Real-Time Conformational Changes and Controlled Orientation of Native Proteins Inside a Protein Nanoreactor

Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS–RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.

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Real-time kinetics measurements of protein induced conformational changes in DNA

2009 IEEE LEOS Annual Meeting Conference Proceedings ◽

10.1109/leos.2009.5343144 ◽

2009 ◽

Author(s):

Philipp S. Spuhler ◽

Jelena Knezevic ◽

Ayca Yalcin ◽

Peter Droge ◽

Ulrich Rant ◽

...

Keyword(s):

Real Time ◽

Conformational Changes

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Real-time visualization of conformational changes within single MloK1 cyclic nucleotide-modulated channels

Nature Communications ◽

10.1038/ncomms12789 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 19

Author(s):

Martina Rangl ◽

Atsushi Miyagi ◽

Julia Kowal ◽

Henning Stahlberg ◽

Crina M. Nimigean ◽

...

Keyword(s):

Real Time ◽

Conformational Changes ◽

Cyclic Nucleotide ◽

High Speed ◽

Function Analysis ◽

Stable Conformation ◽

Mesorhizobium Loti ◽

Force Microscopy ◽

Atomic Force ◽

Real Time Visualization

AbstractEukaryotic cyclic nucleotide-modulated (CNM) ion channels perform various physiological roles by opening in response to cyclic nucleotides binding to a specialized cyclic nucleotide-binding domain. Despite progress in structure-function analysis, the conformational rearrangements underlying the gating of these channels are still unknown. Here, we image ligand-induced conformational changes in single CNM channels from Mesorhizobium loti (MloK1) in real-time, using high-speed atomic force microscopy. In the presence of cAMP, most channels are in a stable conformation, but a few molecules dynamically switch back and forth (blink) between at least two conformations with different heights. Upon cAMP depletion, more channels start blinking, with blinking heights increasing over time, suggestive of slow, progressive loss of ligands from the tetramer. We propose that during gating, MloK1 transitions from a set of mobile conformations in the absence to a stable conformation in the presence of ligand and that these conformations are central for gating the pore.

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Monitoring Conformational Changes During Agonist Release from a GPCR in Real Time: Transmembrane Helix 6 Resets as All-Trans Retinal is Released from Rhodopsin

Biophysical Journal ◽

10.1016/j.bpj.2013.11.636 ◽

2014 ◽

Vol 106 (2) ◽

pp. 102a

Author(s):

Chrisopher T. Schafer ◽

Jay M. Janz ◽

David L. Farrens

Keyword(s):

Real Time ◽

Conformational Changes ◽

Transmembrane Helix

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Voltage clamp fluorimetry studies of mammalian voltage-gated K+ channel gating

Biochemical Society Transactions ◽

10.1042/bst0351080 ◽

2007 ◽

Vol 35 (5) ◽

pp. 1080-1082 ◽

Cited By ~ 14

Author(s):

T.W. Claydon ◽

D. Fedida

Keyword(s):

Ion Channel ◽

Potassium Channel ◽

Real Time ◽

Voltage Clamp ◽

Conformational Changes ◽

Channel Gating ◽

K Channel ◽

Voltage Gated ◽

Kv Channels ◽

Health And Disease

VCF (voltage clamp fluorimetry) provides a powerful technique to observe real-time conformational changes that are associated with ion channel gating. The present review highlights the insights such experiments have provided in understanding Kv (voltage-gated potassium) channel gating, with particular emphasis on the study of mammalian Kv1 channels. Further applications of VCF that would contribute to our understanding of the modulation of Kv channels in health and disease are also discussed.

