Folding Stability Research Articles

C-Terminal β8-α9 Interaction Modulates Thermal Stability and Enzymatic Activity Differently in Hyperthermophilic Esterase EstE1 and Mesophilic Esterase rPPE.

Hydrophobic interactions and hydrogen bonds are 2 types of noncovalent interactions that play distinct roles in the folding and structural stability of proteins. However, the specific roles of these interactions in hydrophobic or hydrophilic environments in α/β-hydrolases are not fully understood. A hyperthermophilic esterase EstE1 in a dimer maintains the C-terminal β8-α9 strand-helix via hydrophobic interactions (Phe276 and Leu299), constituting a closed dimer interface. Moreover, a mesophilic esterase rPPE in a monomer maintains the same strand-helix via a hydrogen bond (Tyr281 and Gln306). Unpaired polar residues (F276Y in EstE1 and Y281A/F and Q306A in rPPE) or reduced hydrophobic interactions (F276A/L299A in EstE1) between the β8-α9 strand-helix decrease thermal stability. EstE1 (F276Y/L299Q) and rPPE WT, both with the β8-α9 hydrogen bond, showed the same thermal stability as EstE1 WT and rPPE (Y281F/Q306L), which possess hydrophobic interactions instead. However, EstE1 (F276Y/L299Q) and rPPE WT exhibited higher enzymatic activity than EstE1 WT and rPPE (Y281F/Q306L), respectively. This suggests that α/β-hydrolases favor the β8-α9 hydrogen bond for catalytic activity in monomers or oligomers. Overall, these findings demonstrate how α/β-hydrolases modulate hydrophobic interactions and hydrogen bonds to adapt to different environments. Both types of interactions contribute equally to thermal stability, but the hydrogen bond is preferred for catalytic activity. IMPORTANCE Esterases hydrolyze short to medium-chain monoesters and contain a catalytic His on a loop between the C-terminal β8-strand and α9-helix. This study explores how hyperthermophilic esterase EstE1 and mesophilic esterase rPPE adapt to different temperatures by utilizing the β8-α9 hydrogen bonds or hydrophobic interactions differently. EstE1 forms a hydrophobic dimer interface, while rPPE forms a monomer stabilized by a hydrogen bond. The study demonstrates that these enzymes stabilize β8-α9 strand-helix differently but achieve similar thermal stability. While the β8-α9 hydrogen bond or hydrophobic interactions contribute equally to thermal stability, the hydrogen bond provides higher activity due to increased catalytic His loop flexibility in both EstE1 and rPPE. These findings reveal how enzymes adapt to extreme environments while maintaining their functions and have implications for engineering enzymes with desired activities and stabilities.

On-Demand Transient Paper Substrate for Selective Disposability of Thin-Film Electronic Devices.

This study demonstrates a novel approach to creating a thin-film electronic device that offers selective or complete disposability only in on-demand conditions while maintaining stable operation reliability during everyday use. The approach involves a transient paper substrate, combined with phase change encapsulation and highly bendable planarization materials, achieved through a simple solution process. The substrate used in this study offers a smooth surface morphology that enables the creation of stable multilayers for thin-film electronic devices. It also exhibits superior waterproof properties, which allows the proof-of-concept organic light-emitting device to function even when submerged in water. Additionally, the substrate provides controlled surface roughness under repeated bending, demonstrating reliable folding stability for 1000 cycles at 10 mm of curvature. Furthermore, a specific component of the electronic device can be selectively made to malfunction through predetermined voltage input, and the entire device can be fully disposed of via Joule-heating-induced combustion.

Comparing the influence of explicit and implicit solvation models on site-specific thermodynamic stability of proteins.

Understanding the molecular basis for protein stability requires a thermodynamic analysis of protein folding. Thermodynamic analysis is often performed by sampling many atomistic conformations using molecular simulations that employ either explicit or implicit water models. However, it remains unclear to what extent thermodynamic results from different solvation models are reliable at the molecular level. In this study, we quantify the influence of both solvation models on folding stability at the individual backbone and side chain resolutions. We assess the residue-specific folding free energy components of a β-sheet protein and a helical protein using trajectories resulting from TIP3P explicit and generalized Born/surface area implicit solvent simulations of model proteins. We found that the thermodynamic discrepancy due to the implicit solvent mostly originates from charged side chains, followed by the under-stabilized hydrophobic ones. In contrast, the contributions of backbone residue in both proteins were comparable for explicit and implicit water models. Our study lays out the foundation for detailed thermodynamic assessment of solvation models in the context of protein simulation.

