Time-resolved methods in biophysics. 8. Frequency domain fluorometry: applications to intrinsic protein fluorescence

Ross, Justin A.; Jameson, David M.

doi:10.1039/b804450n

Cited by 79 publications

(69 citation statements)

References 75 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…27,28 Several mechanisms were proposed to explain the observed heterogeneity of the Trp lifetime in single Trp proteins including the presence of Trp side chain rotamers, quenching by water molecules, electron transfer to the peptide carbonyl group, excited state electron or proton transfer and intersystem crossing. [29][30][31] Interestingly, an overlay of the 15 lowest energy structures of Ca 21 bound DREAM determined by NMR spectroscopy 22 and the structure of the DREAM C-terminal domain (residues 161-256) 23 displays the presence of a single Trp rotamer (t rotamer) in the DREAM structure. However, as shown in Figure 7, a transition from the t to g1 rotamer of Trp169 sidechain is observed during molecular dynamic simulations, which support the idea that the bimodal distribution could arise from two rotameric orientations of Trp169 with the side chain of the g1 rotamer being more solvent exposed (see below).…”

Section: Discussionmentioning

confidence: 99%

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

et al. 2015

View full text Add to dashboard Cite

Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensors (NCS) that transduce the intracellular changes in Ca 21 concentration into a variety of responses including gene expression, regulation of Kv channel activity, and calcium homeostasis. Despite the significant sequence and structural similarities with other NCS members, DREAM shows several features unique among NCS such as formation of a tetramer in the apo-state, and interactions with various intracellular biomacromolecules including DNA, presenilin, Kv channels, and calmodulin. Here we use spectroscopic techniques in combination with molecular dynamics simulation to study conformational changes induced by Ca 21 /Mg 21 association to DREAM. Our data indicate a minor impact of Ca 21 association on the overall structure of the Nand C-terminal domains, although Ca 21 binding decreases the conformational heterogeneity as evident from the decrease in the fluorescence lifetime distribution in the Ca 21 bound forms of the protein. Time-resolved fluorescence data indicate that Ca 21 binding triggers a conformational transition that is characterized by more efficient quenching of Trp residue. The unfolding of DREAM occurs through an partially unfolded intermediate that is stabilized by Ca 21 association to EF-hand 3 and EF-hand 4. The native state is stabilized with respect to the partially unfolded state only in the presence of both Ca 21 and Mg 21 suggesting that, under physiological conditions, Ca 21 free DREAM exhibits a high conformational flexibility that may facilitate its physiological functions.

show abstract

Section: Discussionmentioning

confidence: 99%

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

et al. 2015

View full text Add to dashboard Cite

show abstract

“…This behaviour of tryptophan is presently thought to be a consequence of the combined effect of the three discrete rotamers of tryptophan [28][29][30] and solvent interaction in the 1 L a fluorescing state. [31] The sharpening of the three peaks on immobilisation suggests the rotamers are restricted perhaps due to structural rigidity, which is more pronounced after transferring into organic media. This behaviour parallels what was recently found for rotamers of the single Trp in human serum albumin when stabilised by binding to quercetin.…”

Section: Intrinsic Fluorescence Spectroscopymentioning

confidence: 99%

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

et al. 2009

View full text Add to dashboard Cite

Structure of immobilised enzymes: Circular dichroism, infrared and fluorescence spectroscopic methods are used to characterise in situ structural stability of enzymes immobilised on particles. The enzyme subtilisin Carlsberg exhibits a similar secondary structure in solution and in the immobilised state but on the surface of porous silica gel a rigid tertiary structure is preferred (see picture). We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm−1 to 1632 cm−1, indicating a shift from α‐helical structure to β‐sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to β‐sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.

show abstract

“…This result is not surprising, because most single Trp containing proteins exhibit complex lifetime decays. 48 As shown in Figure 3 and Table III, the results for the single Trp constructs of both apo and iron-bound hTF N-lobe fit reasonably well to Lorentzian distributions, which reflects not only the intrinsic excited state lifetime of the Trp residue but also the dynamics of that residue as it samples different protein environments in both the ground and excited state. 41 Interestingly, the distribution profile for the apo versus iron containing W128Y and W264 becomes very narrow [ Fig.…”

Section: Lifetime Analysismentioning

confidence: 67%

Excited‐state lifetime studies of the three tryptophan residues in the N‐lobe of human serum transferrin

James¹,

Ross²,

Mason³

et al. 2009

Protein Science

Self Cite

View full text Add to dashboard Cite

The energy transfer from the three Trp residues at positions 8, 128, and 264 within the human serum transferrin (hTF) N-lobe to the ligand to metal charge transfer band has been investigated by monitoring changes in Trp fluorescence emission and lifetimes. The fluorescence emission from hTF N-lobe is dominated by Trp264, as revealed by an 82% decrease in the quantum yield when this Trp residue is absent. Fluorescence lifetimes were determined by multifrequency phase fluorometry of mutants containing one or two Trp residues. Decays of these samples are best described by two or three discrete lifetimes or by a unimodal Lorentzian distribution. The discrete lifetimes and the center of the lifetime distribution for samples containing Trp128 and Trp264 are affected by iron. The distribution width narrows on iron removal and is consistent with a decrease in dynamic mobility of the dominant fluorophore, Trp264. Both the quantum yield and the lifetimes are lower when iron is present, however, not proportionally. The greater effect of iron on quantum yields is indicative of nonexcited state quenching, i.e., static quenching. The results of these experiments provide quantitative data strongly suggesting that Fö rster resonance energy transfer is not the sole source of Trp quenching in the N-lobe of hTF.

show abstract

Time-resolved methods in biophysics. 8. Frequency domain fluorometry: applications to intrinsic protein fluorescence

Cited by 79 publications

References 75 publications

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

Excited‐state lifetime studies of the three tryptophan residues in the N‐lobe of human serum transferrin

Contact Info

Product

Resources

About

Time-resolved methods in biophysics. 8. Frequency domain fluorometry: applications to intrinsic protein fluorescence

Cited by 79 publications

References 75 publications

Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

Excited‐state lifetime studies of the three tryptophan residues in the N‐lobe of human serum transferrin

Contact Info

Product

Resources

About

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Ca²⁺ and Mg²⁺ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator