Double Cysteine Mutants Research Articles

Neutron reflection study of the interaction of the eukaryotic pore-forming actinoporin equinatoxin II with lipid membranes reveals intermediate states in pore formation

Equinatoxin II (EqtII), a eukaryotic pore-forming toxin, lyses cell membranes through a mechanism involving the insertion of its N-terminal α-helix into the membrane. EqtII pore formation is dependent on sphingomyelin (SM), although cholesterol (Chol) and membrane microdomains have also been suggested to enhance its activity. We have investigated the mechanism of EqtII binding and insertion by using neutron reflection to determine the structures of EqtII–membrane assemblies in situ. EqtII has several different modes of binding to membranes depending on the lipid composition. In pure dimyristoyl-phosphatidylcholine (DMPC) membranes, EqtII interacts weakly and reversibly with the lipid head groups in an orientation approximately parallel to the membrane surface. The presence of sphingomyelin (SM) gives rise to a more upright orientation of EqtII, but Chol is required for insertion into the core of the membrane. Cooling the EqtII–lipid assembly below the lipid phase transition temperature leads to deep water penetration and a significant reduction in the extension of the protein outside the membrane, indicating that phase-separation plays a role in EqtII pore-formation. An inactive double-cysteine mutant of EqtII in which the α-helix is covalently tethered to the rest of the protein, interacts only reversibly with all the membranes. Releasing the α-helix in situ by reduction of the disulphide bridge, however, causes the mutant protein to penetrate in DMPC–SM–Chol membranes in a manner identical to that of the wild-type protein. Our results help clarify the early steps in pore formation by EqtII and highlight the valuable information on protein–membrane interactions available from neutron reflection measurements.

Contact between the β1 and β2 Segments of α-Synuclein that Inhibits Amyloid Formation.

Conversion of the intrinsically disordered protein α-synuclein (α-syn) into amyloid aggregates is a key process in Parkinson's disease. The sequence region 35-59 contains β-strand segments β1 and β2 of α-syn amyloid fibril models and most disease-related mutations. β1 and β2 frequently engage in transient interactions in monomeric α-syn. The consequences of β1-β2 contacts are evaluated by disulfide engineering, biophysical techniques, and cell viability assays. The double-cysteine mutant α-synCC, with a disulfide linking β1 and β2, is aggregation-incompetent and inhibits aggregation and toxicity of wild-type α-syn. We show that α-syn delays the aggregation of amyloid-β peptide and islet amyloid polypeptide involved in Alzheimer's disease and type 2 diabetes, an effect enhanced in the α-synCC mutant. Tertiary interactions in the β1-β2 region of α-syn interfere with the nucleation of amyloid formation, suggesting promotion of such interactions as a potential therapeutic approach.

The conservative cysteines in transmembrane domain of AtVKOR/LTO1 are critical for photosynthetic growth and photosystem II activity in Arabidopsis.

Thylakoid protein vitamin K epoxide reductase (AtVKOR/LTO1) is involved in oxidoreduction. The deficiency of this compound causes pleiotropic defects in Arabidopsis thaliana, such as severely stunted growth, smaller sized leaves, and delay of flowering. Transgenic complementation of wild-type AtVKOR (VKORWT) to vkor mutant lines ultimately demonstrates that the phenotype changes are due to this gene. However, whether AtVKOR functions in Arabidopsis through its protein oxidoreduction is unknown. To further study the redox-active sites of AtVKOR in vivo, a series of plasmids containing cysteine-mutant VKORs were constructed and transformed into vkor deficient lines. Compared with transgenic AtVKORWT plants, the size of the transgenic plants with a single conservative cysteine mutation (VKORC109A, VKORC116A, VKORC195A, and VKORC198A) were smaller, and two double-cysteine mutations (VKORC109AC116A and VKORC195AC198A) showed significantly stunted growth, similar with the vkor mutant line. However, mutations of two non-conservative cysteines (VKORC46A and VKORC230A) displayed little obvious changes in the phenotypes of Arabidopsis. Consistently, the maximum and actual efficiency of photosystem II (PSII) in double-cysteine mutation plants decreased significantly to the level similar to that of the vkor mutant line both under normal growth light and high light. A significantly decreased amount of D1 protein and increased accumulation of reactive oxygen species were observed in two double-cysteine mutations under high light. All of the results above indicated that the conservative cysteines in transmembrane domains were the functional sites of AtVKOR in Arabidopsis and that the oxidoreductase activities of AtVKOR were directly related to the autotrophic photosynthetic growth and PSII activity of Arabidopsis thaliana.