815-P: Enhanced Disulphide Bond Stability Contributes to the Once Weekly PD Profile of Insulin Icodec

Insulin icodec is a novel basal insulin analogue with a long half-life (196 hours) that is in clinical development for the treatment of diabetes with once weekly dosing. Insulin icodec was developed based on the hypothesis that lowering the insulin receptor affinity in the presence of a strong HSA-binding moiety would result in a long half-life, provided that non-receptor mediated clearance is diminished. The observation that insulin icodec retains its full potency over weeks has been attributed to low non-insulin receptor mediated clearance as well as to limited degradation in circulation, due to the amino acid modifications in insulin icodec that confer proteolytically-stabilization. Here, we report a previously undescribed insulin clearance mechanism, resulting in splitting insulin molecules into A- and B-chains by a thiol-disulfide exchange reaction. We found that the amino acid substitution of Tyr in A14 to Glu provided a major stabilization of insulin against such degradation while only minor improvements were introduced by the Tyr in B16 to His and Phe in B25 to His substitutions. These results correlate with improvements in the folding stability and likely reflect reduced exposure of insulin icodec's disulfide bonds to the thiol-disulfide exchange reaction. Even though insulin icodec's amino acid substitutions significantly limit its susceptibility to this new clearance mechanism, some free B-chain has been observed in plasma samples from minipigs and people with type 2 diabetes. In summary, we have identified thiol-disulfide exchange reactions as an additional insulin clearance mechanism and minimizing insulin icodec's susceptibility to this process has significantly contributed to its long PD, making it suitable for once weekly dosing. Disclosure F.Hubalek: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. C.Cramer: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. H.Helleberg: Employee; Novo Nordisk A/S. E.Johansson: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. G.Schluckebier: Employee; Novo Nordisk. D.B.Steensgaard: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk. J.Sturis: Employee; Novo Nordisk A/S, Other Relationship; Novozymes, Stock/Shareholder; Novo Nordisk A/S. T.Kjeldsen: Employee; Novo Nordisk, Stock/Shareholder; Novo Nordisk.

A Study of a Protein-Folding Machine: Transient Rotation of the Polypeptide Backbone Facilitates Rapid Folding of Protein Domains in All-Atom Molecular Dynamics Simulations.

Molecular dynamics simulations of protein folding typically consider the polypeptide chain at equilibrium and in isolation from the cellular components. We argue that in order to understand protein folding as it occurs in vivo, it should be modeled as an active, energy-dependent process, in which the cellular protein-folding machine directly manipulates the polypeptide. We conducted all-atom molecular dynamics simulations of four protein domains, whose folding from the extended state was augmented by the application of rotational force to the C-terminal amino acid, while the movement of the N-terminal amino acid was restrained. We have shown earlier that such a simple manipulation of peptide backbone facilitated the formation of native structures in diverse α-helical peptides. In this study, the simulation protocol was modified, to apply the backbone rotation and movement restriction only for a short time at the start of simulation. This transient application of a mechanical force to the peptide is sufficient to accelerate, by at least an order of magnitude, the folding of four protein domains from different structural classes to their native or native-like conformations. Our in silico experiments show that a compact stable fold may be attained more readily when the motions of the polypeptide are biased by external forces and constraints.

Expression and Biological Evaluation of an Engineered Recombinant L-asparaginase Designed by In Silico Method Based on Sequence of the Enzyme from Escherichia coli.

