low-FRET State Research Articles

Pore-forming transmembrane domains control ion selectivity and selectivity filter conformation in the KirBac1.1 potassium channel.

Potassium (K+) channels are membrane proteins with the remarkable ability to very selectively conduct K+ ions across the membrane. High-resolution structures have revealed that dehydrated K+ ions permeate through the narrowest region of the pore, formed by the backbone carbonyls of the signature selectivity filter (SF) sequence TxGYG. However, the existence of nonselective channels with similar SF sequences, as well as effects of mutations in other regions on selectivity, suggest that the SF is not the sole determinant of selectivity. We changed the selectivity of the KirBac1.1 channel by introducing mutations at residue I131 in transmembrane helix 2 (TM2). These mutations increase Na+ flux in the absence of K+ and introduce significant proton conductance. Consistent with K+ channel crystal structures, single-molecule FRET experiments show that the SF is conformationally constrained and stable in high-K+ conditions but undergoes transitions to dilated low-FRET states in high-Na+/low-K+ conditions. Relative to wild-type channels, I131M mutants exhibit marked shifts in the K+ and Na+ dependence of SF dynamics to higher K+ and lower Na+ concentrations. These results illuminate the role of I131, and potentially other structural elements outside the SF, in controlling ion selectivity, by suggesting that the physical interaction of these elements with the SF contributes to the relative stability of the constrained K+-induced SF configuration versus nonselective dilated conformations.

Conformational Dynamics of Nonenveloped Circovirus Capsid to the Host Cell Receptor.

SummaryCircovirus, comprising one capsid protein, is the smallest nonenveloped virus and induces lymphopenia. Circovirus can be used to explore the cell adhesion mechanism of nonenveloped viruses. We developed a single-molecule fluorescence resonance energy transfer (smFRET) assay to directly visualize the capsid's conformational feature. The capsid underwent reversible dynamic transformation between three conformations. The cell surface receptor heparan sulfate (HS) altered the dynamic equilibrium of the capsid to the high-FRET state, revealing the HS-binding region. Neutralizing antibodies restricted capsid transition to a low-FRET state, masking the HS-binding domain. The lack of positively charged amino acids in the HS-binding site reduced cell surface affinity and attenuated virus infectivity via conformational changes. These intrinsic characteristics of the capsid suggested that conformational dynamics is critical for the structural changes occurring upon cell surface receptor binding, supporting a dynamics-based mechanism of receptor binding.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

SummaryLanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

Conformational Selection and Submillisecond Dynamics of the Ligand-binding Domain of the N-Methyl-d-aspartate Receptor

The N-methyl-d-aspartate (NMDA) receptors are heteromeric non-selective cation channels that require the binding of glycine and glutamate for gating. Based on crystal structures, the mechanism of partial agonism at the glycine-binding site is thought to be mediated by a shift in the conformational equilibrium between an open clamshell and a closed clamshell-like structure of the bilobed ligand-binding domain (LBD). Using single-molecule Förster resonance energy transfer (smFRET) and multiparameter fluorescence detection, which allows us to study the conformational states and dynamics in the submillisecond time scale, we show that there are at least three conformational states explored by the LBD: the low FRET, medium FRET, and high FRET states. The distance of the medium and low FRET states corresponds to what has been observed in crystallography structures. We show that the high FRET state, which would represent a more closed clamshell conformation than that observed in the crystal structure, is most likely the state initiating activation, as evidenced by the fact that the fraction of the protein in this state correlates well with the extent of activation. Furthermore, full agonist bound LBDs show faster dynamic motions between the medium and high FRET states, whereas they show slower dynamics when bound to weaker agonists or to antagonists.

Single-Molecule FRET Reveals Hidden Complexity in a Protein Energy Landscape

SummaryHere, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated.

Single-Molecule Studies of the ER Calcium Sensor STIM1

The ER membrane (EM) protein STIM1 is the ER Ca2+ sensor that initiates store-operated Ca2+ entry through Orai channels in lymphocytes and many other cells. Communication of ER Ca2+ store depletion to Orai channels in the plasma membrane involves a transition of STIM1 dimers from an inactive to an active state, but the conformational changes underlying this transition remain poorly characterized. We have developed an in-vitro assay to study the conformation and dynamics of STIM1 at the single-molecule level. Purified STIM1 cytosolic fragments were labeled at single sites with Alexa555 and Alexa647 by site-directed mutagenesis and cysteine-maleimide chemistry. These served as donor and acceptor dyes for high-resolution intradimer distance measurements using single-molecule FRET. Labeled STIM1 fragments were encapsulated in 100nm diameter, surface-immobilized liposomes and imaged with a prism-based TIRF microscope.We identified three broad regions within the STIM1 fragments, defined by their characteristic FRET signatures. Within the ER-proximal N-terminal region containing the predicted coiled-coil 1 domain (aa274-339), we observed several transiently stable conformational states, ranging from closely apposed (FRET ∼0.8) to widely separated (FRET ∼0). The different FRET states occurred with similar probability, and transitions between them were frequent. In the region 363-449, encompassing the CRAC activation domain (CAD), stable intermediate to high FRET values (0.6-0.9) were dominant, indicating close apposition of paired sites in the dimer. Interestingly, low-FRET states (0.1-0.4) were also observed at several sites, suggesting the existence of an open conformation with relatively low probability. Finally, C-terminal to the CAD (aa512-660) we found stable, broad low-FRET signals, consistent with the predicted unstructured nature of this region. FRET ratios progressively declined from ∼0.2 to 0 towards the C-terminal end, indicating increasing separation. Further development of this approach will enable direct, high-resolution probing of STIM1 activation under controlled conditions.

