Calorimetric quantification of linked equilibria in cyclodextrin/lipid/detergent mixtures for membrane-protein reconstitution

Textor, Martin; Vargas, Carolyn; Keller, Sandro

doi:10.1016/j.ymeth.2015.01.002

Cited by 15 publications

(10 citation statements)

References 51 publications

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“…Interestingly, in a study of membrane helix-helix interactions, significant energetic differences were observed when helices were in bicelles that resembled micelles and bicelles that closely approximated bilayer properties ( 60 ). ITC also provides direct means to monitor reconstitution of detergent-solubilized proteins into lipid vesicles, potentially opening avenues for rational optimization of the procedure ( 62 , 63 ).…”

Section: Methodsmentioning

confidence: 99%

Isothermal titration calorimetry of ion-coupled membrane transporters

Boudker

2015

Methods

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Binding of ligands, ranging from proteins to ions, to membrane proteins is associated with absorption or release of heat that can be detected by isothermal titration calorimetry (ITC). Such measurements not only provide binding affinities but also afford direct access to thermodynamic parameters of binding - enthalpy, entropy and heat capacity. These parameters can be interpreted in a structural context, allow discrimination between different binding mechanisms and guide drug design. In this review, we introduce advantages and limitations of ITC as a methodology to study molecular interactions of membrane proteins. We further describe case studies where ITC was used to analyze thermodynamic linkage between ions and substrates in ion-coupled transporters. Similar type of linkage analysis will likely be applicable to a wide range of transporters, channels, and receptors.

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Section: Methodsmentioning

confidence: 99%

Isothermal titration calorimetry of ion-coupled membrane transporters

Boudker

2015

Methods

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“…Hence, we performed steady-state Trp fluorescence experiments upon exposing the protein in the presence of the zwitterionic detergent LDAO at a constant detergent concentration of 10 mM to increasing concentrations of the chemical denaturant GdmCl. As the critical micellar concentration of LDAO increases from 1.9 mM in the absence of GdmCl (34) to $42 mM in the presence of 8 M GdmCl, micelles disintegrate when the GdmCl concentration reaches $4.6 M ( Fig. S7).…”

Section: Steady-state Trp Fluorescence Spectroscopymentioning

confidence: 99%

Slow Interconversion in a Heterogeneous Unfolded-State Ensemble of Outer-Membrane Phospholipase A

Krainer

Gracia

Frotscher

et al. 2017

Biophysical Journal

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Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with other cell components. In the case of outer-membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance, because these proteins remain unfolded for extended periods of time during their biogenesis and rely on interactions with binding partners to support proper folding. Using a combination of ensemble and single-molecule spectroscopy, we have scrutinized the unfolded state of outer-membrane phospholipase A (OmpLA) to provide a detailed view of its structural dynamics on timescales from nanoseconds to milliseconds. We find that even under strongly denaturing conditions and in the absence of residual secondary structure, OmpLA populates an ensemble of slowly (>100 ms) interconverting and conformationally heterogeneous unfolded states that lack the fast chain-reconfiguration motions expected for an unstructured, fully unfolded chain. The drastically slowed sampling of potentially folding-competent states, as compared with a random-coil polypeptide, may contribute to the slow in vitro folding kinetics observed for many OMPs. In vivo, however, slow intramolecular long-range dynamics might be advantageous for entropically favored binding of unfolded OMPs to chaperones and, by facilitating conformational selection after release from chaperones, for preserving binding-competent conformations before insertion into the outer membrane.

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“…as detailed elsewhere [7][8][9]. Here, Q denotes the normalized heat of reaction, c D the surfactant concentration in the calorimeter cell, and Dc D the above-mentioned parameter affecting the width of the transition range.…”

Section: Introductionmentioning

confidence: 99%

“…(1), (2), and (4À6) have previously been used [7][8][9] to fit demicellization isotherms that largely differed in terms of CMC, demicellization heat (i.e., signal amplitude), and steepness of the transition. One disadvantage inherent to this earlier approach is that the micellization enthalpy does not show up directly as a fitting parameter but, instead, is calculated from Eqs.…”

Section: Introductionmentioning

confidence: 99%

“…Then, Q demic is obtained from the difference in heat between linear preand post-transition baselines at the CMC [6]. In an alternative approach, a generic sigmoidal function can be fitted to the isotherm to derive the CMC as well as Q demic through nonlinear least-squares fitting [7][8][9]. In both cases, the standard molar micellization enthalpy, DH mic , is then calculated from Q demic .…”

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confidence: 99%

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