Medical usage of L-asparaginase (ASNase), the first-line of acute lymphoblastic leukemia treatment, is linked to allergic responses and toxicities, which necessitates the development of new bio-better ASNases. The aim of the current study was in silico design of a novel ASNase with predicted improved enzymatic properties using strategies encompassing sequence-function analysis of known ASNase mutants. Additionally, current study aimed to show that the new enzyme is active. Based on 21 experimentally reported mutations for ASNase, a virtual library of mutated enzymes with all 7546 possible combinations of up to 4 mutations was generated. Three-dimensional models of proposed mutant enzymes were built and their in silico stabilities were calculated. The most promising mutant was selected for preparing a genetic construct suitable for expression of the designed ASNase in bacterial cells. Computational study predicted that Y176F/S241C double mutation of Escherichia coli ASNase may increase its folding stability. The designed ASNase was expressed in two different E. coli strains (Origami B(DE3) and BL21(DE3)pLysS) and then the soluble fractions prepared from the cell lysates of the host cells were used in enzyme activity assay. Results showed that enzyme activity of soluble fraction from Origami (95.4 ± 7.5 IU/0.1 mL) was four times higher than that of soluble fraction from pLysS (25.8 ± 2.5 IU/0.1 mL). A novel functional double mutant ASNase with predicted improved enzymatic properties was designed and produced in E. coli. The results of the current study suggest a great commercial potential for the identified enzyme in pharmaceutical and industrial applications.

Advancing Multicomponent Strategies to Macrobicyclic Peptides.

Macrocyclization of peptides is typically used to fix specific bioactive conformations and improve their pharmacological properties. Recently, macrobicyclic peptides have received special attention owing to their capacity to mimic protein structures or be key components of peptide-drug conjugates. Here, we describe the development of novel synthetic strategies for two distinctive types of peptide macrobicycles. A multicomponent macrocyclo-dimerization approach is introduced for the production of interconnected β-turns, allowing two macrocyclic rings to be formed and dimerized in one pot. Also, an on-resin double stapling strategy is described for the assembly of lactam-bridged macrobicycles with stable tertiary folds.

A comprehensive examination of ACE2 receptor and prediction of spike glycoprotein and ACE2 interaction based on in silico analysis of ACE2 receptor

The ACE2 receptor plays a vital role not only in the SARS-CoV-induced epidemic but also in various other diseases, including cardiovascular diseases and ARDS. While studies have explored the interactions between ACE2 and SARS-CoV proteins, comprehensive research utilizing bioinformatic tools on the ACE2 protein has been lacking. The one aim of present study was to extensively analyze the regions of the ACE2 protein. After utilizing all bioinformatics tools especially G104 and L108 regions on ACE2 were come forward. The results of our analysis revealed that possible mutations or deletions in the G104 and L108 regions play a critical role in both the biological functioning and the determination of the chemical-physical properties of ACE2. Additionally, these regions were found to be more susceptible to mutations or deletions compared to other regions of the ACE2 protein. Notably, the randomly selected peptide, LQQNGSSVLS (100–109), which includes G104 and L108, exhibited a crucial role in binding the RBD of the spike protein, as supported by docking scores. Furthermore, both MDs and iMODs results provided evidence that G104 and L108 influence the dynamics of ACE2-spike complexes. This study is expected to offer a new perspective on the ACE2-SARS-CoV interaction and other research areas where ACE2 plays a significant role, such as biotechnology (protein engineering, enzyme optimization), medicine (RAS, pulmonary and cardiac diseases), and basic research (structural motifs, stabilizing protein folds, or facilitating important inter molecular contacts, protein’s proper structure and function). Communicated by Ramaswamy H. Sarma

Structure of LARP7 Protein p65–telomerase RNA Complex in Telomerase Revealed by Cryo-EM and NMR

La-related protein 7 (LARP7) are a family of RNA chaperones that protect the 3′-end of RNA and are components of specific ribonucleoprotein complexes (RNP). In Tetrahymena thermophila telomerase, LARP7 protein p65 together with telomerase reverse transcriptase (TERT) and telomerase RNA (TER) form the core RNP. p65 has four known domains—N-terminal domain (NTD), La motif (LaM), RNA recognition motif 1 (RRM1), and C-terminal xRRM2. To date, only the xRRM2 and LaM and their interactions with TER have been structurally characterized. Conformational dynamics leading to low resolution in cryo-EM density maps have limited our understanding of how full-length p65 specifically recognizes and remodels TER for telomerase assembly. Here, we combined focused classification of Tetrahymena telomerase cryo-EM maps with NMR spectroscopy to determine the structure of p65–TER. Three previously unknown helices are identified, one in the otherwise intrinsically disordered NTD that binds the La module, one that extends RRM1, and another preceding xRRM2, that stabilize p65–TER interactions. The extended La module (αN, LaM and RRM1) interacts with the four 3′ terminal U nucleotides, while LaM and αN additionally interact with TER pseudoknot, and LaM with stem 1 and 5′ end. Our results reveal the extensive p65–TER interactions that promote TER 3′-end protection, TER folding, and core RNP assembly and stabilization. The structure of full-length p65 with TER also sheds light on the biological roles of genuine La and LARP7 proteins as RNA chaperones and core RNP components.

