Deep Mutagenesis of a Transporter for Uptake of a Non-Native Substrate Identifies Conformationally Dynamic Regions

The serotonin transporter, SERT, catalyzes serotonin reuptake at the synapse to terminate neurotransmission via an alternating access mechanism, and SERT inhibitors are the most widely prescribed antidepressants. Here, deep mutagenesis is used to determine the effects of nearly all amino acid substitutions on human SERT surface expression and transport of the fluorescent substrate analogue APP+, identifying many mutations that enhance APP+ import. Comprehensive simulations of the entire ion-coupled import process reveal that while binding of the native substrate, serotonin, reduces free energy barriers between conformational states to promote SERT dynamics, the conformational free energy landscape in the presence of APP+ instead resembles Na+ bound-SERT, with a higher free energy barrier for transitioning to an inward-facing state. The deep mutational scan for SERT-catalyzed import of APP+ finds mutations that promote the necessary conformational changes that would otherwise be facilitated by the native substrate. Indeed, hundreds of gain-of-function mutations for APP+ import are found along the permeation pathway, most notably mutations that favor opening of a solvent-exposed intracellular vestibule. The mutagenesis data support the simulated mechanism in which the neurotransmitter and a symported sodium share a common cytosolic exit pathway to achieve coupling. Furthermore, the mutational landscape for SERT surface trafficking, which likely filters out misfolded sequences, reveals that residues along the permeation pathway are mutationally tolerant, providing plausible evolutionary pathways for changes in transporter properties while maintaining folded structure.

Metamorphic proteins: the Janus proteins of structural biology

The structural paradigm that the sequence of a protein encodes for a unique three-dimensional native fold does not acknowledge the intrinsic plasticity encapsulated in conformational free energy landscapes. Metamorphic proteins are a recently discovered class of biomolecules that illustrate this plasticity by folding into at least two distinct native state structures of comparable stability in the absence of ligands or cofactors to facilitate fold-switching. The expanding list of metamorphic proteins clearly shows that these proteins are not mere aberrations in protein evolution, but may have actually been a consequence of distinctive patterns in selection pressure such as those found in virus–host co-evolution. In this review, we describe the structure–function relationships observed in well-studied metamorphic protein systems, with specific focus on how functional residues are sequestered or exposed in the two folds of the protein. We also discuss the implications of metamorphosis for protein evolution and the efforts that are underway to predict metamorphic systems from sequence properties alone.

Charting the Complete Thermodynamic Landscape of Gas Adsorption for a Responsive Metal-Organic Framework

<div>New nanoporous materials are able to revolutionize adsorption and separation processes. In particular, materials with adaptive cavities have high selectivity and may display previously undiscovered phenomena, such as negative gas adsorption (NGA), in which gas is released from the framework upon an increase in pressure. Although the thermodynamic driving force behind this and many other counterintuitive adsorption phenomena have been thoroughly investigated in recent years, several experimental observations remain difficult to explain. This necessitates a comprehensive analysis of gas adsorption akin to the conformational free energy landscapes used to understand the function of proteins. For the first time, we constructed the complete thermodynamic landscape of methane adsorption on DUT-49, a system that demonstrates NGA. Traversing this complex landscape correctly reproduces the experimentally observed structural transitions, the temperature dependence of the NGA phenomenon and the observed hysteresis between adsorption and desorption. The complete thermodynamic description presented here provides unparalleled insight into the process of adsorption and provides a framework to understand other adsorbents that challenge our preconceptions.<br></div>

Charting the Complete Thermodynamic Landscape of Gas Adsorption for a Responsive Metal-Organic Framework

<div>New nanoporous materials are able to revolutionize adsorption and separation processes. In particular, materials with adaptive cavities have high selectivity and may display previously undiscovered phenomena, such as negative gas adsorption (NGA), in which gas is released from the framework upon an increase in pressure. Although the thermodynamic driving force behind this and many other counterintuitive adsorption phenomena have been thoroughly investigated in recent years, several experimental observations remain difficult to explain. This necessitates a comprehensive analysis of gas adsorption akin to the conformational free energy landscapes used to understand the function of proteins. For the first time, we constructed the complete thermodynamic landscape of methane adsorption on DUT-49, a system that demonstrates NGA. Traversing this complex landscape correctly reproduces the experimentally observed structural transitions, the temperature dependence of the NGA phenomenon and the observed hysteresis between adsorption and desorption. The complete thermodynamic description presented here provides unparalleled insight into the process of adsorption and provides a framework to understand other adsorbents that challenge our preconceptions.<br></div>

Comprehensive characterization of oligosaccharide conformational ensembles with conformer classification by free-energy landscape via reproductive kernel Hilbert space

A kernel method enables the comprehensive characterization of conformational ensembles of oligosaccharides in association with the conformational free-energy landscape.