Single-Molecule FRET Studies of the ER Calcium Sensor STIM1

The ER membrane (EM) protein STIM1 is the ER Ca2+ sensor that initiates store-operated Ca2+ entry through Orai channels in lymphocytes and many other cells. Communication of ER Ca2+ store depletion to Orai channels in the plasma membrane involves a transition of STIM1 dimers from an inactive to an active state, but the conformational changes underlying this transition remain poorly characterized. We have developed an in-vitro assay to study the conformation and dynamics of STIM1 at the single-molecule level. Purified STIM1 cytosolic fragments were labeled at single sites with Alexa555 and Alexa647 by site-directed mutagenesis and cysteine-maleimide chemistry. These served as donor and acceptor dyes for high-resolution intradimer distance measurements using single-molecule FRET. Labeled STIM1 fragments were encapsulated in 100nm diameter, surface-immobilized liposomes and imaged with a prism-based TIRF microscope. We identified three broad regions within the STIM1 fragments, defined by their characteristic FRET signatures. Within the ER-proximal N-terminal region containing the predicted coiled-coil 1 domain (aa274-339), we observed several transiently stable conformational states, ranging from closely apposed (FRET ∼0.8) to widely separated (FRET ∼0). The different FRET states occurred with similar probability, and transitions between them were frequent. In the region 363-449, encompassing the CRAC activation domain (CAD), stable intermediate to high FRET values (0.6-0.9) were dominant, indicating close apposition of paired sites in the dimer. Interestingly, low-FRET states (0.1-0.4) were also observed at several sites, suggesting the existence of an open conformation with relatively low probability. Finally, C-terminal to the CAD (aa512-660) we found stable, broad low-FRET signals, consistent with the predicted unstructured nature of this region. FRET ratios progressively declined from ∼0.2 to 0 towards the C-terminal end, indicating increasing separation. Further development of this approach will enable direct, high-resolution probing of STIM1 activation under controlled conditions.

EF-G:A-Site Trna Distance Changes during Translocation

Prokaryotic elongation factor G (EF-G) catalyzes the coupled translocations on the ribosome of mRNA and A- and P-site bound tRNAs during the polypeptide elongation cycle. However, the kinetic pathway of this important step has not been fully elucidated. Here we describe the application of single molecule fluorescence resonance energy transfer (smFRET) to probe the mechanism of this key process in real time. Attaching a fluorescent probe within domain IV of EF-G permits monitoring of FRET efficiencies to sites in both ribosomal protein L11 and A-site tRNA. Two different sites on L11, position 38 or position 87, as well as two different A-site tRNAs, Phe-tRNAPhe or Val-tRNAVal were labeled with Cy5. The EF-G:L11 FRET efficiency is constant during EF-G occupancy of the ribosome (300 - 400 ms), whereas the EF-G:tRNA FRET proceeds from high to intermediate to low FRET values. This indicates that EF-G:A-site tRNA distance increases progressively during translocation, with our results providing the first good estimates of the timing of such changes. For both tRNAs, the EF-G:tRNA pair spends most of its time in the intermediate FRET state, with shorter lifetimes in the high and low FRET states. Each transition in the FRET value corresponds to a relative distance change of about 5 to 7 A, so in total, we capture a 12 A relative movement of A-site tRNA away from EF-G during translocation. EF-G:A-site tRNA FRET is sensitive to buffer conditions, with high Mg2+ and polyamines stabilizing the high FRET state. In addition, the translocation inhibitors viomycin and spectinomycin block formation of the low FRET state, although spectinomycin allows more movement between EF-G and tRNA than viomycin. Supported by NIH grant GM080376 to YEG and BSC and AHA fellowship 12POST8910014 to CC.