Effect of Actin and Nucleotide on the Movement of A1-Type Myosin Essential Light Chain, Detected by Time-Resolved FRET

We have used site-directed time-resolved FRET to detect structural changes of the N-terminal extension on the A1 isoform of ELC, during active interaction with actin. Skeletal muscle myosin subfragment 1 (S1) has two ELC isoforms, A1 and A2, which differ by the presence of 40-45 additional residues at the N-terminus of A1. Removal of ELC from myosin results in a loss of movement of actin filaments, and a reduction in isometric force. It has been proposed that the N-terminal extension of A1 interacts directly with actin to modulate actomyosin kinetics. We have used time-resolved FRET to explore the structural details of this modulation. We recently showed, using probes on actin and ELC, that the amplitude of the myosin power stroke is much greater for A1 than A2. We have now engineered single cysteine (C16, in the N-terminal extension) and double cysteine (C16-C180, in the C-terminal lobe) mutants of A1-ELC and used FRET to determine the distance from C16 to actin 374 or to C180, as affected by the power stroke. Labeled ELCs were exchanged into S1, and the pure isoenzyme (S1A1) was isolated using Talon affinity resin. Labeling and exchange preserved the functional properties of S1A1. Intermolecular FRET between C374 of actin and C16 of A1 showed that the distance increases from 2.9 nm to 3.5 nm upon addition of ATP. Intramolecular FRET between C16 and C180 does not change in the presence of ATP or actin. We conclude that (1) the N-terminus of ELC moves away from actin during the transition between strongly and weakly bound states of acto-S1, and (2) the converter/C-terminal domain of myosin/ELC and the N-terminal extension move as a rigid body during the power stroke.

Characterization of a Bifunctional Spin Label for the Structure and Dynamics of a Membrane Protein using CW-EPR Spectroscopy

Electron Paramagnetic Resonance (EPR) spectroscopy is a powerful structural biology tool when combined with site-directed spin labeling (SDSL) to study the structure and dynamics of peptides and proteins. The most widely used spin label for SDSL is methanthiosulfonate (MTSL), however the flexibility of this spin label introduces greater uncertainties in the EPR measurements obtained for determining structure, side chain dynamics, and backbone motion of membrane protein systems. Most recently, a new bifunctional spin label (BSL) 3,4-Bis-(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy, has been introduced to overcome some of the limitations associated with the MTSL spin label and has been invaluable in determining protein dynamics and inter-residue distances due to its restricted internal motion and fewer size restrictions. While BSL has been successful in providing information about the structure and dynamics of several proteins, the details of its characterization is lacking. The goal of this research is to gain insights into the behavior of the BSL that has been so useful in providing details to characterize a variety of protein systems. Fmoc solid phase peptide synthesis (SPPS) has been used to generate a series of double cysteine mutants of the 23 amino acid α-helical membrane peptide, AChR M2delta at i and i+4 residue positions. BSLs bearing peptides were incorporated into 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) bicelles and multilamellar vesicles. The spectra were collected at different temperatures utilizing continuous wave (CW)-EPR spectroscopy to examine the dynamics of the BSL within a membrane. The EPR spectral lineshape analysis will be conducted to obtain the static and dynamic EPR parameters. This study will provide more accurate insights into the behavior of BSL in order to provide the optimal conditions for other studies that may utilize this promising spin label.

The Conformational Dynamics of a Secondary Multidrug Transporter are Modulated by the Lipid Bilayer Composition

