Application of Fluorescence Spectroscopy for Determining the Structure and Function of Proteins

Jiskoot, Wim; Hlady, Vladimir; Naleway, John J.; Herron, James N.

doi:10.1007/978-1-4899-1079-0_1

Cited by 23 publications

(14 citation statements)

References 157 publications

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“…The significant left shift in Trp fluorescence of NEG-HSA represents a shift in the spectral form from that thought to occur when there is contact of Trp with bulk water (spectral form III) towards that seen when Trp is exposed to water molecules with much reduced mobility (spectral form II) (17). This is consistent with the suggestion that Trp 214 is normally exposed to bulk water by intra-molecular movement and that NEG restricts this movement, also consistent with reduced acrylamide quenching.…”

Section: Discussionmentioning

confidence: 97%

See 1 more Smart Citation

Fluorometric and Mass Spectrometric Analysis of Nonenzymatic Glycosylated Albumin

Zoellner

Hou

Hochgrebe

et al. 2001

Biochemical and Biophysical Research Communications

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Section: Discussionmentioning

confidence: 97%

“…After thorough mixing, Trp fluorescence was determined in emission scans from 300 to 450 nm using an excitation wavelength of 295 nm. This excitation wavelength minimised the contribution of Phe and Tyr to readings (17).…”

Section: Methodsmentioning

confidence: 99%

Fluorometric and Mass Spectrometric Analysis of Nonenzymatic Glycosylated Albumin

Zoellner

Hou

Hochgrebe

et al. 2001

Biochemical and Biophysical Research Communications

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“…For instance, far-UV circular dichroism analysis provides detailed information on the secondary structural elements in a protein and can require as little as 20 μg of protein [64,65]. Intrinsic fluorescence experiments can require even less protein (5-20 μg) and monitor the packing around tryptophan residues and thus provide a probe of tertiary structure in the molecule [66,67].…”

Section: Biophysical Approachesmentioning

confidence: 99%

Preformulation Research: Assessing Protein Solution Behavior during Early Development

Pérez-Ramírez¹,

Guziewicz²,

Simler³

2010

Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals

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“…Folding pathways of proteins are not easy to delineate as intermediate structures may be transient with respect to the measurement timescale. Methods used to determine protein conformation and dynamics include xray crystallography [7], nuclear magnetic resonance (NMR) spectroscopy [8,9], circular dichroism (CD) spectroscopy [10,11], fluorescence spectroscopy [12] as well as ion mobility mass spectrometry (IM-MS) [13][14][15][16][17][18]. Ion mobility mass spectrometry is a gas phase electrophoretic technique which separates a molecule according charge, mass and collision cross section (CCS) as it drifts through a neutral buffer gas [19,20].…”

Section: Introductionmentioning

confidence: 99%

Ion-Ion interactions affect (re)folding dynamics of ubiquitin ions in the gas phase. Distinct folding intermediates formed via charge-reduction pathways

Jhingree¹,

Barran²

2017

Preprint

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Using Ubiquitin as an exemplar protein we examine the effect of net charge reduction post electrospray ionisation by exposure to the electron transfer reagent, 1,3-dicyanobenzene. We monitor the change in gas phase conformation of both precursor and products with ion mobility mass spectrometry (IM-MS). Dramatic conformational rearrangement is seen for low charge state ions upon exposure to the electron transfer reagent. Ions with low charge states sprayed from both native-like and denaturing solvent conditions undergo structural transitions to conformers with cross sections in the range measured for the native structure (TWCCSN2®He, 950-1000 Å2). Thus, we infer that some memory of the solution phase structure is retained in the gas phase. Intermediate structures are seen in the reduction of the [M+6H]6+ ion sprayed from both native-like and denaturing solvents. Further, the reduction pathway of this ion shows compaction to structures with a TWCCSN2®He centred at 1069 Å2 (5+) and 949 Å2 (4+) for ions originating from native-like and denaturing solvents respectively. We propose that charge reduction sites for radical anion localisation (to effect electron transfer) are not easily accessible in the case of ubiquitin molecules originating from native-like solution conditions. This highlights the importance of salt bridge interactions in maintaining the structural integrity of a protein in the gas phase. Most interestingly, two distinct conformer populations are seen for the 6+ charge-reduced product originating from the 7+ and, the 6+ exposed to radical anions (post ESI); we infer that these populations are intermediate in the refolding of ubiquitin in the gas phase, sometimes transient. Overall, we are able to monitor the refolding pathway of ubiquitin in the gas phase as its charge is reduced and show that charge-charge interactions play a significant role in the gas phase conformation adopted; whereby specific conformations are formed.

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Application of Fluorescence Spectroscopy for Determining the Structure and Function of Proteins

Cited by 23 publications

References 157 publications

Fluorometric and Mass Spectrometric Analysis of Nonenzymatic Glycosylated Albumin

Fluorometric and Mass Spectrometric Analysis of Nonenzymatic Glycosylated Albumin

Preformulation Research: Assessing Protein Solution Behavior during Early Development

Ion-Ion interactions affect (re)folding dynamics of ubiquitin ions in the gas phase. Distinct folding intermediates formed via charge-reduction pathways

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