scholarly journals Designed folding pathway of modular coiled-coil-based proteins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jana Aupič ◽  
Žiga Strmšek ◽  
Fabio Lapenta ◽  
David Pahovnik ◽  
Tomaž Pisanski ◽  
...  
Keyword(s):  
Protein Structures ◽  
Coiled Coil ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Folding Pathway ◽  
Single Polypeptide Chain ◽  
Long Range Interactions ◽  
Tandem Repeat Proteins ◽  
Single Polypeptide ◽  
Dynamics Simulations

AbstractNatural proteins are characterised by a complex folding pathway defined uniquely for each fold. Designed coiled-coil protein origami (CCPO) cages are distinct from natural compact proteins, since their fold is prescribed by discrete long-range interactions between orthogonal pairwise-interacting coiled-coil (CC) modules within a single polypeptide chain. Here, we demonstrate that CCPO proteins fold in a stepwise sequential pathway. Molecular dynamics simulations and stopped-flow Förster resonance energy transfer (FRET) measurements reveal that CCPO folding is dominated by the effective intra-chain distance between CC modules in the primary sequence and subsequent folding intermediates, allowing identical CC modules to be employed for multiple cage edges and thus relaxing CCPO cage design requirements. The number of orthogonal modules required for constructing a CCPO tetrahedron can be reduced from six to as little as three different CC modules. The stepwise modular nature of the folding pathway offers insights into the folding of tandem repeat proteins and can be exploited for the design of modular protein structures based on a given set of orthogonal modules.

scholarly journals Design: An assay based on single-polypeptide-chain heterodimeric A2AR/D2R and non-oligomerized fusions for in vivo analysis of their allosteric receptor-receptor interactions

2016 ◽  
Author(s):  
Toshio Kamiya ◽  
Takashi Masuko ◽  
Dasiel Oscar Borroto-Escuela ◽  
Haruo Okado ◽  
Hiroyasu Nakata
Keyword(s):  
Energy Transfer ◽  
Polypeptide Chain ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
R And D ◽  
Antenna Complex ◽  
Single Polypeptide Chain ◽  
Single Polypeptide ◽  
Light Harvesting Antenna

AbstractBackgroundThe adenosine A2A receptor (A2AR) heteromerizes with the dopamine D2 receptor (D2R). In order to explore their functional interaction, we engineered previously stable single-polypeptide-chain (sc) A2AR/D2LR: whether the molecular entity of the striatal A2AR/D2R antagonism, i.e., scA2AR/D2Rs are just A2AR/D2R with the antagonism, remains unresolved.New MethodTo further clarify the heteromerization through the scA2AR/D2LR, we here designed supramolecularly ‘exclusive’ monomers and dimers, using the Cε2 domain of IgE-Fc or apoproteins of the bacterial light-harvesting antenna complex.ResultsA concept of the recptor protein assembly regulation, i.e., the selective monomer/non-obligate dimer formation was obtained. Although none of these new fusions were constructed or tested, we could aim at obtaining heterodimer-specific agents, using the scA2AR/D2R. Whether the resulting designs were explained feasibly and rationally was addressed. The structure and function of the non-obligate dimer were here discussed through scA2AR/D2R, focusing on the procedure of the membrane protein design and methods for transient protein-protein interactions.Summary and OutlookGiven that upon being expressed and allosteric regulation occurs regardless of specific signal to non-specific noise (S/N) ratio, the supramolecular designs, allowing us to express selectively monomer/non-obligate dimer of class A GPCR, are experimentally testable and will be used to confirm in vivo that such low S/N ratio interaction between A2AR and D2LR functions in the dopamine neurotransmission in the striatum.Abbreviations:A2ARadenosine A2A receptor3HA-A2ARA2AR tagged with a triple HA epitopeBchlbacteriochlorophyllBRETbioluminescence resonance energy transferCcarboxy-terminalCDcluster of differentiationD2LR and D2SRthe long and short form of dopamine D2 receptor, respectivelyFabantigen binding fragmentFcFc fragmentFcεRIhigh affinity receptor for IgEFRETfluorescence resonance energy transferG4San amino acid sequence consisting of a four-glycine-repeat followed by a serine residueGABAγ-aminobutyric acidGABABGABA type B receptorGPCRG protein-coupled receptorGttransducinHAhemagglutininHIVhuman immunodeficiency virusICintracellular loopsIgimmunoglobulinLHlight-harvesting antenna complexmAbmonoclonal antibodyMrmolecular weightNamino-terminalPDParkinson’s diseasePSphotosystemRCreaction centerRlucRenilla luciferasescsingle-chainTMtransmembrane3Dthree-dimensional

