scholarly journals Electrostatic effects in proteins are governed by pH-redistribution of the conformational ensemble

2020 ◽  
Author(s):  
Christos M. Kougentakis ◽  
Ananya Majumdar ◽  
E. Bertrand García-Moreno
Keyword(s):  
Conformational Changes ◽  
De Novo ◽  
Energy Transduction ◽  
Conformational Ensemble ◽  
Biological Macromolecules ◽  
Ph Sensing ◽  
Functional Sites ◽  
Ionizable Residues ◽  
Open States

The imperative for charges to be hydrated is one of the most important organizing principles in biology, responsible for the general architecture of biological macromolecules and for energy storage in the form of electrochemical gradients. Paradoxically, many functional sites in proteins have buried ionizable groups1. These groups are tolerated because they are usually buried in the neutral state2. However, when they become charged they can drive structural transitions to open states in which the charge can be stabilized, mostly through interactions with water3. This coupling between the ionization of a buried group and conformational reorganization is precisely the mechanism used by proteins to perform energy transduction4,5,6. By applying this principle to a family of 25 variants of staphylococcal nuclease with internal Lys residues, it was possible to characterize in detail the range of localized partial unfolding events that even a highly stable protein that unfolds cooperatively can undergo in response to H+-binding. Conformational states that constitute vanishingly small populations of the equilibrium native state ensemble of this protein were identified by correlation of structural and thermodynamic data, providing a map of the conformational landscape of this protein with unprecedented detail. The data demonstrate that the apparent pKa values of buried ionizable residues are not determined by the properties of their microenvironment but by the intrinsic propensity of the protein to populate open states in which internal charged residues can be hydrated. The role of buried residues in functional sites in proteins relies on their ability to tune the conformational ensemble for redistribution in response to small changes in pH. These results provide the physical framework necessary for understanding the role of pH-driven conformational changes in driving biological energy transduction4, the identification of pH-sensing proteins in nature7, and for the engineering of pH-sensitive dynamics and function in de novo designed proteins8.

scholarly journals NMDA receptor channel gating control by the pre-M1 helix

2020 ◽  
Vol 152 (4) ◽  
Author(s):  
Miranda J. McDaniel ◽  
Kevin K. Ogden ◽  
Steven A. Kell ◽  
Pieter B. Burger ◽  
Dennis C. Liotta ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Conformational Changes ◽  
Time Course ◽  
Transmembrane Domain ◽  
Single Channel ◽  
De Novo ◽  
Channel Gating ◽  
Open Probability ◽  
Single Channel Currents

The NMDA receptor (NMDAR) is an ionotropic glutamate receptor formed from the tetrameric assembly of GluN1 and GluN2 subunits. Within the flexible linker between the agonist binding domain (ABD) and the M1 helix of the pore-forming transmembrane helical bundle lies a two-turn, extracellular pre-M1 helix positioned parallel to the plasma membrane and in van der Waals contact with the M3 helix thought to constitute the channel gate. The pre-M1 helix is tethered to the bilobed ABD, where agonist-induced conformational changes initiate activation. Additionally, it is a locus for de novo mutations associated with neurological disorders, is near other disease-associated de novo sites within the transmembrane domain, and is a structural determinant of subunit-selective modulators. To investigate the role of the pre-M1 helix in channel gating, we performed scanning mutagenesis across the GluN2A pre-M1 helix and recorded whole-cell macroscopic and single channel currents from HEK293 cell-attached patches. We identified two residues at which mutations perturb channel open probability, the mean open time, and the glutamate deactivation time course. We identified a subunit-specific network of aromatic amino acids located in and around the GluN2A pre-M1 helix to be important for gating. Based on these results, we are able to hypothesize about the role of the pre-M1 helix in other NMDAR subunits based on sequence and structure homology. Our results emphasize the role of the pre-M1 helix in channel gating, implicate the surrounding amino acid environment in this mechanism, and suggest unique subunit-specific contributions of pre-M1 helices to GluN1 and GluN2 gating.

scholarly journals Exploring the Release of Toxic Oligomers from α-Synuclein Fibrils with Antibodies and STED Microscopy

