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1.
Biophys J ; 90(6): 2179-91, 2006 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-16387771

RESUMO

Fitting of photon-count number histograms is a way of analysis of fluorescence intensity fluctuations, a successor to fluorescence correlation spectroscopy. First versions of the theory for calculating photon-count number distributions have assumed constant emission intensity by a molecule during a counting time interval. For a long time a question has remained unanswered: to what extent is this assumption violated in experiments? Here we present a theory of photon-count number distributions that takes account of intensity fluctuations during a counting time interval. Theoretical count-number distributions are calculated via a numerical solution of Master equations (ME), which is a set of differential equations describing diffusion, singlet-triplet transitions, and photon emission. Detector afterpulsing and dead-time corrections are also included. The ME-theory is tested by fitting a series of photon-count number histograms corresponding to different lengths of the counting time interval. Compared to the first version of fluorescence intensity multiple distribution analysis theory introduced in 2000, the fit quality is significantly improved. It is discussed how a theory of photon-count number distributions, which assumes constant emission intensity during a counting time interval, may also yield a good fit quality. We argue that the spatial brightness distribution used in calculations of the fit curve is not the true spatial brightness distribution. Instead, a number of dynamic processes, which cause fluorescence intensity fluctuations, are indirectly taken into account via the profile adjustment parameters.


Assuntos
Algoritmos , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Modelos Químicos , Fótons , Radiometria/métodos , Espectrometria de Fluorescência/métodos , Simulação por Computador , Doses de Radiação
2.
Biophys J ; 83(5): 2812-25, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12414713

RESUMO

Tyrosine ring dynamics of the gastrointestinal hormone motilin was studied using two independent physical methods: fluorescence polarization anisotropy decay and NMR relaxation. Motilin, a 22-residue peptide, was selectively (13)C labeled in the ring epsilon-carbons of the single tyrosine residue. To eliminate effects of differences in peptide concentration, the same motilin sample was used in both experiments. NMR relaxation rates of the tyrosine ring C(epsilon)-H(epsilon) vectors, measured at four magnetic field strengths (9.4, 11.7, 14.1, and 18.8 Tesla) were used to map the spectral density function. When the data were analyzed using dynamic models with the same number of components, the dynamic parameters from NMR and fluorescence are in excellent agreement. However, the estimated rotational correlation times depend on the choice of dynamic model. The correlation times estimated from the two-component model-free approach and the three-component models were significantly different (1.7 ns and 2.2 ns, respectively). Various earlier studies of protein dynamics by NMR and fluorescence were compared. The rotational correlation times estimated by NMR for samples with high protein concentration were on average 18% longer for folded monomeric proteins than the corresponding times estimated by fluorescence polarization anisotropy decay, after correction for differences in viscosity due to temperature and D(2)O/H(2)O ratio.


Assuntos
Anisotropia , Motilina/química , Tirosina/química , Animais , Fenômenos Biofísicos , Biofísica , Espectroscopia de Ressonância Magnética/métodos , Modelos Teóricos , Peptídeos/química , Prótons , Espectrometria de Fluorescência/métodos , Temperatura , Fatores de Tempo
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