LmrP, a Major Facilitator Superfamily (MFS) multidrug transporter from Lactococcus lactis, acts as a secondary antiporter, catalysing the extrusion of a spectrum of hydrophobic drugs by dissipating a proton gradient. It has evolved to bind and export structurally diverse cytotoxic substances from within the membrane, in contrast to the majority of MFS transporters specialized in the transport of a single soluble substrate, captured from the aqueous medium. Its transport mechanism could thus differ from the alternating access model of the lactose permease, the actual paradigm for all MFS transporters. Recently, we have shown that the protonation of conserved acidic residues is the driving force of the conformational transition, and that substrate binding initiates the cycle by catalysing proton passage (Masureel, Martens et al. 2014). While this analysis was performed on detergent solubilized protein, we now aim to investigate the role of the lipid bilayer in the conformational cycle. For this purpose, we performed Double Electron Electron Resonance (DEER) distance measurements on a library of labeled double-cysteine mutants monitoring both sides of the transporter. Spin labeled mutants were reconstituted in nanodiscs and the distance changes monitored upon changes in the pH or addition of substrate(s). We found that the pH-dependent equilibrium between the inside-open and outside-open conformations is strongly shifted in nanodiscs compared to detergent, suggesting that the pKa of the acidic residues is modified by the lipids. By combining specific lipidic compositions and mutagenesis, we are investigating whether such change in the dynamics are the results of direct phospholipid-protein interaction. Specific structural effects of given lipids are observed: small amounts of cardiolipin allow the conformational transition to occur at physiological pH. These findings could shed light on the mechanism of MFS multidrug transporters in general.

Conformationally Sensitive Proximity of Extracellular Loops 2 and 4 of the γ-Aminobutyric Acid (GABA) Transporter GAT-1 Inferred from Paired Cysteine Mutagenesis

The sodium- and chloride-coupled GABA transporter GAT-1 is a member of the neurotransmitter:sodium:symporters, which are crucial for synaptic transmission. Structural work on the bacterial homologue LeuT suggests that extracellular loop 4 closes the extracellular solvent pathway when the transporter becomes inward-facing. To test whether this model can be extrapolated to GAT-1, cysteine residues were introduced at positions 359 and 448 of extracellular loop 4 and transmembrane helix 10, respectively. Treatment of HeLa cells, expressing the double cysteine mutant S359C/K448C with the oxidizing reagent copper(II)(1,10-phenantroline)3, resulted in a significant inhibition of [(3)H]GABA transport. However, transport by the single cysteine mutant S359C was also inhibited by the oxidant, whereas its activity was almost 4-fold stimulated by dithiothreitol. Both effects were attenuated when the conserved cysteine residues, Cys-164 and/or Cys-173, were replaced by serine. These cysteines are located in extracellular loop 2, the role of which in the structure and function of the eukaryotic neurotransmitter:sodium:symporters remains unknown. The inhibition of transport of S359C by the oxidant was markedly reduced under conditions expected to increase the proportion of inward-facing transporters, whereas the reactivity of the mutants to a membrane-impermeant sulfhydryl reagent was not conformationally sensitive. Our data suggest that extracellular loops 2 and 4 come into close proximity to each other in the outward-facing conformation of GAT-1.

In Situ Time-Resolved FRET Reveals Effects of Sarcomere Length on Cardiac Thin-Filament Activation

During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca2+ and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca2+-induced opening of N-cTnC and is presumed to also destabilize cTnI–actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca2+-sensitizing N-cTnC–cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca2+-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca2+ and 1.80 and 2.2-μm sarcomere length. Mg2+-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca2+] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive feedback regulation is a key determinant in the length-dependent Ca2+ sensitivity of myofilament activation and consequently the mechanism underlying the Frank-Starling law of the heart.

Disulfide Bond Requirements for Active Wnt Ligands

Secreted Wnt lipoproteins are cysteine-rich and lipid-modified morphogens that bind to the Frizzled (FZD) receptor and LDL receptor-related protein 6 (LRP6). Wnt engages FZD through protruding thumb and index finger domains, which are each assembled from paired β strands secured by disulfide bonds and grasp two sides of the FZD ectodomain. The importance of Wnt disulfide bonds has been assumed but uncharacterized. We systematically analyzed cysteines and associated disulfide bonds in the prototypic Wnt3a. Our data show that mutation of any individual cysteine of Wnt3a results in covalent Wnt oligomers through ectopic intermolecular disulfide bond formation and diminishes/abolishes Wnt signaling. Although individual cysteine mutations in the amino part of the saposin-like domain and in the base of the index finger are better tolerated and permit residual Wnt3a secretion/activity, those in the amino terminus, the thumb, and at the tip of the index finger are incompatible with secretion and/or activity. A few select double cysteine mutants based on the disulfide bond pattern restore Wnt secretion/activity. Further, a double cysteine mutation at the index finger tip results in a Wnt3a with normal secretion but minimal FZD binding and dominant negative properties. Our results experimentally validate predictions from the Wnt crystal structure and highlight critical but different roles of the saposin-like and cytokine-like domains, including the thumb and the index finger in Wnt folding/secretion and FZD binding. Finally, we modified existing expression vectors for 19 epitope-tagged human WNT proteins by removal of a tag-supplied ectopic cysteine, thereby generating tagged WNT ligands active in canonical and non-canonical signaling.