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Unlocking the secrets of mutable collagenous tissue

The Biochemist ◽

10.1042/bio04001008 ◽

2018 ◽

Vol 40 (1) ◽

pp. 8-11

Author(s):

Himadri S. Gupta ◽

Greg Szulgit ◽

Maurice R. Elphick ◽

Jingyi Mo

Keyword(s):

Real Time ◽

Conformational Changes ◽

Molecular Level ◽

Sea Urchins ◽

Collagen Fibrils ◽

Sea Cucumbers ◽

X Ray ◽

Collagenous Tissue ◽

Key Factor ◽

Shed Light

The mutable collagenous tissue (MCT) of echinoderms (e.g. sea cucumbers, starfish and sea urchins) is unique because of its ability to ‘switch’ mechanical states rapidly and reversibly – from stiff to soft and vice versa. This kind of tissue in humans, for example, in skin, tendons and ligaments, does not have this property. So what are the molecular-level secrets by which MCT achieves this transformative ability? New real-time ultrastructural investigations are beginning to shed light on this question. Synchrotron X-ray measurements of dynamic molecular conformational changes point to the key factor being the gel-like matrix between the collagen fibrils. These findings could have applications for developing treatments for collagen-based disorders.

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The Improvement of Nanoemulsion Stability and Antioxidation via Protein-Chlorogenic Acid-Dextran Conjugates as Emulsifiers

Nanomaterials ◽

10.3390/nano10061094 ◽

2020 ◽

Vol 10 (6) ◽

pp. 1094 ◽

Cited By ~ 2

Author(s):

Chang Liu ◽

Hua Jin ◽

Yue Yu ◽

Jingying Sun ◽

Huanyu Zheng ◽

...

Keyword(s):

Chlorogenic Acid ◽

Conformational Changes ◽

Soybean Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Protein Isolate ◽

Soybean Protein Isolate ◽

Molecular Weights ◽

Native Proteins ◽

Fluorescence Measurements

In this experiment, the peanut protein isolate (PPI), soybean protein isolate (SPI), rice bran protein isolate (RBPI), and whey protein isolate (WPI) were modified by linking chlorogenic acid covalently and linking dextran by Maillard reaction to prepare protein-chlorogenic acid-dextran (PCD) conjugates. As for structures, conformational changes of conjugates were determined by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Fourier transform infrared (FT-IR), and fluorescence measurements. The molecular weights of PCD conjugates became larger, the structure became disorder, and the amino acid residues inside the protein were exposed to the polar environment when compared to protein-chlorogenic acid (PC) and native proteins (NPs). As for properties, the interfacial tension reduced and antioxidant activity of PCD conjugates enhanced in varying degrees. Based on this, PCD conjugates were used as emulsifiers in order to investigate the properties of nanoemulsions and compared with PC conjugates and NPs. The mean droplet diameters (MDD) results showed that the nanoemulsions that were stabilized by PCD conjugates had the smallest particle sizes and exhibited uniformly dispersed spherical shapes. The storage and oxidative stabilities of PCD conjugates were also significantly improved. In comparison, nanoemulsion that was stabilized by PPI-chlorogenic acid-dextran conjugate had the smallest particle size and optimal stability among four protein stabilized nanoemulsions.

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Native proteins trap high-energy transit conformations

Science Advances ◽

10.1126/sciadv.1501188 ◽

2015 ◽

Vol 1 (9) ◽

pp. e1501188 ◽

Cited By ~ 9

Author(s):

Andrew E. Brereton ◽

P. Andrew Karplus

Keyword(s):

Protein Folding ◽

Conformational Changes ◽

Protein Function ◽

Protein Structures ◽

High Energy ◽

Native Proteins ◽

Regulate Protein ◽

The Right ◽

And Function ◽

Open Question

During protein folding and as part of some conformational changes that regulate protein function, the polypeptide chain must traverse high-energy barriers that separate the commonly adopted low-energy conformations. How distortions in peptide geometry allow these barrier-crossing transitions is a fundamental open question. One such important transition involves the movement of a non-glycine residue between the left side of the Ramachandran plot (that is, ϕ < 0°) and the right side (that is, ϕ > 0°). We report that high-energy conformations with ϕ ~ 0°, normally expected to occur only as fleeting transition states, are stably trapped in certain highly resolved native protein structures and that an analysis of these residues provides a detailed, experimentally derived map of the bond angle distortions taking place along the transition path. This unanticipated information lays to rest any uncertainty about whether such transitions are possible and how they occur, and in doing so lays a firm foundation for theoretical studies to better understand the transitions between basins that have been little studied but are integrally involved in protein folding and function. Also, the context of one such residue shows that even a designed highly stable protein can harbor substantial unfavorable interactions.

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