PubMed Health⌕ Search

Biomedical subjects

Pinghua Ge

Publications and source records attributed to Pinghua Ge.

9 recordsLinked to original sources
PubMed2006

Distance measurements reveal a common topology of prokaryotic voltage-gated ion channels in the lipid bilayer.

Voltage-dependent ion channels are fundamental to the physiology of excitable cells because they underlie the generation and propagation of the action potential and excitation-contraction coupling. To understand how ion channels work, it is important to determine their structures in different conformations in a membrane environment. The validity of the crystal structure for the prokaryotic K(+) channel, K(V)AP, has been questioned based on discrepancies with biophysical data from functional eukaryotic channels, underlining the need for independent structural data under native conditions. We investigated the structural organization of two prokaryotic voltage-gated channels, NaChBac and K(V)AP, in liposomes by using luminescence resonance energy transfer. We describe here a transmembrane packing representation of the voltage sensor and pore domains of the prokaryotic Na channel, NaChBac. We find that NaChBac and K(V)AP share a common arrangement in which the structures of the Na and K selective pores and voltage-sensor domains are conserved. The packing arrangement of the voltage-sensing region as determined by luminescence resonance energy transfer differs significantly from that of the K(V)AP crystal structure, but resembles that of the eukaryotic K(V)1.2 crystal structure. However, the voltage-sensor domain in prokaryotic channels is closer to the pore domain than in the K(V)1.2 structure. Our results indicate that prokaryotic and eukaryotic channels that share similar functional properties have similar helix arrangements, with differences arising likely from the later introduction of additional structural elements.

Bacterial Proteins↗
PubMed2005

Small vertical movement of a K+ channel voltage sensor measured with luminescence energy transfer.

Voltage-gated ion channels open and close in response to voltage changes across electrically excitable cell membranes. Voltage-gated potassium (Kv) channels are homotetramers with each subunit constructed from six transmembrane segments, S1-S6 (ref. 2). The voltage-sensing domain (segments S1-S4) contains charged arginine residues on S4 that move across the membrane electric field, modulating channel open probability. Understanding the physical movements of this voltage sensor is of fundamental importance and is the subject of controversy. Recently, the crystal structure of the KvAP channel motivated an unconventional 'paddle model' of S4 charge movement, indicating that the segments S3b and S4 might move as a unit through the lipid bilayer with a large (15-20-A) transmembrane displacement. Here we show that the voltage-sensor segments do not undergo significant transmembrane translation. We tested the movement of these segments in functional Shaker K+ channels by using luminescence resonance energy transfer to measure distances between the voltage sensors and a pore-bound scorpion toxin. Our results are consistent with a 2-A vertical displacement of S4, not the large excursion predicted by the paddle model. This small movement supports an alternative model in which the protein shapes the electric field profile, focusing it across a narrow region of S4 (ref. 6).

Animals↗
PubMed2003

An actin-dependent conformational change in myosin.

Conformational changes within myosin lead to its movement relative to an actin filament. Several crystal structures exist for myosin bound to various nucleotides, but none with bound actin. Therefore, the effect of actin on the structure of myosin is poorly understood. Here we show that the swing of smooth muscle myosin lever arm requires both ADP and actin. This is the first direct observation that a conformation of myosin is dependent on actin. Conformational changes within myosin were monitored using fluorescence resonance energy transfer techniques. A cysteine-reactive probe is site-specifically labeled on a 'cysteine-light' myosin variant, in which the native reactive cysteines were removed and a cysteine engineered at a desired position. Using this construct, we show that the actin-dependent ADP swing causes an 18 A change in distance between a probe on the 25/50 kDa loop on the catalytic domain and a probe on the regulatory light chain, corresponding to a 23 degrees swing of the light-chain domain.

Actins↗
PubMed2002

Thiol-reactive lanthanide chelates for phasing protein X-ray diffraction data.