Abstract 224: Formation of Cholesterol-Enriched Nascent HDL Requires Helices 5 and 7

Heart disease claims nearly 400,000 individuals in the US per year. Large population studies have repeatedly demonstrated an inverse relationship between HDL concentration and risk. Despite the strong correlation, pharmacological trials that raise plasma HDL have shown little efficacy, suggesting that HDL function should be monitored. Studies were initiated into the mechanism of cholesterol efflux leading to nascent HDL (nHDL) formation. The most productive cholesterol removal occurs when lipid-poor apoA-I, the main structural protein in nHDL, forms particles after interacting with ABCA1. ApoA-I acquires cholesterol and phospholipid at the cell surface forming 10-12 nm diameter nHDL whose composition resembles lipid rafts. Because lipidated apoA-I has a significantly different 3-D conformation compared to its lipid-poor counterpart, the structural modifications leading to nHDL formation were investigated. First, we completed a 3-D solution structure of lipid-poor apoA-I using lysine specific chemical cross-linking in conjunction with specifically engineered double cysteine-containing apoA-I mutants. Strategically placed cysteines form disulfide bonds (locked) when residues are within ~0.5 nm. If no disulfide bond formed (unlocked) then the two cysteines were separated by more than ~0.5 nm. Both methods rely on mass spectrometry to identify amino acid sequence and distance constraints within each 10 repeating helices comprising 80% of full-length apoA-I. By comparing lipid-poor apoA-I conformation to our previously described 3-D structure of apoA-I on 10-12 nm nHDL, we formulated a series of hypothetical helical domain transitions that might drive protein-lipid interaction and particle formation. To test, 10 different double cysteine mutants were employed in either their “locked” (oxidized) or “unlocked” (reduced) state by evaluating their ability to form recombinant HDL using synthetic phospholipid or to form nHDL using HEK cells. After determining HDL particle yield, size and composition from all locked and unlocked double cysteine apoA-I mutants we conclude that both the N- and C-terminal ends of apoA-I are essential for the first steps in lipid acquisition, but that the central helices 5, 6 and 7 are essential for nHDL formation.

Crystal Structure and Self-Interaction of the Type VI Secretion Tail-Tube Protein from Enteroaggregative Escherichia coli

The type VI secretion system (T6SS) is a widespread machine used by bacteria to control their environment and kill or disable bacterial species or eukaryotes through toxin injection. The T6SS comprises a central tube formed of stacked hexamers of hemolysin co-regulated proteins (Hcp) and terminated by a trimeric valine-glycine repeat protein G (VgrG) component, the cell puncturing device. A contractile tail sheath, formed by the TssB and TssC proteins, surrounds this tube. This syringe-like machine has been compared to an inverted phage, as both Hcp and VgrG share structural homology with tail components of Caudovirales. Here we solved the crystal structure of a tryptophan-substituted double mutant of Hcp1 from enteroaggregative Escherichia coli and compared it to the structures of other Hcps. Interestingly, we observed that the purified Hcp native protein is unable to form tubes in vitro. To better understand the rationale for observation, we measured the affinity of Hcp1 hexamers with themselves by surface plasmon resonance. The intra-hexamer interaction is weak, with a KD value of 7.2 µM. However, by engineering double cysteine mutants at defined positions, tubes of Hcp1 gathering up to 15 stacked hexamers formed in oxidative conditions. These results, together with those available in the literature regarding TssB and TssC, suggest that assembly of the T6SS tube differs significantly from that of Sipho- or Myoviridae.

Association of α-Synuclein with Lipid Vesicles. Stopped-Flow Kinetics of Concerted Binding and Conformational Change

