Using modern approaches to sedimentation velocity to detect conformational changes in proteins

Brautigam, Chad A.; Tso, Shih Chia; Deka, Ranjit K.; Liu, Wei Z.; Norgard, Michael V.

doi:10.1007/s00249-020-01453-w

Cited by 7 publications

(4 citation statements)

References 57 publications

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“…The major discrepancy is in the a/b ratio derived from the Perrin (P) function, which severely overestimated the degree of extension in the molecules in reference to the estimate by the Scheraga-Mandelkern β function and hydrodynamic modeling. Hydrodynamic predictions tend to be especially useful when evaluating the plausibility of several different conformational models, rather than describing the exact shape and dimensions of a given molecule in solution (Brautigam et al 2020; Marx et al 2020; Monsen et al 2021; Rocco and Byron 2015b; Yarawsky et al 2022). Despite minor inconsistencies in the hydrodynamic predictions and experimental data, it is very clear that there is increasing extension from BSA to Brpt1.5 and Brpt5.5.…”

Section: Resultsmentioning

confidence: 99%

Strong non-ideality effects at low protein concentrations: considerations for elongated proteins

Yarawsky

Dinu

Harding

et al. 2022

Preprint

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A recent investigation was aimed at obtaining structural information on a highly extended protein via SEC-MALS-SAXS. Significantly broadened elution peaks were observed, reminiscent of a phenomenon known as viscous fingering. This phenomenon is usually observed above 50 mg/mL for proteins like bovine serum albumin (BSA). Interestingly, the highly extended protein (Brpt5.5) showed viscous fingering at concentrations lower than 5 mg/mL. The current study explores this and other non-ideal behavior, emphasizing the presence of these effects at relatively lower concentrations for extended proteins. BSA, Brpt5.5, and a truncated form of Brpt5.5 referred to as Brpt1.5 are studied systematically using size-exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation (AUC), and viscosity. The viscous fingering effect is quantified using two approaches and is found to correlate well with the intrinsic viscosity of the proteins - Brpt5.5 exhibits the most severe effect and is the most extended protein tested in the study. By AUC, the hydrodynamic non-ideality was measured for each protein via global analysis of a concentration series. Compared to BSA, both Brpt1.5 and Brpt5.5 showed significant non-ideality that could be easily visualized at concentrations at or below 5 mg/mL and 1 mg/mL, respectively. A variety of relationships were examined for their ability to differentiate the proteins by shape using information from AUC and/or viscosity. Furthermore, these relationships were also tested in the context of hydrodynamic modeling. The importance of considering non-ideality when investigating the structure of extended macromolecules is discussed.

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Section: Resultsmentioning

confidence: 99%

Strong non-ideality effects at low protein concentrations: considerations for elongated proteins

Yarawsky

Dinu

Harding

et al. 2022

Preprint

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“…The SV-AUC (sedimentation velocity-AUC) approach has been widely used to study purity, aggregation, and conformational changes of biomolecules. Brautigam and colleagues compared the c(s) distribution method with the difference sedimentation velocity (DSV) method to investigate the detection of ligand-induced conformational changes in proteins (Brautigam et al (2020)). They also developed a new programme (DiSECT) to automate DSV data analysis and compared the benefits as well as challenges of each method.…”

Section: General Methodsmentioning

confidence: 99%

Analytical ultracentrifugation: still the gold standard that offers multiple solutions

Dobson

Patel

2020

Eur Biophys J

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Understanding the nature of macromolecules and their interactions in solution underpins many fields, including biology, chemistry and materials science. The 24th International Analytical Ultracentrifugation Workshop and Symposium (AUC2019, held in Christchurch, New Zealand, August 2019), brought together 77 international delegates to highlight recent developments in the field. There was a focus on analytical ultracentrifugation, although we recognise that this is but one of the key methods in the biophysicist's toolkit. Many of the presentations showcased the versatility of analytical ultracentrifugation and how such experiments are integrated with other solution techniques, such as small-angle X-ray scattering, cryo-electron microscopy, isothermal titration calorimetry and more. This special issue emphasises a wide range of themes covered in the meeting, including carbohydrate chemistry, protein chemistry, polymer science, and macromolecular interactions.

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“…The availability of absorbance, refractive and fluorescence detection allows AUC's application to a broad range of samples and solvents. Size distribution analysis by boundary sedimentation velocity AUC (SV-AUC) offers access to a wide range of sample characterization information that includes the following: structural heterogeneity (e.g., fragmentation, aggregation and chemical composition) [13], concentration [15], interactions [14], changes in conformation [16] and sample consistency [10] with few solvent matrix limitations, minimum sample manipulation and easy method development [7]. Importantly, AUC readily offers significant and unique possibilities for characterizing the new classes of very large, complex molecular biopharmaceutical gene therapy delivery systems that are now being actively developed, such as viral vectors [17,18], liposomes and nanoparticles [19][20][21][22][23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Boundary convection during sedimentation velocity in the Optima analytical ultracentrifuge

Berkowitz¹,

Laue

2021

Analytical Biochemistry

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Using modern approaches to sedimentation velocity to detect conformational changes in proteins

Cited by 7 publications

References 57 publications

Strong non-ideality effects at low protein concentrations: considerations for elongated proteins

Strong non-ideality effects at low protein concentrations: considerations for elongated proteins

Analytical ultracentrifugation: still the gold standard that offers multiple solutions

Boundary convection during sedimentation velocity in the Optima analytical ultracentrifuge

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