scholarly journals Universal nature of collapsibility in the context of protein folding and evolution

2018 ◽  
Author(s):  
D. Thirumalai ◽  
Himadri S. Samanta ◽  
Hiranmay Maity ◽  
Govardhan Reddy
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Saxs Data ◽  
X Ray ◽  
Model Free ◽  
X Ray Scattering ◽  
Theoretical Predictions ◽  
Helical Proteins ◽  
Universal Nature

AbstractTheory and simulations predicted sometime ago that the sizes of unfolded states of globular proteins should decrease continuously as the denaturant concentration is shifted from a high to a low value. However, small angle X-ray scattering (SAXS) data were used to assert the opposite, while interpretation of single molecule Forster resonance energy transfer experiments (FRET) supported the theoretical predictions. The disagreement between the two experiments is the SAXS-FRET controversy. By harnessing recent advances in SAXS and FRET experiments and setting these findings in the context of a general theory and simulations, we establish that compaction of unfolded states is universal. The theory also predicts that proteins rich in β-sheets are more collapsible than α-helical proteins. Because the extent of compaction is small, experiments have to be accurate and their interpretations should be as model free as possible. Theory also suggests that collapsibility itself could be a physical restriction on the evolution of foldable sequences, and provides a physical basis for the origin of multi-domain proteins.

scholarly journals Comment on “Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water”

Science ◽  
2018 ◽  
Vol 361 (6405) ◽  
pp. eaar7101 ◽  
Author(s):  
Robert B. Best ◽  
Wenwei Zheng ◽  
Alessandro Borgia ◽  
Karin Buholzer ◽  
Madeleine B. Borgia ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Small Angle ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Förster Resonance Energy Transfer ◽  
Unfolded Proteins ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

Riback et al. (Reports, 13 October 2017, p. 238) used small-angle x-ray scattering (SAXS) experiments to infer a degree of compaction for unfolded proteins in water versus chemical denaturant that is highly consistent with the results from Förster resonance energy transfer (FRET) experiments. There is thus no “contradiction” between the two methods, nor evidence to support their claim that commonly used FRET fluorophores cause protein compaction.

scholarly journals The structural ensemble of a Holliday junction determined by X-ray scattering interference

2020 ◽  
Vol 48 (14) ◽  
pp. 8090-8098
Author(s):  
Thomas Zettl ◽  
Xuesong Shi ◽  
Steve Bonilla ◽  
Steffen M Sedlak ◽  
Jan Lipfert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genetic Recombination ◽  
Resonance Energy ◽  
Holliday Junction ◽  
Full Description ◽  
Conformational Ensemble ◽  
X Ray ◽  
X Ray Scattering ◽  
Wide Range ◽  
Ray Scattering

Abstract The DNA four-way (Holliday) junction is the central intermediate of genetic recombination, yet key aspects of its conformational and thermodynamic properties remain unclear. While multiple experimental approaches have been used to characterize the canonical X-shape conformers under specific ionic conditions, the complete conformational ensemble of this motif, especially at low ionic conditions, remains largely undetermined. In line with previous studies, our single-molecule Förster resonance energy transfer (smFRET) measurements of junction dynamics revealed transitions between two states under high salt conditions, but smFRET could not determine whether there are fast and unresolvable transitions between distinct conformations or a broad ensemble of related states under low and intermediate salt conditions. We therefore used an emerging technique, X-ray scattering interferometry (XSI), to directly probe the conformational ensemble of the Holliday junction across a wide range of ionic conditions. Our results demonstrated that the four-way junction adopts an out-of-plane geometry under low ionic conditions and revealed a conformational state at intermediate ionic conditions previously undetected by other methods. Our results provide critical information to build toward a full description of the conformational landscape of the Holliday junction and underscore the utility of XSI for probing conformational ensembles under a wide range of solution conditions.

scholarly journals Integrating multiple experimental data to determine conformational ensembles of an intrinsically disordered protein

2020 ◽  
Author(s):  
Gregory-Neal W. Gomes ◽  
Mickaël Krzeminski ◽  
Ashley Namini ◽  
Erik. W. Martin ◽  
Tanja Mittag ◽  
...  
Keyword(s):  
Experimental Data ◽  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Intrinsically Disordered Protein ◽  
Disordered Proteins ◽  
Saxs Data ◽  
Conformational Ensembles ◽  
Intrinsically Disordered

AbstractIntrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes structural characterization challenging, but of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. We have used an integrative modelling approach to understand how conformational restraints imposed by the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET) reach concordance on structural ensembles for Sic1 and phosphorylated Sic1 (pSic1). To resolve apparent discrepancies between smFRET and SAXS, we integrated SAXS data with non-smFRET (NMR) data and reserved the new smFRET data for Sic1 and pSic1 as an independent validation. The consistency of the SAXS/NMR restrained ensembles with smFRET, which was not guaranteed a priori, indicates that the perturbative effects of NMR or smFRET labels on the Sic1 and pSic1 ensembles are minimal. Furthermore, the mutual agreement with such a diverse set of experimental data suggest that details of the generated ensembles can now be examined with a high degree of confidence to reveal distinguishing features of Sic1 vs. pSic1. From the experimentally well supported ensembles, we find they are consistent with independent biophysical models of Sic1’s ultrasensitive binding to its partner Cdc4. Our results underscore the importance of integrative modelling in calculating and drawing biological conclusions from IDP conformational ensembles.

scholarly journals Lateral gate dynamics of the bacterial translocon during cotranslational membrane protein insertion

2021 ◽  
Vol 118 (26) ◽  
pp. e2100474118
Author(s):  
Evan Mercier ◽  
Xiaolin Wang ◽  
Manisankar Maiti ◽  
Wolfgang Wintermeyer ◽  
Marina V. Rodnina
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Biological Significance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Phospholipid Bilayer ◽  
Membrane Phospholipids ◽  
Model Free ◽  
Lateral Gate

During synthesis of membrane proteins, transmembrane segments (TMs) of nascent proteins emerging from the ribosome are inserted into the central pore of the translocon (SecYEG in bacteria) and access the phospholipid bilayer through the open lateral gate formed of two helices of SecY. Here we use single-molecule fluorescence resonance energy transfer to monitor lateral-gate fluctuations in SecYEG embedded in nanodiscs containing native membrane phospholipids. We find the lateral gate to be highly dynamic, sampling the whole range of conformations between open and closed even in the absence of ligands, and we suggest a statistical model-free approach to evaluate the ensemble dynamics. Lateral gate fluctuations take place on both short (submillisecond) and long (subsecond) timescales. Ribosome binding and TM insertion do not halt fluctuations but tend to increase sampling of the open state. When YidC, a constituent of the holotranslocon, is bound to SecYEG, TM insertion facilitates substantial opening of the gate, which may aid in the folding of YidC-dependent polytopic membrane proteins. Mutations in lateral gate residues showing in vivo phenotypes change the range of favored states, underscoring the biological significance of lateral gate fluctuations. The results suggest how rapid fluctuations of the lateral gate contribute to the biogenesis of inner-membrane proteins.

scholarly journals Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions

2019 ◽  
Vol 116 (25) ◽  
pp. 12301-12310 ◽  
Author(s):  
Ivan Peran ◽  
Alex S. Holehouse ◽  
Isaac S. Carrico ◽  
Rohit V. Pappu ◽  
Osman Bilsel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Random Coil ◽  
Coarse Grained ◽  
Unfolded Proteins ◽  
Time Resolved ◽  
Conformational Preferences ◽  
Unfolded State ◽  
Theoretical Predictions

Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

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