Life ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 431
Author(s):  
Alessandra Bigi ◽  
Emilio Ermini ◽  
Serene W. Chen ◽  
Roberta Cascella ◽  
Cristina Cecchi
Keyword(s):  
Conformational Changes ◽  
De Novo ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Phospholipid Bilayer ◽  
Sted Microscopy ◽  
Cell Dysfunction ◽  
Intrinsically Disordered ◽  
Neuronal Membranes

α-Synuclein (αS) is an intrinsically disordered and highly dynamic protein involved in dopamine release at presynaptic terminals. The abnormal aggregation of αS as mature fibrils into intraneuronal inclusion bodies is directly linked to Parkinson’s disease. Increasing experimental evidence suggests that soluble oligomers formed early during the aggregation process are the most cytotoxic forms of αS. This study investigated the uptake by neuronal cells of pathologically relevant αS oligomers and fibrils exploiting a range of conformation-sensitive antibodies, and the super-resolution stimulated emission depletion (STED) microscopy. We found that prefibrillar oligomers promptly penetrate neuronal membranes, thus resulting in cell dysfunction. By contrast, fibril docking to the phospholipid bilayer is accompanied by αS conformational changes with a progressive release of A11-reactive oligomers, which can enter into the neurons and trigger cell impairment. Our data provide important evidence on the role of αS fibrils as a source of harmful oligomers, which resemble the intermediate conformers formed de novo during aggregation, underling the dynamic and reversible nature of protein aggregates responsible for α-synucleinopathies.

scholarly journals Conformational dynamics and role of the acidic pocket in ASIC pH-dependent gating

2017 ◽  
Vol 114 (14) ◽  
pp. 3768-3773 ◽  
Author(s):  
Sabrina Vullo ◽  
Gaetano Bonifacio ◽  
Sophie Roy ◽  
Niklaus Johner ◽  
Simon Bernèche ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Conformational Dynamics ◽  
Ph Sensing ◽  
Central Channel ◽  
Ph Sensors ◽  
Pain Sensation ◽  
Acid Sensing Ion Channels ◽  
Acidic Residues ◽  
Ph Dependent

Acid-sensing ion channels (ASICs) are proton-activated Na+ channels expressed in the nervous system, where they are involved in learning, fear behaviors, neurodegeneration, and pain sensation. In this work, we study the role in pH sensing of two regions of the ectodomain enriched in acidic residues: the acidic pocket, which faces the outside of the protein and is the binding site of several animal toxins, and the palm, a central channel domain. Using voltage clamp fluorometry, we find that the acidic pocket undergoes conformational changes during both activation and desensitization. Concurrently, we find that, although proton sensing in the acidic pocket is not required for channel function, it does contribute to both activation and desensitization. Furthermore, protonation-mimicking mutations of acidic residues in the palm induce a dramatic acceleration of desensitization followed by the appearance of a sustained current. In summary, this work describes the roles of potential pH sensors in two extracellular domains, and it proposes a model of acidification-induced conformational changes occurring in the acidic pocket of ASIC1a.

Role of spectrin in membrane fusion and in shape change of human erythrocyte ghost

1978 ◽  
Vol 36 (2) ◽  
pp. 328-329
Author(s):  
Hideo Hayashi ◽  
Yoshikazu Hirai ◽  
John T. Penniston
Keyword(s):  
Conformational Changes ◽  
Human Erythrocyte ◽  
Shape Change ◽  
Membrane Surface ◽  
Slight Modification ◽  
Active Role ◽  
Main Role ◽  
Bank Blood ◽  
Human Erythrocyte Ghost

Spectrin is a membrane associated protein most of which properties have been tentatively elucidated. A main role of the protein has been assumed to give a supporting structure to inside of the membrane. As reported previously, however, the isolated spectrin molecule underwent self assemble to form such as fibrous, meshwork, dispersed or aggregated arrangements depending upon the buffer suspended and was suggested to play an active role in the membrane conformational changes. In this study, the role of spectrin and actin was examined in terms of the molecular arrangements on the erythrocyte membrane surface with correlation to the functional states of the ghosts.Human erythrocyte ghosts were prepared from either freshly drawn or stocked bank blood by the method of Dodge et al with a slight modification as described before. Anti-spectrin antibody was raised against rabbit by injection of purified spectrin and partially purified.