Dynamics of the N-Terminal Domain of Myosin V Monitored by FRET

The N-terminus is a highly variable region among the myosin superfamily and has been associated with alterations in rotation of the lever arm and step size. We examined the structural dynamics of the N-terminal region of myosin V using steady-state and stopped-flow FRET. We introduced a tetra-cysteine site at the extreme N-terminus of a Myosin V 1IQ construct and labeled it with a bisarsenical fluorescin derivative (FlAsH) (MV NT-FLAsH). Energy transfer between FlAsH and mant or deac labeled nucleotides (dmantATP, dmantADP and deacATP) was monitored. Steady-state FRET experiments with the mant-FlAsH pair demonstrated a high FRET state in the presence of dmantATP and a low FRET state in the presence of dmantADP. Sequential-mix, in which MV NT-FLAsH is first mixed with dmantATP, aged to allow formation of the M.ADP.Pi state, and then mixed with saturating actin allowed us to explore the actin-acitvated product release steps. Biphasic transients with rates corresponding to the fast and slow rate of dmantADP release were observed. The sequential mix experiments with deacATP yielded three phases; a fast, actin dependent, phosphate release phase followed by the two phases of ADP release. Our results suggest the FRET signal monitors a structural change in the N-terminus associated with the sequential release of products. A high FRET state upon binding of ATP is associated with formation of the pre-powerstroke state of the lever arm and a low or no FRET state in the presence of ADP suggests formation of the post-powerstroke state. Our results allow us to hypothesize that movement of the N-terminal domain follows the movement of the converter/lever arm. Further experiments will explore the dynamics of the N-terminal domain and examine how its motion correlates with the conformation of the nucleotide binding pocket and lever arm.

Kinetics of the Triplex-Duplex Transition in DNA

The kinetics of triplex folding/unfolding is investigated by the single-molecule fluorescence resonance energy transfer (FRET) technique. In neutral pH conditions, the average dwell times in both high-FRET (folded) and low-FRET (unfolded) states are comparable, meaning that the triplex is marginally stable. The dwell-time distributions are qualitatively different: while the dwell-time distribution of the high-FRET state should be fit with at least a double-exponential function, the dwell-time distribution of the low-FRET state can be fit with a single-exponential function. We propose a model where the folding can be trapped in metastable states, which is consistent with the FRET data. Our model also accounts for the fact that the relevant timescales of triplex folding/unfolding are macroscopic.

Single-Molecule Fluorescence Measurements of Ribosomal Translocation Dynamics

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.

DNA Methylation Induces a More Compact and Rigid Nucleosome Structure

Cytosine methylation in CpG dinucleotides is an important epigenetic modification in eukaryotes with roles in regulating a variety of genome transactions. There have been many studies of DNA methyltransferases and methyl-CpG binding proteins elucidating their roles in various genome activities. However, less is known about how methylated CpGs directly affect nucleosome structure. We implemented a single molecule FRET coupled with anisotropy that can simultaneously measure dynamic distance changes and flexibility of the two ends of a nucleosomal DNA. Using the method, we monitored effects of DNA methylation on the structure of mononucleosomes. In the absence of methylation, most nucleosomes displayed two low FRET states (FRET efficiency <0.5). When treated with a CpG methyltansferase, we observed a >40-fold increase in the number of nucleosomes that made excursions to a high FRET state (FRET efficiency >0.7). Moreover, based on the anisotropy measurements, a stronger association between the DNA ends and the histone octamer was observed from the nucleosomes in the high FRET state. The increased FRET and anisotropy after DNA methylation strongly suggest a more compact and rigid nucleosomal structure and provide a basic biophysical understanding of how DNA methylation may contribute to the formation of a repressive and transcriptionally inactive chromatin structure.

Nucleosome Dynamics Studied by Free Solution Single Molecule FRET

To help understanding the mechanism of nucleosome opening and closing, we studied the disassembly of mononucleosomes by quantitative single-molecule FRET, using the SELEX-generated Widom 601 positioning sequence labeled with donor and acceptor fluorophores. Reversible dissociation was induced by increasing NaCl concentration. At least 3 species with different FRET were identified and assigned to structures: (i) the most stable high-FRET species corresponding to the intact nucleosome, (ii) a less stable mid-FRET species that we attribute to a first intermediate with a partially unwrapped DNA and less histones, and (iii) a low-FRET species characterized by a very broad FRET distribution, representing highly unwrapped structures and free DNA formed at the expense of the other 2 species. Selective FCS analysis indicates that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 A measured for the high-FRET species corresponds to a compact conformation close to the known crystallographic structure. The coexistence and interconversion of these species is first demonstrated under non-invasive conditions. A geometric model of the DNA unwinding predicts the presence of the observed FRET species. The different structures of these species in the disassembly pathway map the energy landscape indicating major barriers for 10-bp and minor ones for 5-bp DNA unwinding steps. Further information about stepwise dissociation, and exchange of histones between nucleosomes is obtained by FRET between DNA and labeled histones.