Alpha-synuclein (AS), a 140aa intrinsically disordered protein, self-associates into oligomeric forms and aggregates into amyloid fibrils in Parkinson's disease. Certain mutations affect these processes and accelerate disease pathogenesis. The physiological roles of AS are a matter of speculation. Membrane binding is undoubtedly involved and the protein acquires α-helical structure in the process (1).We have studied the thermodynamics and kinetics of AS-membrane association utilizing vesicles (SUVs) of differing composition. Functionally neutral single cysteine mutants of AS were labeled with a polarity sensitive excited-state intramolecular proton transfer (ESIPT) probe (MFE). Double cysteine mutants were labeled with a FRET pair (Alexa Fluor488, Alexa Fluor568) at a series of selected positions in the primary sequence. Kinetic studies were conducted by stopped-flow, using 5-20 nM protein concentrations and increasing levels of SUVs (generally 20-200 µM) Signal changes indicative of membrane association were observed: increased intensity and shape change of dual band ESIPT emission, and altered FRET with the Alexa dyes. The analysis revealed a two-step reaction sequence in the time range <10 s. We attribute the first step to binding, and from the dependence on lipid concentration determined the second order rate constants and corresponding spectroscopic parameters. The second concentration independent step (1-10 s range) presumably arises from conformational changes in the protein (α-helix formation) and its accommodation to or perturbation of the lipid microenvironment (ESIPT dye).Accompanying thermodynamic measurements led to estimates of dissociation constants as a function of membrane composition, charge, and shape (SUVs, LUVs). A new experimental protocol (slopes), implemented in a microplate reader, circumvented technical problems usually manifested in titrations of protein with lipid.[1] Pfefferkorn C M, Jiang, Lee J C (2012). Biophysics of α-synuclein membrane interactions. Biochim Biophys Acta1818:162-171.

State-Dependent Movement between the First and Last External Loops of the Na/K Pump α Subunit

Previously we reported that conserved Na/K pump α-subunit residues D128 (loop L1-2, shark-α1 numbering) and R979 (L9-10) reach atomic proximity in E2P (Artigas, 2009. Biophys J. 96(3):145a). L1-2 is expected to move throughout the pump cycle. We used double cysteine substitutions to address the relative displacement of L1-2 with respect to R979. We mutated R979C and concomitantly introduced a cysteine at each of the residues within L1-2, from Q118 (external end of TM1) to L132 (within TM2), and analyzed the functional effect of reducing and oxidizing reagents on these double cysteine mutants heterologously expressed in Xenopus oocytes. The pump current (IP) induced by 3 mM K under two-electrode voltage clamp in the absence of Na was measured; first without redox treatment, then after 15 min in 10 mM TCEP (a reducing agent) and subsequently, following oxidation with cupper phenanthroline (100 μM Cu: 300 μM Phe, applied in N-Methyl D-glucamine). TCEP increased the IP of E122C/R979C (20%), E124C/R979C (20%), P125C/R979C (38%), Q126C/R979C (52%) and D128C/R979C (130%), whilst Cu:Phe induced similar effects, in the opposite direction than TCEP, with a maximum reduction of IP (>80%) in D128C/R981C. The effect of Cu:Phe on the charge movement without K in the presence of Na was also studied. Cu:Phe abolished charge movement of D128C/R981C and modified the characteristics of the transients of the other double cysteine mutants that showed effects of crosslinking in IP. Our results indicate that residue 128 (at the end of a rigid TM2-helix) must separate from R979 in order for TM2 to perform its required motions, while crosslinking residues further in the flexible loop does not block IP, but modifies the E1-E2 conformational equilibrium in the presence of Na. Supported by R15NS081570-01A1.

Functional Consequences of Cysteine Mutations at the Kainate Receptor Dimer Interface

The cation-binding pocket is integral to the stability of the kainate receptor dimer interface, allowing proper activation of the receptor. Interestingly, a double cysteine mutation (Y521C/L783C GluK2) across the interface eliminates the need for occupancy of this pocket. We therefore hypothesized that this mutation greatly disturbs the electrostatic environment of the dimer interface and functions solely because of disulfide bond formation. Molecular dynamics simulations of Y521C/L783C showed disrupted interactions across the dimer interface mainly due to increased water accessibility. This occurred with or without a disulfide bond in the double cysteine simulations, consistent with our hypothesis. We next investigated the cysteine mutations in isolation and found that single cysteine or serine mutations at Y521 yielded macroscopic currents from outside-out patches that decayed much slower than wild-type but had similar amplitudes (>1 nA). However, L783C alone yielded no discernible current and responses from L783S were greatly reduced (l10 pA currents). Analysis of the likely interactions that occur at L783 show that it is part of a hydrophobic pocket that prevents water access to the dimer interface. Simulations further support this idea as they demonstrate that both cysteine and serine substitution at the 783 position allow increased hydration of the interface. One final prediction follows that in electrophysiological experiments if the disulfide bond is cleaved then the exposed cysteines should disrupt their surrounding environment thereby disturbing receptor function. Indeed, in oxidizing conditions there was an 80% decrease in the current amplitude that was recovered in reducing conditions. In conclusion, we propose that the excess water in the dimer interface interrupts the normal interactions required for activation. Altogether, these experiments are consistent with the hypothesis that the electrostatic environment of the dimer interface is perturbed by the cysteine mutations at these residues.

