scholarly journals NMR pseudocontact shifts in a symmetric protein homotrimer

2020 ◽  
Vol 74 (8-9) ◽  
pp. 413-419
Author(s):  
Thomas Müntener ◽  
Raphael Böhm ◽  
Kenneth Atz ◽  
Daniel Häussinger ◽  
Sebastian Hiller
Keyword(s):  
Protein Complexes ◽  
Single Point ◽  
Paramagnetic Nmr ◽  
Pseudocontact Shift ◽  
Point Model ◽  
Pseudocontact Shifts ◽  
Functional Roles ◽  
Functional Studies ◽  
Predicted Values ◽  
Symmetric Systems

Abstract NMR pseudocontact shifts are a valuable tool for structural and functional studies of proteins. Protein multimers mediate key functional roles in biology, but methods for their study by pseudocontact shifts are so far not available. Paramagnetic tags attached to identical subunits in multimeric proteins cause a combined pseudocontact shift that cannot be described by the standard single-point model. Here, we report pseudocontact shifts generated simultaneously by three paramagnetic Tm-M7PyThiazole-DOTA tags to the trimeric molecular chaperone Skp and provide an approach for the analysis of this and related symmetric systems. The pseudocontact shifts were described by a “three-point” model, in which positions and parameters of the three paramagnetic tags were fitted. A good correlation between experimental data and predicted values was found, validating the approach. The study establishes that pseudocontact shifts can readily be applied to multimeric proteins, offering new perspectives for studies of large protein complexes by paramagnetic NMR spectroscopy.

scholarly journals Multiple conformations facilitate PilT function in the type IV pilus

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Matthew McCallum ◽  
Samir Benlekbir ◽  
Sheryl Nguyen ◽  
Stephanie Tammam ◽  
John L. Rubinstein ◽  
...  
Keyword(s):  
Single Particle ◽  
Protein Complexes ◽  
Comprehensive Model ◽  
Type Iv Pilus ◽  
Electron Cryomicroscopy ◽  
X Ray ◽  
Functional Studies ◽  
Type Iv ◽  
Coevolution Analysis ◽  
Multiple Conformations

AbstractType IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C2 symmetry; however, most of these ATPases crystallize with either C3 or C6 symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C2,C3, and C6 conformations. The physiologic importance of these conformations is validated by coevolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data, we propose a comprehensive model of PilT function with broad implications for PilT/VirB11-like family members.

scholarly journals Structural and functional insights into transmembrane AMPA receptor regulatory protein complexes

2019 ◽  
Vol 151 (12) ◽  
pp. 1347-1356 ◽  
Author(s):  
Edward C. Twomey ◽  
Maria V. Yelshanskaya ◽  
Alexander I. Sobolevsky
Keyword(s):  
Protein Complexes ◽  
Regulatory Protein ◽  
Regulatory Proteins ◽  
Specific Gene ◽  
Modular Architecture ◽  
Auxiliary Subunits ◽  
Functional Studies ◽  
Excitatory Neurotransmission ◽  
Synaptic Complexes ◽  
The Brain

Fast excitatory neurotransmission is mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptor (AMPAR). AMPARs initiate depolarization of the postsynaptic neuron by allowing cations to enter through their ion channel pores in response to binding of the neurotransmitter glutamate. AMPAR function is dramatically affected by auxiliary subunits, which are regulatory proteins that form various complexes with AMPARs throughout the brain. The most well-studied auxiliary subunits are the transmembrane AMPAR regulatory proteins (TARPs), which alter the assembly, trafficking, localization, kinetics, and pharmacology of AMPARs. Recent structural and functional studies of TARPs and the TARP-fold germ cell-specific gene 1-like (GSG1L) subunit have provided important glimpses into how auxiliary subunits regulate the function of synaptic complexes. In this review, we put these recent structures in the context of new functional findings in order to gain insight into the determinants of AMPAR regulation by TARPs. We thus reveal why TARPs display a broad range of effects despite their conserved modular architecture.

