NUTS and BOLTS: Applications of fluorescence-detected sedimentation

Kroe, Rachel R.; Laue, Thomas M.

doi:10.1016/j.ab.2008.11.033

Cited by 80 publications

(102 citation statements)

References 20 publications

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“…In this context, the measurement of the concentration dependence of the sedimentation coefficient may provide an improved, more realistic model of self-association. It is not yet clear whether this approach would be applicable to systems other than TTR; however, the ϳ2 S decrease in s when determined in serum as opposed to dilute buffer is consistent with observations for other similarly sized proteins (41).…”

Section: Discussionsupporting

confidence: 48%

“…Although there is a slight slope to the plateau and some residual signal above the boundary at lower radial positions, these features were not fit by the Lamm equation, as evidenced by the lack of additional peaks in the c(s) distribution. There is no evidence that the boundary is distorted by the Johnston-Ogston effect (40,41). Despite the qualitative agreement between the sedimentation coefficient determined by Lamm equation modeling and that determined by boundary midpoint migration (Fig.…”

Section: Figure 2 Example Of C(s) Analysis Of F-rttr Sedimentation Vmentioning

confidence: 74%

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The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

Kingsbury

Laue

Klimtchuk

et al. 2008

Journal of Biological Chemistry

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Transthyretin (TTR) is normally a stable plasma protein.However, in cases of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril and the factors that promote this process are largely undetermined. Most models involve the dissociation of the native TTR tetramer as the initial step. It is largely accepted that the TTR gene mutations associated with TTR-related amyloidosis lead to the expression of variant proteins that are intrinsically unstable and prone to aggregation. It has been suggested that amyloidogenicity may be conferred to wild-type TTR (the form deposited in SSA) by chemical modification of the lone cysteine residue (Cys 10 ) through mixed disulfide bonds. S-Sulfonation and S-cysteinylation are prevalent TTR modifications physiologically, and studies have suggested their ability to modulate the structure of TTR under denaturing conditions. In the present study, we have used fluorescencedetected sedimentation velocity to determine the effect of S-sulfonate and S-cysteine on the quaternary structural stability of fluorophore-conjugated recombinant TTR under nondenaturing conditions. We determined that S-sulfonation stabilized TTR tetramer stability by a factor of 7, whereas S-cysteinylation enhanced dissociation by 2-fold with respect to the unmodified form. In addition, we report the direct observation of tetramer stabilization by the potential therapeutic compound diflunisal. Finally, as proof of concept, we report the sedimentation of TTR in serum and the qualitative assessment of the resulting data.

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Section: Discussionsupporting

confidence: 48%

Section: Figure 2 Example Of C(s) Analysis Of F-rttr Sedimentation Vmentioning

confidence: 74%

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

Kingsbury

Laue

Klimtchuk

et al. 2008

Journal of Biological Chemistry

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“…Analytical ultracentrifugation was performed in a Beckman ProteomeLab XL-A (Beckman Instruments) equipped with a fluorescence detector (Aviv Biomedical, Lakewood, NJ) (29). Runs were performed at 42,000 rpm with a Ti-50 Rotor (Beckman Instruments).…”

Section: Methodsmentioning

confidence: 99%

The Charged Linker Region Is an Important Regulator of Hsp90 Function

Hainzl¹,

Lapina

Büchner

et al. 2009

Journal of Biological Chemistry

148

108

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Hsp90 is an ATP-dependent molecular chaperone which assists the maturation of a large set of target proteins. Members of the highly conserved Hsp90 family are found from bacteria to higher eukaryotes, with homologues in different organelles. The core architecture of Hsp90 is defined by the N-terminal ATP binding domain followed by the middle domain and the C-terminal dimerization domain. A long, highly charged linker between the N-terminal domain and the middle domain is a feature characteristic for Hsp90s of eukaryotic organisms. We set out to clarify the function of this linker by studying the effects of deletions in this region in vivo and in vitro. Here we show that increasing deletions in the charged linker region lead to defects ranging from mild temperature sensitivity to a lethal phenotype. The lethal deletion variants investigated in this study still exhibit ATPase activity. Deletion of the charged linker ultimately causes a loss of Hsp90 regulation by co-chaperones, as the sensitivity for Aha1-mediated ATPase acceleration declines, and binding of p23/Sba1 is lost in non-viable deletion constructs. In vivo client assays additionally demonstrated that the deletion of the linker had a pronounced effect on the ability of Hsp90 to facilitate client activation. A partial reconstruction of the linker sequence showed that the supplementation by artificial sequences can rescue the functionality of Hsp90 and restore the conformational flexibility of the protein, required for the processing of client proteins. Hsp903 is an ATP-dependent molecular chaperone present in the cytosol of eubacteria and eucaryotes. It regulates the maturation and activation of numerous proteins involved in signal transduction, cell cycle control, hormone signaling, and transcription (1-3). Recent crystal structures of HtpG from Escherichia coli and Hsp90 from Saccharomyces cerevisiae show that the overall structural organization of the proteins is highly conserved (4 -6). Both prokaryotic and eukaryotic Hsp90 proteins consist of an N-terminal ATP binding domain, a middle domain involved in client protein binding, and a C-terminal dimerization domain. During the ATPase cycle, large conformational changes in the Hsp90 dimer lead to the transient dimerization of the N-terminal domains and their association with the middle domains (7-12). Despite the conservation of the basic molecular architecture and the ATPase mechanism, there are major differences between Hsp90 from prokaryotes and eukaryotes. The most striking difference is the emergence of a large set of co-chaperones in eucaryotes that bind to Hsp90 and seem to modulate and expand its properties (13). Furthermore, in contrast to procaryotes, Hsp90 is an essential protein in eucaryotes (14, 15) and it had been shown in S. cerevisiae that ATP hydrolysis by Hsp90 is required for sustaining its essential function (16 -18). Finally, eukaryotic Hsp90 contains additional structural elements compared with prokaryotic Hsp90; that is, a long charged linker between the N terminus and the middle...

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“…As the rotor spins, each cell passes through the optical paths of detectors capable of measuring the concentration of molecules at closely spaced radial intervals in the cell. There are three commercially available optical detectors for the XLI to measure the concentration distributions: an absorbance spectrophotometer and Rayleigh interferometer from Beckman Coulter mentioned on 10-5, and the fluorescence detector from Aviv Biomedical (Lakewood, NJ) [25]. All subsequent analysis of sedimentation data relies on the quantity and quality of data available from these detectors.…”

Section: Optical Systemsmentioning

confidence: 99%

Applications of Sedimentation Velocity Analytical Ultracentrifugation

Laue

2010

Formulation and Process Development Strategies for Manufacturing Biopharmaceuticals

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NUTS and BOLTS: Applications of fluorescence-detected sedimentation

Cited by 80 publications

References 20 publications

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

The Charged Linker Region Is an Important Regulator of Hsp90 Function

Applications of Sedimentation Velocity Analytical Ultracentrifugation

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