Lanthanides can contribute a large anomalous component to X-ray scattering when present and ordered in a target crystal. This large anomalous signal is a useful source of phase information in X-ray crystallographic studies of biological macromolecules. Thiol-reactive lanthanide chelates were tested as a means of incorporation of lanthanides into protein crystals. Two compounds, each capable of being loaded with a lanthanide of choice, were synthesized: diethylenetriaminepentaacetic 3-(2-pyridyldithio)propionyl hydrazide (DTPA-PDPH) and 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic 3-(2-pyridyldithio)propionyl hydrazide (DOTA-PDPH). A cysteine mutant of the 34 kDa phosphate-binding protein (PBP-A197C) from Escherichia coli was used as a test case. PBP-A197C was labeled with DTPA-PDPH loaded with dysprosium. Characteristics of DTPA-PDPH enabled spectroscopic monitoring of the labeling reaction. Complete labeling of PBP-A197C was confirmed by mass spectrometry and SDS-PAGE analysis. Labeled PBP-A197C (PBP-A197C-DTPA-Dy) crystallized identically to unlabeled protein. X-ray diffraction data were collected from PBP-A197C-DTPA-Dy crystals in-house with a Cu Kalpha rotating-anode source and with a tuneable synchrotron source (ALS 5.0.2). Synchrotron data were collected at energies corresponding to the Dy L(III) edge f" peak and a high-energy remote. Each data set was treated as an independent SAD experiment. A large anomalous signal was present in the data collected in-house and at the synchrotron. The Dy site was easily located in anomalous difference Patterson maps calculated from each of the data sets. In each case, SAD phasing resulted in high-quality electron-density maps, as evidenced by the success of automated model building. The generality of the method was analyzed with several other test proteins. Labeling of some of these proteins with thiol-reactive lanthanide chelates was deleterious to protein solubility or crystallization. In two of the cases the lanthanide chelate was disordered in the crystals. These results suggest that this method may not be well suited for high-throughput crystallography. However, for difficult cases requiring a large anomalous signal, thiol-reactive lanthanide chelates may prove to be a valuable tool.

Chelating Agents↗
PubMed2002

Nickel complexes of bidentate [S2] and [SN] borato ligands.

Nickel(II) complexes of the monoanionic borato ligands [Ph2B(CH2SCH3)2] (abbreviated Ph2Bt), [Ph2B(CH2S(t)Bu)2] (Ph2Bt(tBu)), [Ph2B(1-pyrazolyl)(CH2SCH3)], and [Ph2B(1-pyrazolyl)(CH2S(t)Bu)] have been prepared and characterized. While [Ph2Bt] formed the square planar homoleptic complex, [Ph2Bt]2Ni, the larger [S2] ligand with tert-butyl substituents, [Ph2BttBu], yielded an unexpected organometallic derivative, [Ph2Bt(tBu)]Ni(eta2-CH2SBut), resulting from B-C bond rupture. The analogous thiametallacycle derived from the [S3] ligand, [PhB(CH2S(t)Bu)3] (PhTt(tBu)), has been structurally authenticated (Schebler, P. J.; Mandimutsira, B. S.; Riordan, C. G.; Liable-Sands, L.; Incarvito, C. D.; Rheingold, A. L. J. Am. Chem. Soc. 2001, 123, 331). The [SN] borato ligands formed exclusively the cis stereoisomers upon reaction with Ni(II) sources, [Ph2B(1-pyrazolyl)(CH2SR)]2Ni. Analysis of the Ni(II/I) reduction potentials by cyclic voltammetry revealed a approximately 600 mV anodic shift upon replacement of two thioether donors ([Ph2Bt]2Ni) with two pyrazolyl donors ([Ph2B(1-pyrazolyl)(CH2SCH3)]2Ni) consistent with the all thioether environment stabilizing the lower oxidation state of nickel.

Journal Article↗
PubMed1996

Poly(2-thienyl)borates: An Investigation into the Coordination of Thiophene and Its Derivatives.