Single-point model of MIMO-UWB indoor systems using time-reversal transmission

2017 ◽  
Author(s):  
Eleni Bogdani ◽  
Demosthenes Vouyioukas ◽  
Nikolaos Nomikos ◽  
Dimitrios N. Skoutas ◽  
Charalambos Skianis
Keyword(s):  
Time Reversal ◽  
Single Point ◽  
Point Model

scholarly journals Identification of productive and futile encounters in an electron transfer protein complex

2017 ◽  
Vol 114 (10) ◽  
pp. E1840-E1847 ◽  
Author(s):  
Witold Andrałojć ◽  
Yoshitaka Hiruma ◽  
Wei-Min Liu ◽  
Enrico Ravera ◽  
Masaki Nojiri ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Paramagnetic Nmr ◽  
Conformational Ensemble ◽  
X Ray Diffraction ◽  
Cytochrome P450cam ◽  
Transfer Protein ◽  
Nmr Data ◽  
Electron Transfer Protein ◽  
Potential Map ◽  
Positively Charged

Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble of more dynamic orientations: the encounter states. The structure of the stereospecific complex between cytochrome P450cam and putidaredoxin was solved recently by X-ray diffraction as well as paramagnetic NMR spectroscopy. Other than the stereospecific complex, the NMR data clearly show the presence of additional states in the complex in solution. In these encounter states, populated for a small percentage of the time, putidaredoxin assumes multiple orientations and samples a large part of the surface of cytochrome P450cam. To characterize the nature of the encounter states, an extensive paramagnetic NMR dataset has been analyzed using the Maximum Occurrence of Regions methodology. The analysis reveals the location and maximal spatial extent of the additional states needed to fully explain the NMR data. Under the assumption of sparsity of the size of the conformational ensemble, several minor states can be located quite precisely. The distribution of these minor states correlates with the electrostatic potential map around cytochrome P450cam. Whereas some minor states are on isolated positively charged patches, others are connected to the stereospecific site via positively charged paths. The existence of electrostatically favorable pathways between the stereospecific interaction site and the different minor states or lack thereof suggests a means to discriminate between productive and futile encounter states.

Solution structure of the Taf14 YEATS domain and its roles in cell growth of Saccharomyces cerevisiae

2011 ◽  
Vol 436 (1) ◽  
pp. 83-90 ◽  
Author(s):  
Wen Zhang ◽  
Jiahai Zhang ◽  
Xuecheng Zhang ◽  
Chao Xu ◽  
Xiaoming Tu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Transcriptional Activation ◽  
Solution Structure ◽  
Protein Complexes ◽  
Domain Family ◽  
Functional Studies ◽  
C Terminus ◽  
Eukaryotic Species ◽  
Negative Role

Chromatin modifications play important roles in cellular biological process. A novel conserved domain family, YEATS, has been discovered in a variety of eukaryotic species ranging from yeasts to humans. Taf14, which is involved in a few protein complexes of chromatin remodelling and gene transcription, and is essential for keeping chromosome stability, regular cell growth and transcriptional regulation, contains a YEATS domain at its N-terminus. In the present study, we determined the solution structure of the Taf14 YEATS domain using NMR spectroscopy. The Taf14 YEATS domain adopts a global fold of an elongated β-sandwich, similar to the Yaf9 YEATS domain. However, the Taf14 YEATS domain differs significantly from the Yaf9 YEATS domain in some aspects, which might indicate different structural classes of the YEATS domain family. Functional studies indicate that the YEATS domain is critical for the function of Taf14 in inhibiting cell growth under stress conditions. In addition, our results show that the C-terminus of Taf14 is responsible for its interaction with Sth1, which is an essential component of the RSC complex. Taken together, this implies that Taf14 is involved in transcriptional activation of Saccharomyces cerevisiae and the YEATS domain of Taf14 might play a negative role in cell growth.