Single Molecule Study of Disassembly of SNARE Complex by NSF/α−SNAP

In eukaryotic cells, material exchange between distinct organelles within the cytosol is required for many biochemical processes. The phospholipid vesicles containing the materials are transported to the target membrane and merged. All known forms of intracellular membrane fusion or vesicle fusion involve a highly conserved family of proteins termed SNAREs (Soluble N-ethyl maleimide sensitive-factor Attachment Protein Receptors). The assembly and dissociation of SNARE proteins drives membrane fusion. A good example is chemical release in the synapse, which is essential for inter-communication between neurons. Although it has been known that the ATPase activity of NSF (N-ethyl maleimide Sensitive-Factor) is involved in the disassembly of SNARE complex, the molecular details of the reaction are not understood. In order to investigate the molecular mechanism, we applied a fluorescence-based in vitro approach at the single molecule level. SNARE complexes from three neuronal proteins (Syntanxin1a, Snap-25, and Synaptobrevin) were labeled with suitably chosen FRET dye pairs based on the available structural information about the SNARE complex. For the particular labeling pairs, high FRET corresponds to assembled complex, low FRET corresponds to dissociated complex. To test the labels, we used a conventional bulk FRET assay and found a progressive temporal change from high FRET to low FRET states, indicating complex dissociation into each individual protein in a Mg2+, ATP, and α-SNAP dependent manner. Using a single molecule TIRF optical setup, the same reactions were monitored, and likewise show a change in FRET signal corresponding to complex disassembly. We are studying the disassembly reaction for a number of different labeling pairs along the SNARE helical bundle. Single molecule kinetic studies are underway to understand the molecular mechanism of NSF catalyzed SNARE protein disassembly.

Nucleosome disassembly intermediates characterized by single-molecule FRET

The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessibility for transcription and replication. To help understanding the mechanism of nucleosome opening and closing in these processes, we studied the disassembly of mononucleosomes by quantitative single-molecule FRET with high spatial resolution, using the SELEX-generated "Widom 601" positioning sequence labeled with donor and acceptor fluorophores. Reversible dissociation was induced by increasing NaCl concentration. At least 3 species with different FRET were identified and assigned to structures: (i) the most stable high-FRET species corresponding to the intact nucleosome, (ii) a less stable mid-FRET species that we attribute to a first intermediate with a partially unwrapped DNA and less histones, and (iii) a low-FRET species characterized by a very broad FRET distribution, representing highly unwrapped structures and free DNA formed at the expense of the other 2 species. Selective FCS analysis indicates that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 A measured for the high-FRET species corresponds to a compact conformation close to the known crystallographic structure. The coexistence and interconversion of these species is first demonstrated under non-invasive conditions. A geometric model of the DNA unwinding predicts the presence of the observed FRET species. The different structures of these species in the disassembly pathway map the energy landscape indicating major barriers for 10-bp and minor ones for 5-bp DNA unwinding steps.

Temperature Dependent Energy Transfer Measurements Reveal Flexibility in the Upper 50 kDa Domain of Myosin V

Our previous work has demonstrated that labeling myosin V in the upper 50 kDa domain with the biarsenical dye FlAsH can serve as an acceptor for fluorescence resonance energy transfer studies with mant labeled nucleotides and IAEDANS actin. These FRET studies suggest that myosin V can adopt a conformation in which the nucleotide binding pocket and the actin binding cleft are in a closed conformation. Our studies suggest the upper 50 kDa domain may be highly flexible in certain nucleotide-states which allows tight binding to nucleotide and actin. Molecular geometric simulations demonstrate the upper 50 kDa domain is most flexible in the myosin V.ADP state, consistent with this state having a high affinity for ADP and actin. Currently, we examined the temperature dependence of the FRET signal between mantADP and MV FlAsH. We found that at low temperature (4-15°C) a high FRET state dominates (closed pocket) while at high temperature (30-37°C) a low FRET state dominates (open pocket). This transition is reversible suggesting a temperature-dependent conformational change. We also found that FlAsH labeled G440A MV, a non-hydrolyzable mutant, has a similar temperature-dependent transition in the presence of mantATP. In contrast, the transition does not occur in the presence of mantADP.BeFx or with the non-hydrolyzable E44A MV mutant in the presence of mantATP. Our results suggest coordination of the gamma-phosphate of ATP rigidifies the upper 50 kDa domain which results in a weak actin affinity state (open actin binding cleft and closed nucleotide binding pocket). However, upon phosphate release the upper 50kDa domain becomes more flexible which allows myosin to adopt a conformation in which it has a high affinity for both nucleotide and actin (closed nucleotide binding pocket and actin binding cleft).