Posttransplant CMV infection and the role of immunosuppression (Sponsor: Novartis Pharma GmbH, Nürnberg)

2016 ◽  
Vol 04 (01) ◽  
pp. 4-10
Keyword(s):  
Lower Incidence ◽  
Paradigm Shift ◽  
Mtor Inhibitor ◽  
De Novo ◽  
Expert Panel ◽  
Risk Of Infection ◽  
Cmv Infection ◽  
Expert Meeting ◽  
Selection Of

AbstractImmunosuppression permits graft survival after transplantation and consequently a longer and better life. On the other hand, it increases the risk of infection, for instance with cytomegalovirus (CMV). However, the various available immunosuppressive therapies differ in this regard. One of the first clinical trials using de novo everolimus after kidney transplantation [1] already revealed a considerably lower incidence of CMV infection in the everolimus arms than in the mycophenolate mofetil (MMF) arm. This result was repeatedly confirmed in later studies [2–4]. Everolimus is now considered a substance with antiviral properties. This article is based on the expert meeting “Posttransplant CMV infection and the role of immunosuppression”. The expert panel called for a paradigm shift: In a CMV prevention strategy the targeted selection of the immunosuppressive therapy is also a key element. For patients with elevated risk of CMV, mTOR inhibitor-based immunosuppression is advantageous as it is associated with a significantly lower incidence of CMV events.

The Role of Hydrophobicity in the Stability and pH-Switchability of (RXDX)4 and Coumarin-RXDX Conjugate β-Sheets

2020 ◽  
Author(s):  
Ryan Weber ◽  
Martin McCullagh
Keyword(s):  
Biological Imaging ◽  
Ph Sensing ◽  
Hydrophobic Residue ◽  
Ideal Candidate ◽  
Self Assembling ◽  
Sensing Applications ◽  
Β Sheets ◽  
The Stability ◽  
Dynamics Simulations

<p>pH-switchable, self-assembling materials are of interest in biological imaging and sensing applications. Here we propose that combining the pH-switchability of RXDX (X=Ala, Val, Leu, Ile, Phe) peptides and the optical properties of coumarin creates an ideal candidate for these materials. This suggestion is tested with a thorough set of all-atom molecular dynamics simulations. We first investigate the dependence of pH-switchabiliy on the identity of the hydrophobic residue, X, in the bare (RXDX)<sub>4</sub> systems. Increasing the hydrophobicity stabilizes the fiber which, in turn, reduces the pH-switchabilty of the system. This behavior is found to be somewhat transferable to systems in which a single hydrophobic residue is replaced with a coumarin containing amino acid. In this case, conjugates with X=Ala are found to be unstable and both pHs while conjugates with X=Val, Leu, Ile and Phe are found to form stable β-sheets at least at neutral pH. The (RFDF)<sub>4</sub>-coumarin conjugate is found to have the largest relative entropy value of 0.884 +/- 0.001 between neutral and acidic coumarin ordering distributions. Thus, we posit that coumarin-(RFDF)<sub>4</sub> containing peptide sequences are ideal candidates for pH-sensing bioelectronic materials.</p>

249 ROLE OF DE NOVO LIPOGENESIS IN GROWING VERY LOW BIRTH WEIGHT BREASTFED INFANTS.

2004 ◽  
Vol 52 (Suppl 1) ◽  
pp. S122.6-S123
Author(s):  
M. Garg ◽  
C. Bell ◽  
L. Rogers ◽  
S. Bassilian ◽  
W. N.P. Lee
Keyword(s):  
Birth Weight ◽  
Low Birth Weight ◽  
Very Low Birth Weight ◽  
De Novo ◽  
De Novo Lipogenesis ◽  
Breastfed Infants

scholarly journals Correlation of membrane protein conformational and functional dynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Raghavendar Reddy Sanganna Gari ◽  
Joel José Montalvo‐Acosta ◽  
George R. Heath ◽  
Yining Jiang ◽  
Xiaolong Gao ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Conformational Changes ◽  
Single Channel ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
High Temporal Resolution ◽  
Dependent Manner ◽  
Functional Dynamics ◽  
Ph Dependent ◽  
Open States

AbstractConformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.

scholarly journals LncRNA CRNDE attenuates chemoresistance in gastric cancer via SRSF6-regulated alternative splicing of PICALM

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Feifei Zhang ◽  
Hui Wang ◽  
Jiang Yu ◽  
Xueqing Yao ◽  
Shibin Yang ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Alternative Splicing ◽  
Noncoding Rna ◽  
De Novo ◽  
Acquired Resistance ◽  
Genetic Alterations ◽  
Clinical Samples ◽  
Autophagy Flux ◽  
Resistance To Chemotherapy

AbstractDe novo and acquired resistance, which are mainly mediated by genetic alterations, are barriers to effective routine chemotherapy. However, the mechanisms underlying gastric cancer (GC) resistance to chemotherapy are still unclear. We showed that the long noncoding RNA CRNDE was related to the chemosensitivity of GC in clinical samples and a PDX model. CRNDE was decreased and inhibited autophagy flux in chemoresistant GC cells. CRNDE directly bound to splicing protein SRSF6 to reduce its protein stability and thus regulate alternative splicing (AS) events. We determined that SRSF6 regulated the PICALM exon 14 skip splice variant and triggered a significant S-to-L isoform switch, which contributed to the expression of the long isoform of PICALM (encoding PICALML). Collectively, our findings reveal the key role of CRNDE in autophagy regulation, highlighting the significance of CRNDE as a potential prognostic marker and therapeutic target against chemoresistance in GC.

scholarly journals Discovery of novel community-relevant small proteins in a simplified human intestinal microbiome

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Hannes Petruschke ◽  
Christian Schori ◽  
Sebastian Canzler ◽  
Sarah Riesbeck ◽  
Anja Poehlein ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Intestinal Microbiota ◽  
De Novo ◽  
Bacterial Species ◽  
Intestinal Microbiome ◽  
Single Strain ◽  
Small Proteins ◽  
Human Intestinal Microbiota ◽  
Wide Range

Abstract Background The intestinal microbiota plays a crucial role in protecting the host from pathogenic microbes, modulating immunity and regulating metabolic processes. We studied the simplified human intestinal microbiota (SIHUMIx) consisting of eight bacterial species with a particular focus on the discovery of novel small proteins with less than 100 amino acids (= sProteins), some of which may contribute to shape the simplified human intestinal microbiota. Although sProteins carry out a wide range of important functions, they are still often missed in genome annotations, and little is known about their structure and function in individual microbes and especially in microbial communities. Results We created a multi-species integrated proteogenomics search database (iPtgxDB) to enable a comprehensive identification of novel sProteins. Six of the eight SIHUMIx species, for which no complete genomes were available, were sequenced and de novo assembled. Several proteomics approaches including two earlier optimized sProtein enrichment strategies were applied to specifically increase the chances for novel sProtein discovery. The search of tandem mass spectrometry (MS/MS) data against the multi-species iPtgxDB enabled the identification of 31 novel sProteins, of which the expression of 30 was supported by metatranscriptomics data. Using synthetic peptides, we were able to validate the expression of 25 novel sProteins. The comparison of sProtein expression in each single strain versus a multi-species community cultivation showed that six of these sProteins were only identified in the SIHUMIx community indicating a potentially important role of sProteins in the organization of microbial communities. Two of these novel sProteins have a potential antimicrobial function. Metabolic modelling revealed that a third sProtein is located in a genomic region encoding several enzymes relevant for the community metabolism within SIHUMIx. Conclusions We outline an integrated experimental and bioinformatics workflow for the discovery of novel sProteins in a simplified intestinal model system that can be generically applied to other microbial communities. The further analysis of novel sProteins uniquely expressed in the SIHUMIx multi-species community is expected to enable new insights into the role of sProteins on the functionality of bacterial communities such as those of the human intestinal tract.

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