A Multi-Point Tire Model for Studying Bridge–Vehicle Coupled Vibration

2016 ◽  
Vol 16 (08) ◽  
pp. 1550047 ◽  
Author(s):  
Lu Deng ◽  
Ran Cao ◽  
Wei Wang ◽  
Xinfeng Yin
Keyword(s):  
Numerical Simulation ◽  
Single Point ◽  
Point Contact ◽  
Road Surface ◽  
Dynamic Responses ◽  
Tire Model ◽  
Point Model ◽  
Disk Model ◽  
The Road ◽  
Dynamic Amplification

The contact between a vehicle tire and the road surface has been usually assumed as a single-point contact in the numerical simulation of vehicle–bridge interacted vibrations. In reality, the tire contacts the road surface through a patch instead of a single point. According to some recent studies, the single-point tire model may overestimate the dynamic amplification of bridge responses due to vehicle loadings. A new tire model, namely, the multi-point tire model, is therefore proposed in this paper with the purpose of improving the accuracy of numerical simulation results over the single-point model, while maintaining a certain level of simplicity for applications. A series of numerical simulations are carried out to compare the effect of the proposed tire model with those of the existing single-point model and disk model on the bridge dynamic responses. The proposed tire model is also verified against the field test results. The results show that the proposed multi-point tire model can predict the bridge dynamic responses with better accuracy than the single-point model, especially under distressed bridge deck conditions, and is computationally more efficient and simpler for application than the disk model.

scholarly journals Synthesis of Multi-Protein Complexes through Charge-Directed Sequential Activation of Tyrosine Residues

2021 ◽  
Author(s):  
Casey S. Mogilevsky ◽  
Marco Lobba ◽  
Daniel D. Brauer ◽  
Alan Marmelstein ◽  
Johnathan Maza ◽  
...  
Keyword(s):  
Protein Design ◽  
Protein Complexes ◽  
Single Point ◽  
Tyrosine Residues ◽  
Starting Point ◽  
Sequential Activation ◽  
Substrate Tolerance ◽  
A Charge ◽  
Site Selective ◽  
Biology Research

Site-selective protein-protein coupling has long been a goal of chemical biology research. In recent years, that goal has been realized to varying degrees through a number of techniques, including the use of tyrosinase-based coupling strategies. Early publications utilizing tyrosinase from <i>Agaricus bisporus</i> showed the potential to convert tyrosine residues into <i>ortho</i>-quinone functional groups, but this enzyme is challenging to produce recombinantly and suffers from some limitations in substrate scope. Initial screens of several tyrosinase candidates revealed that the tyrosinase from <i>Bacillus megaterium</i> (megaTYR) as an enzyme that possesses a broad substrate tolerance. We use the expanded substrate preference as a starting point for protein design experiments and show that single point mutants of megaTYR are capable of activating tyrosine residues in various sequence contexts. We leverage this new tool to enable the construction of protein trimers via a charge-directed sequential activation of tyrosine residues (CDSAT).

scholarly journals Identification of recombinant Fabs for structural and functional characterization of HIV-host factor complexes

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0250318
Author(s):  
Natalia Sevillano ◽  
Evan M. Green ◽  
Jörg Votteler ◽  
Dong Young Kim ◽  
Xuefeng Ren ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Functional Characterization ◽  
Phage Display Library ◽  
Host Factor ◽  
Host Protein ◽  
Rapid Screening ◽  
Functional Studies ◽  
Negative Stain ◽  
Immunization Strategies