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.

scholarly journals Rational design of protein-specific folding modifiers

2020 ◽  
Author(s):  
Anirban Das ◽  
Anju Yadav ◽  
Mona Gupta ◽  
R Purushotham ◽  
Vishram L. Terse ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rational Design ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Force Measurements ◽  
Folding Nucleus ◽  
Dynamics Simulations ◽  
General Method

AbstractProtein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

scholarly journals An improved open-channel structure of MscL determined from FRET confocal microscopy and simulation

2010 ◽  
Vol 136 (4) ◽  
pp. 483-494 ◽  
Author(s):  
Ben Corry ◽  
Annette C. Hurst ◽  
Prithwish Pal ◽  
Takeshi Nomura ◽  
Paul Rigby ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Conformational Changes ◽  
Brownian Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Channel Structure ◽  
Paramagnetic Resonance ◽  
Physiological Processes ◽  
Lipid Environment ◽  
Dynamics Simulations

Mechanosensitive channels act as molecular transducers of mechanical force exerted on the membrane of living cells by opening in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing, blood pressure regulation, and osmoregulation. Here, we determine the likely structure of the open state of the mechanosensitive channel of large conductance using a combination of patch clamp, fluorescence resonance energy transfer (FRET) spectroscopy, data from previous electron paramagnetic resonance experiments, and molecular and Brownian dynamics simulations. We show that structural rearrangements of the protein can be measured in similar conditions as patch clamp recordings while controlling the state of the pore in its natural lipid environment by modifying the lateral pressure distribution via the lipid bilayer. Transition to the open state is less dramatic than previously proposed, while the N terminus remains anchored at the surface of the membrane where it can either guide the tilt of or directly translate membrane tension to the conformation of the pore-lining helix. Combining FRET data obtained in physiological conditions with simulations is likely to be of great value for studying conformational changes in a range of multimeric membrane proteins.

scholarly journals Protection against Fatal Sindbis Virus Encephalitis by Beclin, a Novel Bcl-2-Interacting Protein

1998 ◽  
Vol 72 (11) ◽  
pp. 8586-8596 ◽  
Author(s):  
Xiao Huan Liang ◽  
Linda K. Kleeman ◽  
Hui Hui Jiang ◽  
Gerald Gordon ◽  
James E. Goldman ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Sindbis Virus ◽  
Stop Codon ◽  
Coiled Coil ◽  
Resonance Energy Transfer ◽  
Premature Stop Codon ◽  
Resonance Energy ◽  
Cellular Protein ◽  
Interacting Protein ◽  
Induced Apoptosis

ABSTRACT bcl-2, the prototypic cellular antiapoptotic gene, decreases Sindbis virus replication and Sindbis virus-induced apoptosis in mouse brains, resulting in protection against lethal encephalitis. To investigate potential mechanisms by which Bcl-2 protects against central nervous system Sindbis virus infection, we performed a yeast two-hybrid screen to identify Bcl-2-interacting gene products in an adult mouse brain library. We identified a novel 60-kDa coiled-coil protein, Beclin, which we confirmed interacts with Bcl-2 in mammalian cells, using fluorescence resonance energy transfer microscopy. To examine the role of Beclin in Sindbis virus pathogenesis, we constructed recombinant Sindbis virus chimeras that express full-length human Beclin (SIN/beclin), Beclin lacking the putative Bcl-2-binding domain (SIN/beclinΔBcl-2BD), or Beclin containing a premature stop codon near the 5′ terminus (SIN/beclinstop). The survival of mice infected with SIN/beclin was significantly higher (71%) than the survival of mice infected with SIN/beclinΔBcl-2BD (9%) or SIN/beclinstop (7%) (P < 0.001). The brains of mice infected with SIN/beclin had fewer Sindbis virus RNA-positive cells, fewer apoptotic cells, and lower viral titers than the brains of mice infected with SIN/beclinΔBcl-2BD or SIN/beclinstop. These findings demonstrate that Beclin is a novel Bcl-2-interacting cellular protein that may play a role in antiviral host defense.

