Biology subjects

Pedersen, M. C.

Publications and source records attributed to Pedersen, M. C..

4 recordsLinked to original sources

bioRxiv2022

Refining structural models of membrane proteins withdisordered domains in phospholipid nanodiscs

Small-angle scattering can be used to derive structural information about membrane proteins reconstituted in suitable carrier systems enabling solubilization of the membrane proteins in question. Since the studies are done in solution, there is no need for crystallization or deposition on sample grids, and it is in principle possible to obtain structural information about intrinsically disordered regions which cannot be resolved by crystallography or the quantitative link to which is hard to establish using e.g. electron microscopy methods. In this study, tetramers of the gated spinach aquaporin SoPIP2;1 were reconstituted into nanodiscs and small-angle x-ray scattering data were recorded. From these data, we refine structural models of the entire nanodisc-membrane protein complex including the flexible regions using newly developed models based on Fast Debye sums. We introduce software for these computations available via online repositories and discuss the implications and limitations of these methods. Author summaryWhen it comes to investigating the structure and function of the proteins, a particular class of proteins are known to be cumbersome and problematic: membrane proteins that reside in the cell membrane and regulate and facilitate a number of critical biological processes. Such proteins can often not be studied by conventional means as they unravel and denature structurally or even precipitate in solution. To add insult to injury, such membrane proteins also often contain parts that are intrinsically disordered rendering them irresolvable by e.g. traditional crystallographic techniques and hard to describe structurally. Here, we present a combined computational and experimental approach (as well as the necessary software) to analyze and determine the structure of such proteins in close-to-native conditions in so-called nanodiscs, a biological carrier systems, using small-angle scattering and molecular simulations.

biophysics↗

bioRxiv2021

Mg2+-dependent conformational equilibria in CorA: an integrated view on transport regulation

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg2+ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg2+-bound closed structure nor the Mg2+-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg2+. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg2+ but that conducting states become more populated in Mg2+-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.

biophysics↗

bioRxiv2020

Order and disorder - an integrative structure of the full-length human growth hormone receptor

Despite the many physiological and pathophysiological functions of the human growth hormone receptor (hGHR), a detailed understanding of its modus operandi is hindered by the lack of structural information of the entire receptor at the molecular level. Due to its relatively small size (70 kDa) and large content of structural disorder (>50%), this membrane protein falls between the cracks of conventional high-resolution structural biology methods. Here, we study the structure of the full-length hGHR in nanodiscs with small angle-X-ray scattering (SAXS) as the foundation. We developed an approach in which we combined SAXS, X-ray diffraction and NMR spectroscopy obtained on the individual domains and integrated the data through molecular dynamics simulations to interpret SAXS data on the full-length hGHR in nanodiscs. The structure of the hGHR was determined in its monomeric state and provides the first experimental model of any full-length cytokine receptor in a lipid membrane. Combined, our results highlight that the three domains of the hGHR are free to reorient relative to each other, resulting in a broad structural ensemble. Our work exemplifies how integrating experimental data from several techniques computationally, may enable the characterization of otherwise inaccessible structures of membrane proteins with long disordered regions, a widespread phenomenon in biology. To understand orchestration of cellular signaling by disordered chains, the hGHR is archetypal and its structure emphasizes that we need to take a much broader, ensemble view on signaling.

biophysics↗

bioRxiv2019

PSX: Protein-Solvent Exchange - Software for calculation of deuterium-exchange effects in SANS measurements from protein coordinates

Recent developments in neutron scattering instrumentation and sample handling have enabled studies of more complex biological samples and measurements at shorter exposure times. The experiments are typically conducted in D2O-based buffers to emphasize or diminish scattering from a particular components or to minimize background noise in the experiment. To extract most information from such experiments it is thus desirable to determine accurate estimates of how and when closely bound hydrogen atoms from the biomolecule exchange with the deuterium in the solvent. We introduce and document software, PSX, for exploring the effect of hydrogen-deuterium exchange for proteins solubilized in D2O as well as the underlying bioinformatical models. The software aims to be generally applicable for any atomistic structure of a protein and its surrounding environment, and thus captures effects of both heterogenous exchange rates throughout the protein structure and by varying the experimental conditions such as pH and temperature. The paper concludes with examples of applications and estimates of the effect in typical scenarios emerging in small-angle neutron scattering on biological macromolecules in solution. Our analysis suggests that the common assumption of 90% exchange is in many cases an overestimate with the rapid sample handling systems currently available, which leads to fitting and calibration issues when analysing the data. Source code for the presented software is available from an online repository in which it is published under version 3 of the GNU publishing license.

biophysics↗