scholarly journals Suppressor Mutation Analysis of the Sensory Rhodopsin I-Transducer Complex: Insights into the Color-Sensing Mechanism

1998 ◽  
Vol 180 (8) ◽  
pp. 2033-2042 ◽  
Author(s):  
Kwang-Hwan Jung ◽  
John L. Spudich
Keyword(s):  
Conformational Transition ◽  
Uv Light ◽  
Transmembrane Helix ◽  
Selection Procedure ◽  
Halobacterium Salinarum ◽  
Sensory Rhodopsin ◽  
Two Photon ◽  
Suppressor Mutations ◽  
Photon Excitation ◽  
Sensory Rhodopsin I

ABSTRACT The molecular complex containing the phototaxis receptor sensory rhodopsin I (SRI) and transducer protein HtrI (halobacterial transducer for SRI) mediates color-sensitive phototaxis responses in the archaeonHalobacterium salinarum. One-photon excitation of the complex by orange light elicits attractant responses, while two-photon excitation (orange followed by near-UV light) elicits repellent responses in swimming cells. Several mutations in SRI and HtrI cause an unusual mutant phenotype, called orange-light-inverted signaling, in which the cell produces a repellent response to normally attractant light. We applied a selection procedure for intragenic and extragenic suppressors of orange-light-inverted mutants and identified 15 distinct second-site mutations that restore the attractant response. Two of the 3 suppressor mutations in SRI are positioned at the cytoplasmic ends of helices F and G, and 12 suppressor mutations in HtrI cluster at the cytoplasmic end of the second HtrI transmembrane helix (TM2). Nearly all suppressors invert the normally repellent response to two-photon stimulation to an attractant response when they are expressed with their suppressible mutant alleles or in an otherwise wild-type strain. The results lead to a model for control of flagellar reversal by the SRI-HtrI complex. The model invokes an equilibrium between the A (reversal-inhibiting) and R (reversal-stimulating) conformers of the signaling complex. Attractant light and repellent light shift the equilibrium toward the A and R conformers, respectively, and mutations are proposed to cause intrinsic shifts in the equilibrium in the dark form of the complex. Differences in the strength of the two-photon signal inversion and in the allele specificity of suppression are correlated, and this correlation can be explained in terms of different values of the equilibrium constant (K eq) for the conformational transition in different mutants and mutant-suppressor pairs.

Application of two-photon chromophore excitation to laser scanning microscopy

1991 ◽  
Vol 49 ◽  
pp. 404-405
Author(s):  
David W. Piston ◽  
James H. Strickler ◽  
Watt W. Webb
Keyword(s):  
Laser Scanning ◽  
Uv Light ◽  
Excited Electronic State ◽  
Laser Scanning Confocal Microscopy ◽  
Photochemical Reactions ◽  
Laser Scanning Microscopy ◽  
Scanning Confocal Microscopy ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

The non-linear optical technique of two-photon excitation of fluorescence and photochemical reactions makes possible new applications that are not possible using linear one-photon excitation in laser scanning confocal microscopy. The two-photon excitation effect arises from the simultaneous absorption of two red photons, which causes the transition to an excited electronic state with its normal absorption in the ultraviolet. In our fluorescence experiments, this excited state is the same singlet state, S1, that is populated during a conventional fluorescence experiment, and thus exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) that are observed in typical biological microscopy studies. Likewise, photochemical reactions such as light induced polymerization and photolytic uncaging that are normally catalyzed by UV light can be generated using two-photon excitation. In practice, twophoton excitation is made possible by the very high local instantaneous intensity that is provided by a combination of the diffraction limited focusing in the microscope and the temporal concentration of a subpicosecond mode-locked laser.

scholarly journals Time-Resolved Absorption and Photothermal Measurements with Sensory Rhodopsin I from Halobacterium salinarum

1999 ◽  
Vol 76 (4) ◽  
pp. 2183-2191 ◽  
Author(s):  
Aba Losi ◽  
Silvia E. Braslavsky ◽  
Wolfgang Gärtner ◽  
John L. Spudich
Keyword(s):  
Halobacterium Salinarum ◽  
Sensory Rhodopsin ◽  
Time Resolved ◽  
Sensory Rhodopsin I

scholarly journals Collimated UV light generation by two-photon excitation to a Rydberg state in Rb vapor

2019 ◽  
Vol 44 (11) ◽  
pp. 2931 ◽  
Author(s):  
Mark Lam ◽  
Sambit B. Pal ◽  
Thibault Vogt ◽  
Christian Gross ◽  
Martin Kiffner ◽  
...  
Keyword(s):  
Rydberg State ◽  
Uv Light ◽  
Two Photon ◽  
Photon Excitation ◽  
Rb Vapor ◽  
Light Generation ◽  
Two Photon Excitation

New Photointermediates in the Two Photon Signaling Pathway of Sensory Rhodopsin-I†

Biochemistry ◽  
2000 ◽  
Vol 39 (49) ◽  
pp. 15101-15109 ◽  
Author(s):  
Trevor E. Swartz ◽  
Istvan Szundi ◽  
John L. Spudich ◽  
Roberto A. Bogomolni
Keyword(s):  
Signaling Pathway ◽  
Sensory Rhodopsin ◽  
Two Photon ◽  
Sensory Rhodopsin I

Imaging of Cellular Dynamics by Two-Photon Excitation Microscopy

1995 ◽  
Vol 1 (1) ◽  
pp. 25-34 ◽  
Author(s):  
David W. Piston ◽  
Brian D. Bennett ◽  
Guangtao Ying
Keyword(s):  
Uv Light ◽  
Excited Electronic State ◽  
Red Light ◽  
Three Dimensional ◽  
Focal Volume ◽  
Optical Sectioning ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Two Photon Excitation

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons (∼700 nm) can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet (∼350 nm). In the fluorescence experiments described here, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g., wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. Three properties of two-photon excitation give this method its advantage over conventional optical sectioning microscopies: (1) the excitation is limited to the focal volume, thus providing inherent three-dimensional resolution and minimizing photobleaching and photodamage; (2) the two-photon technique allows imaging of UV fluorophores with only conventional visible light optics; (3) red light is far less damaging to most living cells and tissues than UV light and permits deeper sectioning, because both absorbance and scattering are reduced. Many cell biological applications of two-photon excitation microscopy have been successfully realized, demonstrating the wide ranging power of this technique.

Two-photon excitation microscopy in cellular biophysics

1995 ◽  
Vol 53 ◽  
pp. 62-63
Author(s):  
David W. Piston ◽  
Brian D. Bennett ◽  
Robert G. Summers
Keyword(s):  
Confocal Microscopy ◽  
Laser Beam ◽  
Duty Cycle ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10-5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

Two-photon excitation fluorescence microscopy in living systems

1993 ◽  
Vol 51 ◽  
pp. 154-155 ◽  
Author(s):  
David W. Piston
Keyword(s):  
Confocal Microscopy ◽  
Fluorescence Microscopy ◽  
Singlet State ◽  
Excited Electronic State ◽  
Input Power ◽  
Two Photon ◽  
Simultaneous Absorption ◽  
Photon Excitation ◽  
Very High ◽  
Two Photon Excitation

Two-photon excitation fluorescence microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In our fluorescence experiments, the final excited state is the same singlet state that is populated during a conventional fluorescence experiment. Thus, the fluorophore exhibits the same emission properties (e.g. wavelength shifts, environmental sensitivity) used in typical biological microscopy studies. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10−5 maintains the average input power on the order of 10 mW, only slightly greater than the power normally used in confocal microscopy.

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