scholarly journals CHARMM-DYES: Parameterization of Fluorescent Dyes for Use with the CHARMM Forcefield

2020 ◽  
Author(s):  
Robert Shaw ◽  
Tristan Johnston-Wood ◽  
Benjamin Ambrose ◽  
Timothy Craggs ◽  
Grant Hill
Keyword(s):  
Quantum Chemical ◽  
Spectroscopic Data ◽  
Fluorescent Dyes ◽  
Dipole Moments ◽  
Complex Structure ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Spectroscopic Properties ◽  
Chemical Calculation ◽  
Dynamics Simulations

<div><div><div><p>We present CHARMM-compatible forcefield parameters for a series of fluorescent dyes from the Alexa, Atto and Cy families, commonly used in F¨orster resonance energy transfer (FRET) experiments. These dyes are routinely used in experiments to resolve the dynamics of proteins and nucleic acids at the nanoscale. However, little is known about the accuracy of the theoretical approximations used in determining the dynamics from the spectroscopic data. Molecular dynamics simulations can provide valuable insights into these dynamics at an atomistic level, but this requires accurate parameters for the dyes. The complex structure of the dyes, and the importance of this in determining their spectroscopic properties, means that parameters generated by analogy to existing parameters do not give meaningful results. Through validation relative to quantum chemical calculation and experiment, the new parameters are shown to significantly outperform those that can be generated automatically, giving better agreement in both the charge distributions and structural properties. These improvements, in particular with regards to orientation of the dipole moments on the dyes, are vital for accurate simulation of FRET processes.</p></div></div></div>

scholarly journals Cohesin-dockerin code in cellulosomal dual binding modes and its allosteric regulation by proline isomerization

2019 ◽  
Author(s):  
Andrés Manuel Vera ◽  
Albert Galera-Prat ◽  
Michał Wojciechowski ◽  
Bartosz Różycki ◽  
Douglas Vinson Laurents ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Modes ◽  
Prolyl Isomerase ◽  
Proline Isomerization ◽  
Allosteric Control ◽  
Dynamics Simulations ◽  
Enzyme Complexes

AbstractCellulose is the most abundant organic molecule on Earth and represents a renewable and practically everlasting feedstock for the production of biofuels and chemicals. Self-assembled owing to the high-affinity cohesin-dockerin interaction, cellulosomes are huge multi-enzyme complexes with unmatched efficiency in the degradation of recalcitrant lignocellulosic substrates. The recruitment of diverse dockerin-borne enzymes into a multicohesin protein scaffold dictates the three-dimensional layout of the complex, and interestingly two alternative binding modes have been proposed. Using single-molecule Fluorescence Resonance Energy Transfer, molecular dynamics simulations and NMR measurements on a range of cohesin-dockerin pairs, we directly detect varying distributions between these binding modes that follow a built-in cohesin-dockerin code. Surprisingly, we uncover a prolyl isomerase-modulated allosteric control mechanism, mediated by the isomerization state of a single proline residue, which regulates the distribution and kinetics of binding modes. Overall, our data provide a novel mechanistic understanding of the structural plasticity and dynamics of cellulosomes.

scholarly journals Nanometer-accuracy distance measurements between fluorophores at the single-molecule level

2019 ◽  
Vol 116 (10) ◽  
pp. 4275-4284 ◽  
Author(s):  
Stefan Niekamp ◽  
Jongmin Sung ◽  
Walter Huynh ◽  
Gira Bhabha ◽  
Ronald D. Vale ◽  
...  
Keyword(s):  
Single Molecule ◽  
Open Source Software ◽  
Single Molecules ◽  
Coiled Coil ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Distance Measurements ◽  
Single Molecule Level ◽  
Wide Range ◽  
Different Color

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil “stalk” of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

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