Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins

Bismuto, Ettore; Irace, Gaetano; Colonna, Giovanni; Jameson, David M.; Gratton, Enrico

doi:10.1016/0167-4838(87)90324-4

Cited by 13 publications

(13 citation statements)

References 26 publications

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“…Fluorescent molecules, such as ANS, TNS, and DANCA, have been used in a number of protein systems to investigate dynamic characteristics of the protein matrix [43,52–54]. In the excited state, the dipole moments of such fluorophores associated with proteins can cause rearrangements of charges and dipoles in the protein matrix, e.g., due to charged or polar amino acid side chains or even the dipoles associated with the peptide bonds.…”

Section: Resultsmentioning

confidence: 99%

“…If the dynamics of the protein allows for dipolar relaxation of the protein matrix then distinct alterations in the fluorophore’s emission properties can result, analogous to the case of dipolar relaxation of solvent molecules described above. Bismuto et al, [43] compared the emission properties of TNS associated with both sperm whale and tuna apomyoglobin to ascertain if this probe revealed differences in the heme binding sites of these phylogenetically related proteins. In the original publication, it was documented that the phase and modulation lifetimes for TNS/tuna myoglobin decreased with increasing frequencies, indicative of heterogeneous emitting population, while for TNS/sperm whale myoglobin the phase lifetime became longer than the modulation lifetime with increasing frequency (going negative at the highest frequency).…”

Section: Resultsmentioning

confidence: 99%

“…This section involves the reanalysis of data previously reported [43]. Briefly, lifetime data were obtained on a multifrequency phase and modulation fluorometer [44] with modulation frequencies between 10–150 MHz.…”

Section: Methodsmentioning

confidence: 99%

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Applications of phasors to in vitro time-resolved fluorescence measurements

Štefl

James

Ross

et al. 2011

Analytical Biochemistry

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The phasor method of treating fluorescence lifetime data provides a facile and convenient approach to characterize lifetime heterogeneity and to detect the presence of excited state reactions, such as solvent relaxation and Förster Resonance Energy Transfer. The method utilizes a plot of M sin(Φ) versus M cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. A principle advantage of the phasor method is that it provides a model-less approach to time-resolved data, amenable to visual inspection. Although the phasor approach has been recently applied to Fluorescence Lifetime Imaging Microscopy it has not been extensively utilized for cuvette studies. In the present study we explore the applications of the method to in vitro samples. The phasors of binary and ternary mixtures of fluorescent dyes demonstrates the utility of the method for investigating complex mixtures. Data from excited state reactions, such as dipolar relaxation in membrane and protein systems and also energy transfer from the tryptophan residue to the chromophore in EGFP, are also presented.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Applications of phasors to in vitro time-resolved fluorescence measurements

Štefl

James

Ross

et al. 2011

Analytical Biochemistry

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“…The distributions are very broad, the widths being 4.1 and 10.0 ns for ANS bound to the native and acidic, high-salt proteins, respectively. The distribution centers are also different (18.9 and 11.9 ns for neutral and acidic apomyoglobin, respectively), probably owing to differences in the accessibility of the ANS fluorophore to solvent in the two apomyoglobin structural forms and to differences in dipolar relaxation rates (Bismuto et al, 1987(Bismuto et al, , 1993. Figures 3 and 4 show the frequency dependence of phase and modulation at increasing pressure for neutral and acidic, highsalt ANS-apomyoglobin complexes respectively.…”

Section: 00mentioning

confidence: 99%

Pressure‐induced perturbation of ANS‐apomyoglobin complex: Frequency domain fluorescence studies on native and acidic compact states

Bismuto¹,

Irace²,

Sirangelo³

et al. 1996

Protein Science

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The pressure dependence of the flexibility of the 8-anilino-1-naphthalene sulfonate (ANS)-apomyoglobin complex was investigated in the range between atmospheric pressure and 2.4 kbar by frequency domain fluorometry. We examined two structural states: native and acidic compact. The conformational dynamics of the ANSapomyoglobin complex were deduced by studying the emission decay of ANS, which can form a noncovalent complex with the apoprotein in both the native and the acidic compact forms. Because the free fluorophore has a very short lifetime (less than 75 ps), its contribution can be separated from the long-lived emission. The latter arises from ANS molecules bound to the protein and provides information on the structural and dynamic characteristics of the macromolecule. The fluorescence emission decay of the ANS-apomyoglobin complex at neutral pH has a broad fluorescence lifetime distribution (width at half-maximum = 4.1 ns). The small changes in the fluorescence distribution parameters that occur with changes in pressure indicate that the ANS-apomyoglobin complex at neutral pH holds its compactness even at 2.4 kbar. A small contraction of molecular volume has been detected at low pressure, followed by a slight swelling with an increase in flexibility at higher pressures. The heterogeneity of ANS fluorescence in the acidic compact state of apomyoglobin is even greater than that in the native form (distribution width = 10 ns); moreover, the acidic compact state appears more expanded and accessible to solvent molecules than the native state, as suggested by the distribution center, which is 11 ns for the former and 19 ns for the latter. The lifetime distribution center remains constant with increasing pressure, which suggests that no other binding site is formed at high pressure.

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“…It is important to emphasize that the analysis presented here is based on a simple model and only yields an apparent spectral relaxation time. For many systems undergoing spectral relaxation the correlation functions are typically non-exponential and characterized by the sum of two or more exponentially-decaying processes2829. The interpretation of faster or slower dynamics could therefore also be interpreted as changes in the proportion of faster processes relative to slower processes.…”

Section: Discussionmentioning

confidence: 99%

Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells

Lajevardipour

Chon

Chattopadhyay

et al. 2016

Sci Rep

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Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

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Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins

Cited by 13 publications

References 26 publications

Applications of phasors to in vitro time-resolved fluorescence measurements

Applications of phasors to in vitro time-resolved fluorescence measurements

Pressure‐induced perturbation of ANS‐apomyoglobin complex: Frequency domain fluorescence studies on native and acidic compact states

Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells

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