Viral infection and pathogenesis is mediated by host protein—viral protein complexes that are important targets for therapeutic intervention as they are potentially less prone to development of drug resistance. We have identified human, recombinant antibodies (Fabs) from a phage display library that bind to three HIV-host complexes. We used these Fabs to 1) stabilize the complexes for structural studies; and 2) facilitate characterization of the function of these complexes. Specifically, we generated recombinant Fabs to Vif-CBF-β-ELOB-ELOC (VCBC); ESCRT-I complex and AP2-complex. For each complex we measured binding affinities with KD values of Fabs ranging from 12–419 nM and performed negative stain electron microscopy (nsEM) to obtain low-resolution structures of the HIV-Fab complexes. Select Fabs were converted to scFvs to allow them to fold intracellularly and perturb HIV-host protein complex assembly without affecting other pathways. To identify these recombinant Fabs, we developed a rapid screening pipeline that uses quantitative ELISAs and nsEM to establish whether the Fabs have overlapping or independent epitopes. This pipeline approach is generally applicable to other particularly challenging antigens that are refractory to immunization strategies for antibody generation including multi-protein complexes providing specific, reproducible, and renewable antibody reagents for research and clinical applications. The curated antibodies described here are available to the scientific community for further structural and functional studies on these critical HIV host-factor proteins.

scholarly journals New approach for membrane protein reconstitution into peptidiscs and basis for their adaptability to different proteins

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Gabriella Angiulli ◽  
Harveer Singh Dhupar ◽  
Hiroshi Suzuki ◽  
Irvinder Singh Wason ◽  
Franck Duong Van Hoa ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Structural Basis ◽  
New Approach ◽  
Functional Studies ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Membrane Protein Complexes ◽  
Membrane Protein Reconstitution

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present ‘on-gradient’ reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional ‘on-bead’ approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

scholarly journals Differential Effects of Modified Batrachotoxins on Voltage-gated Sodium Channel Fast and Slow Inactivation

2021 ◽  
Author(s):  
Tim M. G. MacKenzie ◽  
Fayal Abderemane-Ali ◽  
Catherine E. Garrison ◽  
Daniel L. Minor Jr. ◽  
Justin Du Bois
Keyword(s):  
De Novo ◽  
Protein Complexes ◽  
Transmembrane Protein ◽  
Ion Selectivity ◽  
Allosteric Modulators ◽  
Slow Inactivation ◽  
Voltage Gated ◽  
Functional Studies ◽  
Voltage Gated Sodium Channels ◽  
Initiation And Propagation

Voltage-gated sodium channels (Na<sub>V</sub>s), large transmembrane protein complexes responsible for the initiation and propagation of action potentials, are targets for a number of acute poisons. Many of these agents act as allosteric modulators of channel activity and serve as powerful chemical tools for understanding channel function. Batrachotoxin (BTX) is a steroidal amine derivative most commonly associated with poison dart frogs and is unique as a Na<sub>V</sub> ligand in that it alters every property of the channel, including threshold potential of activation, inactivation, ion selectivity, and ion conduction. Structure-function studies with BTX are limited, however, by the inability to access preparative quantities of this compound from natural sources. We have addressed this problem through <i>de novo</i> synthesis of BTX, which gives access to modified toxin structures. In this report, we detail electrophysiology studies of three BTX C20-ester derivatives against recombinant Na<sub>V</sub> subtypes (rat Na<sub>V</sub>1.4 and human Na<sub>V</sub>1.5). Two of these compounds, BTX-B and BTX-<sup>c</sup>Hx, are functionally equivalent to BTX, hyperpolarizing channel activation and blocking both fast and slow inactivation. BTX-yne—a C20-<i>n</i>-heptynoate ester—is a conspicuous outlier, eliminating fast but not slow inactivation. This unique property qualifies BTX-yne as the first reported Na<sub>V</sub> modulator that separates inactivation processes. These findings are supported by functional studies with bacterial Na<sub>V</sub>s (BacNa<sub>V</sub>s) that lack a fast inactivation gate. The availability of BTX-yne should advance future efforts aimed at understanding Na<sub>V</sub> gating mechanisms and designing allosteric regulators of Na<sub>V</sub> activity.

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