The synthesis, characterization, and reactivity of a new sulfur-rich tridentate ligand, tetrakis(2-thienyl)borate (1(-)()), are reported along with a molecular orbital analysis of its coordination to a metal center. Unlike the analogous tetrakis((methylthio)methyl)borate (2(-)()), 1(-)() does not coordinate Mo(CO)(3) when reacted with (C(7)H(8))Mo(CO)(3). The sulfur atoms in both ligands are oriented to coordinate the metal in a pyramidal eta(1) sulfur-bound mode. Approximate molecular orbital calculations are used to compare the metal-ligand interactions in these related species, and the results indicate that the magnitude and polarizability of the electronic charge density of the lone pairs on the sulfur atoms dictate the coordination strength of the ligands. Simple Mulliken atomic charges and orbital occupation numbers are used to determine the extent of charge delocalization. While the conjugation of the sulfur lone pair electrons with adjacent pi bonds in the ligands decreases the corresponding Lewis basicity, the contribution from the aromaticity in the thienyl groups is negligible. During the course of these studies, the structure of K[1] was determined by X-ray diffraction. K[1]: monoclinic space group C2/c, with a = 16.00(2) Å, b = 7.680(7) Å, c = 16.22(2) Å, beta = 118.520(7) degrees, V = 1750(3) Å(3), Z = 4, R(F) = 0.0494, and R(w)(F(2)()) = 0.122. The crystal lattice contains one-dimensional chains of 1(-)() bridged by K ions.

Journal Article↗
PubMedDate not supplied

Thiol-reactive luminescent lanthanide chelates: part 2.

Luminescent lanthanide complexes have unusual spectroscopic characteristics, including millisecond excited-state lifetime and sharply spiked emission spectra. These characteristics make them valuable alternatives to conventional organic fluorescent probes in detection applications and for measuring nanometer-scale conformational changes in biomolecules via resonance energy transfer. Our group has previously reported the syntheses and application of various luminescence complexes that have polyaminocarboxylate chelates coupled to a carbostyril antenna and thiol or amine-reactive groups. Here we report the syntheses of new thiol-reactive forms of DTPA-cs124 chelates, including two iodoacetamide forms (phenylalanine-iodoacetamide and ethylenediamine iodoacetamide) and two methane-thio-sulfonate forms (ethylmethanethiosulfonate and carboxyethylmethanethiosulfonate). In addition, we have developed an improved synthesis of a previously reported maleimide form.

Chelating Agents↗
PubMedDate not supplied

Carbostyril derivatives as antenna molecules for luminescent lanthanide chelates.

Luminescent lanthanide complexes consisting of a lanthanide-binding chelate and organic-based antenna molecule have unusual emission properties, including millisecond excited state lifetimes and sharply spiked spectra, compared to standard organic fluorophores. We have previously used carbostyril (cs124, 7-amino-4-methyl-2(1H)-quinolinone) as an antenna molecule (Li and Selvin, J. Am. Chem. Soc., 1995) attached to a polyaminocarboxylate chelate such as DTPA. Here, we report the chelate syntheses of DTPA conjugated with cs124 derivatives substituted on the 1-, 3-, 4-, 5-, 6-, and 8-position. Among them, the DTPA chelate of cs124-6-SO(3)H has similar lifetime and brightness for both Tb(3+) and Eu(3+) compared to the corresponding DTPA-cs124 complexes, yet it is significantly more soluble in water. The Tb(3+) complex of DTPA-cs124-8-CH(3) has significantly longer lifetime compared to DTPA-cs124 (1.74 vs 1.5 ms), indicating higher lanthanide quantum yield resulting from the elimination of back emission energy transfer from Tb(3+) to the antenna molecule. Thiol-reactive forms of chelates were made for coupling to proteins. These lanthanide complexes are anticipated to be useful in a variety of fluorescence-based bioassays.

Chelating Agents↗