The Fitness Effects of Codon Composition of the Horizontally Transferred Antibiotic Resistance Genes Intensify at Sub-lethal Antibiotic Levels.

The rampant variability in codon bias existing between bacterial genomes is expected to interfere with horizontal gene transfer (HGT), a phenomenon that drives bacterial adaptation. However, delineating the constraints imposed by codon bias on functional integration of the transferred genes is complicated by multiple genomic and functional barriers controlling HGT, and by the dependence of the evolutionary outcomes of HGT on the host's environment. Here, we designed an experimental system in which codon composition of the transferred genes is the only variable triggering fitness change of the host. We replaced Escherichia coli's chromosomal folA gene encoding dihydrofolate reductase, an essential enzyme that constitutes a target for trimethoprim, with combinatorial libraries of synonymous codons of folA genes from trimethoprim-sensitive Listeria grayi and trimethoprim-resistant Neisseria sicca. The resulting populations underwent selection at a range of trimethoprim concentrations, and the ensuing changes in variant frequencies were used to infer the fitness effects of the individual combinations of codons. We found that when HGT causes overstabilization of the 5'-end mRNA, the fitness contribution of mRNA folding stability dominates over that of codon optimality. The 5'-end overstabilization can also lead to mRNA accumulation outside of the polysome, thus preventing the decay of the foreign transcripts despite the codon composition-driven reduction in translation efficiency. Importantly, the fitness effects of mRNA stability or codon optimality become apparent only at sub-lethal levels of trimethoprim individually tailored for each library, emphasizing the central role of the host's environment in shaping the codon bias compatibility of horizontally transferred genes.

Integrative Approach to Dissect the Drug Resistance Mechanism of the H172Y Mutation of SARS-CoV-2 Main Protease.

Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of Paxlovid, a drug approved by the U.S. Food and Drug Administration for high-risk COVID-19 patients. Recently, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvir's inhibitory activity. As the COVID-19 cases skyrocket in China and the selective pressure of antiviral therapy builds in the US, there is an urgent need to characterize and understand how the H172Y mutation confers drug resistance. Here, we investigated the H172Y Mpro's conformational dynamics, folding stability, catalytic efficiency, and inhibitory activity using all-atom constant pH and fixed-charge molecular dynamics simulations, alchemical and empirical free energy calculations, artificial neural networks, and biochemical experiments. Our data suggest that the mutation significantly weakens the S1 pocket interactions with the N-terminus and perturbs the conformation of the oxyanion loop, leading to a decrease in the thermal stability and catalytic efficiency. Importantly, the perturbed S1 pocket dynamics weaken the nirmatrelvir binding in the P1 position, which explains the decreased inhibitory activity of nirmatrelvir. Our work demonstrates the predictive power of the combined simulation and artificial intelligence approaches, and together with biochemical experiments, they can be used to actively surveil continually emerging mutations of SARS-CoV-2 Mpro and assist the optimization of antiviral drugs. The presented approach, in general, can be applied to characterize mutation effects on any protein drug targets.

Twistable and tailorable Cu-doped SnO2@PANI textile for wearable ammonia sensing

Herein, a twistable and tailorable NH3 sensor utilizing hollow porous Cu-doped SnO2@PANI (PCS) composite was assembled on a flexible non-woven fabric substrate by in-situ polymerization technique and its gas sensing performance was tested at room temperature (∼25 °C). The results indicated that the well-designed PCS sensor to 50 ppm NH3 exhibited six times higher response (25.4) than the pristine PANI sensor (4.5). The PCS flexible sensor still possessed a response (1.53) for 10 ppb NH3, and the detection limit could reach 5.2 ppb for NH3. Meanwhile, the PCS flexible sensor demonstrated excellent flexibility with response decreases of only 1.97 % after 1000 times bending (120°), and 2.11 % after 100 times folding (150°) and long-term stability through 42 days of monitoring (maintaining 98.4 % response). Furthermore, the PCS sensor displayed insensitivity to relative humidity (20 % − 75 % RH), and the low sensing temperature could reach − 15 °C. The enhanced sensing performance of the PCS sensor was attributed to the synergistic effect of the vital role of abundant oxygen vacancy and − OH groups, as well as the hollow porous interconnected structure and the p-n heterojunction between Cu-doped SnO2 and PANI. This work provides more opportunities to design smart wearable devices for detecting ppb-level NH3 at room temperature.