Probing Structure and Conformational Changes in the Extracellular Loops of CFTR

CFTR is a member of the ABC transporter family, and as such bears closer homology to transporters than to other channels. However, it is unknown whether, like transporters, the transmembrane domains of CFTR undergo an inward-to-outward facing transition upon NBD dimerization. To test this, we made cysteine substitutions in the extracellular loops (ECL) 1 and 4, which appose each other in inward-facing but not outward-facing structures of ABC transporters. We found that the macroscopic current of D110C/K892C-CFTR channels (but not corresponding single mutants) was increased in the presence of DTT. Multichannel patch recordings revealed that DTT increased NPO of double cysteine mutant channels, indicating that they were locked into a closed state by a spontaneous disulfide bond prior to DTT. To further understand the kinetics of the interaction, we applied Cd2+ trapping and found that D110C alone is transiently bound by Cd2+, causing a rapidly reversible 33 +/- 4.6% inhibition of current. By contrast, Cd2+ inhibited D110C/K892C-CFTR irreversibly and to a greater extent (72.5 +/-6.8%) under identical cumulative exposure. Results were consistent in 2 mM, 20 uM and 200 nM Cd2+. K892C was reversibly modified to a small extent (1.5 +/- 0.9%) and E115C (near the end of ECL1) was not functionally modified by Cd2+, but was labeled by MTS-TAMRA, indicating accessibility. The data suggest: 1) ECL1 and ECL4 come into close proximity in a closed state of CFTR; 2) K892 is located away from the conductance pathway of open CFTR; and 3) ECL1 is oriented such that D110 is closer to the pore than E115. Experiments are currently investigating the single channel effect of Cd2+ on ECL mutants and state-dependent kinetics of Cd2+ coordination by D110C/K892C and other ECL pairs. (NIH R01-DK 056481).

Probing the Structural Topology of a Membrane Peptide in Mechcanically Aligned Lipid Bilayers using Bifunctional Spin Labeling EPR Spectroscopy

Electron Paramagnetic Resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a powerful structural biology tool for studying the structural and dynamic properties of peptides, proteins, and nucleic acids. The most commonly used spin label for SDSL is methanthiosufonate (MTSL), however the flexibility of this spin label can introduce greater uncertainties in the EPR measurements for determining structure, side chain orientations and backbone motion of membrane protein systems. Another spin label, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) has been found to be a useful alternative given its rigid structure, however it is very challenging to introduce this spin label into biological protein systems. The goal of this research is to develop an improved biophysical method for studying the structural and dynamic properties of membrane proteins using EPR spectroscopy that will overcome the limitations associated with MTSL and TOAC. A recently discovered bifunctional spin label (BSL) 3,4-Bis-(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy, will be utilized. Fmoc solid phase peptide synthesis (SPPS) will be used to generate a double cysteine mutant of the 23 amino acid α-helical membrane peptide, AChR M2δ. Once labeled with BSL and incorporated into 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) bilayers, alignment techniques utilizing EPR spectroscopy will be performed to examine the structural topology. This study will provide a structural biology tool that can be used to obtain very accurate and precise EPR measurements to answer several structural and dynamics related questions on membrane protein systems.

Characterization of HIV-1 Capsid with Fluorescence Spectroscopy and Microscopy

Wild type HIV-1 CA protein can spontaneously assemble into long, hollow tubes at high salt (1M NaCl) and high protein (10mg/ml) concentration. These tubes are structural mimics of the mature capsid, and Cryo EM has revealed that they are composed of CA hexamers. Although WT CA tubes are unstable under low salt and low protein concentrations, a specific double cysteine mutation that efficiently cross-links the CA hexamers, dramatically stabilizes the assemblies under a variety of conditions. We are interested in studying the structure of these CA tubes under physiological conditions.

