 8 μs and sample consumption of femtomoles. This device enables us to access conformational changes under conditions far from equilibrium and at previously inaccessible time scales. In this paper, we discuss the design and optimization of the mixer using modeling of convective diffusion phenomena and a characterization of the mixer performance using microparticle image velocimetry, dye quenching, and Förster resonance energy-transfer (FRET) measurements of single-stranded DNA. We also demonstrate the feasibility of measuring fast protein folding kinetics using FRET with acyl-CoA binding protein.In protein folding, important structural events occur on a microsecond time scale. 1 To study their kinetics, folding reactions must be initiated at even shorter time scales. Photochemical initiation 2 and changes in temperature, 3 pressure, 4,5 or chemical potential, as in salt or chemical denaturant concentration changes, 6-9 all provide the perturbation of protein conformational equilibria necessary to initiate folding. Laser temperature-jump relaxation provides the best temporal resolution with dead times in the nanosecond range, 3 but only a small number of proteins denature reversibly at elevated temperature. Temperature-jump techniques are also limited to conditions near the equilibrium unfolding transition where marginally stable folding intermediates are less likely to accumulate. Pressure relaxation initiates the folding process by exploiting a change in the volume between the folded and unfolded conformers. Pressure changes up to 20 MPa in under 100 μs have been created with piezoelectric devices to monitor rate-limiting barrier crossing in the cold shock protein with Förster resonance energy transfer (FRET). 10 However, in pressure-jump experiments, the folding equilibrium is shifted only marginally at low denaturant concentrations where collapse occurs, and chain collapse is therefore not easily measured via FRET as refolding amplitudes are small. 10-12In comparison to temperature-and pressure-jump relaxation techniques, folding experiments based on changes in chemical potential, via rapid mixing of protein solutions in to and out of chaotrope solvents, are more versatile. The technique is applicable to a wide range of proteins as most unfold reversibly in the presence of chemical denaturants such as urea 7 and guanidine hydrochloride (GdCl). 6 Further, mixer-based experiments are not limited to proteins near the folding transition state. Until recently, the main limitation of mixer-based experiments was