Protein Folding Stability Profiling of Colorectal Cancer Chemoresistance Identifies Functionally Relevant Biomarkers.

Reported here is the application of three protein folding stability profiling techniques (including the stability of proteins from rates of oxidation, thermal protein profiling, and limited proteolysis approaches) to identify differentially stabilized proteins in six patient-derived colorectal cancer (CRC) cell lines with different oxaliplatin sensitivities and eight CRC patient-derived xenografts (PDXs) derived from two of the patient derived cell lines with different oxaliplatin sensitivities. Compared to conventional protein expression level analyses, which were also performed here, the stability profiling techniques identified both unique and novel proteins and cellular components that differentiated the sensitive and resistant samples including 36 proteins that were differentially stabilized in at least two techniques in both the cell line and PDX studies of oxaliplatin resistance. These 36 differentially stabilized proteins included 10 proteins previously connected to cancer chemoresistance. Two differentially stabilized proteins, fatty acid synthase and elongation factor 2, were functionally validated in vitro and found to be druggable protein targets with biological functions that can be modulated to improve the efficacy of CRC chemotherapy. These results add to our understanding of CRC oxaliplatin resistance, suggest biomarker candidates for predicting oxaliplatin sensitivity in CRC, and inform new strategies for overcoming chemoresistance in CRC.

Assessing variants of uncertain significance implicated in hearing loss using a comprehensive deafness proteome

Hearing loss is the leading sensory deficit, affecting ~ 5% of the population. It exhibits remarkable heterogeneity across 223 genes with 6328 pathogenic missense variants, making deafness-specific expertise a prerequisite for ascribing phenotypic consequences to genetic variants. Deafness-implicated variants are curated in the Deafness Variation Database (DVD) after classification by a genetic hearing loss expert panel and thorough informatics pipeline. However, seventy percent of the 128,167 missense variants in the DVD are “variants of uncertain significance” (VUS) due to insufficient evidence for classification. Here, we use the deep learning protein prediction algorithm, AlphaFold2, to curate structures for all DVD genes. We refine these structures with global optimization and the AMOEBA force field and use DDGun3D to predict folding free energy differences (∆∆GFold) for all DVD missense variants. We find that 5772 VUSs have a large, destabilizing ∆∆GFold that is consistent with pathogenic variants. When also filtered for CADD scores (> 25.7), we determine 3456 VUSs are likely pathogenic at a probability of 99.0%. Of the 224 genes in the DVD, 166 genes (74%) exhibit one or more missense variants predicted to cause a pathogenic change in protein folding stability. The VUSs prioritized here affect 119 patients (~ 3% of cases) sequenced by the OtoSCOPE targeted panel. Approximately half of these patients previously received an inconclusive report, and reclassification of these VUSs as pathogenic provides a new genetic diagnosis for six patients.

Mega-scale experimental analysis of protein folding stability in biology and protein design

Mega-scale experimental analysis of protein folding stability in biology and protein design

Mega-scale experimental analysis of protein folding stability in biology and protein design

Mega-scale experimental analysis of protein folding stability in biology and protein design

CAG-Repeat RNA Hairpin Folding and Recruitment to Nuclear Speckles with a Pivotal Role of ATP as a Cosolute.

A hallmark of Huntington's disease (HD) is a prolonged polyglutamine sequence in the huntingtin protein and, correspondingly, an expanded cytosine, adenine, and guanine (CAG) triplet repeat region in the mRNA. A majority of studies investigating disease pathology were concerned with toxic huntingtin protein, but the mRNA moved into focus due to its recruitment to RNA foci and emerging novel therapeutic approaches targeting the mRNA. A hallmark of CAG-RNA is that it forms a stable hairpin in vitro which seems to be crucial for specific protein interactions. Using in-cell folding experiments, we show that the CAG-RNA is largely destabilized in cells compared to dilute buffer solutions but remains folded in the cytoplasm and nucleus. Surprisingly, we found the same folding stability in the nucleoplasm and in nuclear speckles under physiological conditions suggesting that CAG-RNA does not undergo a conformational transition upon recruitment to the nuclear speckles. We found that the metabolite adenosine triphosphate (ATP) plays a crucial role in promoting unfolding, enabling its recruitment to nuclear speckles and preserving its mobility. Using in vitro experiments and molecular dynamics simulations, we found that the ATP effects can be attributed to a direct interaction of ATP with the nucleobases of the CAG-RNA rather than ATP acting as "a fuel" for helicase activity. ATP-driven changes in CAG-RNA homeostasis could be disease-relevant since mitochondrial function is affected in HD disease progression leading to a decline in cellular ATP levels.