Monitoring Cardiac Troponin Structural Changes using In-Situ Time-Resolved FRET: Implications on the Regulatory Roles of Cross-Bridges and Sarcomere Length

During cardiac thin filament activation, the N-domain of Ca2+-binding cardiac troponin C (N-cTnC) interacts with the actomyosin inhibitory troponin I (cTnI) subunit, which concomitantly opens the cTnC N-domain and leads to force generation. Recently, we used in situ steady state FRET measurements based on N-cTnC opening to determine that strongly bound cross-bridges (XBs) stabilize this Ca2+-sensitizing N-cTnC-cTnI interaction. However, the method was unable to determine how N-cTnC opening is affected by sarcomere length (SL). In this study, we used time-resolved in situ FRET to monitor the effects of Ca2+-occupancy, XB state, and SL on N-cTnC opening in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM) modified double-cysteine mutant cTnC(13C/51C)AEDANS-DDPM was reconstituted into skinned muscle fibers to examine the N domain of cTnC (N-cTnC) opening. To study the effect of SL on structural transitions of cTnC, we monitored the protein structural transitions at low and high [Ca2+] and SL 1.8 and 2.2 μm. Mg2+-ADP and sodium orthovanadate (Vi) were used to examine the effects of non-cyclng strong and weak XBs, respectively. We found that strongly bound XBs alter structural transitions of cardiac troponin only at 2.2 μm. On the other hand, Vi blunted the SL dependent opening of N-cTnC such that weak XBs have no effect on N-cTnC at either [Ca2+] or SL. In addition, distance distribution analysis indicated that N-cTnC adopts four unique conformations associated with the four states of thin filament regulation, and that N-cTnC conformational equilibria are caused by cycling XBs. Based on our findings, we conclude that the observed dependence of myosin positive feedback regulation on SL is an important determinant of the Frank-Starling law of the heart.

Conformational variation of the translocon enhancing chaperone SecDF

The Sec translocon facilitates transportation of newly synthesized polypeptides from the cytoplasm to the lumen/periplasm across the phospholipid membrane. Although the polypeptide-conducting machinery is formed by the SecYEG-SecA complex in bacteria, its transportation efficiency is markedly enhanced by SecDF. A previous study suggested that SecDF assumes at least two conformations differing by a 120° rotation in the spatial orientation of the P1 head subdomain to the rigid base, and that the conformational dynamics plays a critical role in polypeptide translocation. Here we addressed this hypothesis by analyzing the 3D structure of SecDF using electron tomography and single particle reconstruction. Reconstruction of wt SecDF showed two major conformations; one resembles the crystal structure of full-length SecDF (F-form structure), while the other is similar to the hypothetical structural variant based on the crystal structure of the isolated P1 domain (I-form structure). The transmembrane domain of the I-form structure has a scissor like cleft open to the periplasmic side. We also report the structure of a double cysteine mutant designed to constrain SecDF to the I-form. This reconstruction has a protrusion at the periplasmic end that nicely fits the orientation of P1 in the I-from. These results provide firm evidence for the occurrence of the I-form in solution and support the proposed F- to I-transition of wt SecDF during polypeptide translocation.

A Large-Scale Allosteric Transition in Cytochrome P450 3A4 Revealed by Luminescence Resonance Energy Transfer (LRET)

Effector-induced allosteric transitions in cytochrome P450 3A4 (CYP3A4) were investigated by luminescence resonance energy transfer (LRET) between two SH-reactive probes attached to various pairs of distantly located cysteine residues, namely the double-cysteine mutants CYP3A4(C64/C468), CYP3A4(C377/C468) and CYP3A4(C64/C121). Successive equimolar labeling of these proteins with the phosphorescent probe erythrosine iodoacetamide (donor) and the near-infrared fluorophore DY-731 maleimide (acceptor) allowed us to establish donor/acceptor pairs sensitive to conformational motions. The interactions of all three double-labeled mutants with the allosteric activators α-naphthoflavone and testosterone resulted in an increase in the distance between the probes. A similar effect was elicited by cholesterol. These changes in distance vary from 1.3 to 8.5 Å, depending on the position of the donor/acceptor pair and the nature of the effector. In contrast, the changes in the interprobe distance caused by such substrates as bromocriptine or 1-pyrenebutanol were only marginal. Our results provide a decisive support to the paradigm of allosteric modulation of CYP3A4 and indicate that the conformational transition caused by allosteric effectors increases the spatial separation between the beta-domain of the enzyme (bearing residues Cys64 and Cys377) and the alpha-domain, where Cys121 and Cys468 are located.