514 Understanding Structural and Dynamic Effects of the EWS-FLI1 interactome on the EWS Low Complexity Domain Function

OBJECTIVES/GOALS: The EWSR1-FLI1 gene fusion is implicated as a source of oncogenic activity in the majority of Ewing sarcoma (EwS) cases (>70%). Our studies will provide unparalleled insight into the transformative mechanics of EWS-FLI1 METHODS/STUDY POPULATION: EWS-FLI1 is an intrinsically disordered protein (IDP). IDPs do not form stable secondary or tertiary folds in biological conditions and ofter contain proline-rich regions and other multivalent binding regions. These regions interact with numerous partners resulting in highly dynamic and complex protein-protein interactions. Recent advances in proximity labelling techniques, such as the use of a biotin ligase fused to a protein of interest are exceptionally well suited to identifying IDP interactomes since they do not rely on the binding affinities but rather the distance between interacting proteins. Combining novel discovery proteomics with Nuclear Magnetic Resonance (NMR) approaches will provide unparalleled insight into the transformative mechanics of EWS-FLI1. RESULTS/ANTICIPATED RESULTS: Using the proximity labeling technique TurboID, which limits the number of false-positive interactions, we have shown that EWSR1 and EWS-FLI1 act within the spliceosome (responsible for mRNA splicing and processing). Using bioinformatics techniques, we show EWS-FLI1 interacts with peptidyl-prolyl isomerase (PPI) proteins, specifically PPIL1, through the EWS-FLI1 N-terminal region (EWS-LCD). We used NMR to provide insight into the interaction between PPIL1 and EWS-LCD, showing that EWS-LCD interacts with the catalytic region of PPIL1. We anticipate that PPIL1 isomerizes EWS-FLI1 to modulate its activity, and that EWS-FLI1 interacts with the spliceosome through formation of a biological condensate. However, future proximity labeling and NMR studies are needed to verify this activity. DISCUSSION/SIGNIFICANCE: Our studies will yield actionable insights regarding the protein-protein interfaces in EWS-FLI1 that can be targeted to attenuate the oncogenic activity of EWS-FLI1. More broadly, our results will be applicable toward understanding the etiology of other pediatric cancers and for guiding the development of novel targeted treatments.

Slow conformational changes in the rigid and highly stable chymotrypsin inhibitor 2.

Slow conformational changes are often directly linked to protein function. It is however less clear how such processes may perturb the overall folding stability of a protein. We previously found that the stabilizing double mutant L49I/I57V in the small protein chymotrypsin inhibitor 2 from barley led to distributed increased nanosecond and faster dynamics. Here we asked what effects the L49I and I57V substitutions, either individually or together, have on the slow conformational dynamics of CI2. We used 15 N CPMG spin relaxation dispersion experiments to measure the kinetics, thermodynamics and structural changes associated with slow conformational change in CI2. These changes result in an excited state that is populated to 4.3% at 1 °C. As the temperature is increased the population of the excited state decreases. Structural changes in the excited state are associated with residues that interact with water molecules that have well defined positions and are found at these positions in all crystal structures of CI2. The substitutions in CI2 have only little effect on the structure of the excited state whereas the stability of the excited state to some extent follows the stability of the main state. The minor state is thus most populated for the most stable CI2 variant and least populated for the least stable variant. We hypothesize that the interactions between the substituted residues and the well-ordered water molecules links subtle structural changes around the substituted residues to the region in the protein that experience slow conformational changes. This article is protected by copyright. All rights reserved.

Two Cases of TMEM151A-Associated Paroxysmal Dyskinesia in a Single-Center Series of PRRT2-Negative Patients.

Two Cases of TMEM151A-Associated Paroxysmal Dyskinesia in a Single-Center Series of PRRT2-Negative Patients.