αB-Crystallin inhibits the cell toxicity associated with amyloid fibril formation by κ-casein and the amyloid-β peptide

Amyloid fibril formation is associated with diseases such as Alzheimer’s, Parkinson’s, and prion diseases. Inhibition of amyloid fibril formation by molecular chaperone proteins, such as the small heat-shock protein αB-crystallin, may play a protective role in preventing the toxicity associated with this form of protein misfolding. Reduced and carboxymethylated κ-casein (RCMκ-CN), a protein derived from milk, readily and reproducibly forms fibrils at physiological temperature and pH. We investigated the toxicity of fibril formation by RCMκ-CN using neuronal model PC12 cells and determined whether the inhibition of fibril formation altered its cell toxicity. To resolve ambiguities in the literature, we also investigated whether fibril formation by amyloid-β1–40 (Aβ_1–40), the peptide associated with Alzheimer’s disease, was inhibited by αB-crystallin and if this affected the toxicity of Aβ. To this end, either RCMκ-CN or Aβ_1–40 was incubated at neutral pH to induce fibril formation before treating PC12 cells and assessing cell viability. Incubated (fibrillar) RCMκ-CN was more toxic to PC12 cells than native RCMκ-CN with the highest level of toxicity being associated with mature fibrils and protofibrils. Furthermore, the toxicity of RCMκ-CN was attenuated when its fibril formation was inhibited, either through the chaperone action of αB-crystallin or when it interacted with its natural binding partners in milk, α_S- and β-casein. Likewise, incubating Aβ_1–40 with αB-crystallin inhibited both Aβ_1–40 fibril formation and the associated cell toxicity. Importantly, by inhibiting fibril formation, αB-crystallin prevents the cell toxicity associated with protein misfolding.     

Characterization of excitation beam on second-harmonic generation in fibrillous type I collagen

Following our established theoretical model to deal with the second-harmonic generation (SHG) excited by a linearly polarized focused beam in type I collagen, in this paper, we further quantitatively characterize the differences between SHG emissions in type I collagen excited by collimated and focused beams. The effects of the linear polarization angle (α) and the fibril polarity characterized by the hyperpolarizability ratio ρ on SHG emission has been compared under collimated and focused beam excitation, respectively. In particular, SHG emission components along the i axis ( I_2w,i )\left( {I_{2\omega {,}i} } \right) (i = x,y,z), the induced SHG emission deviation angle γ _ij , and the detected SHG signals (I _2ω,ij ) in the ij plane by rotating the applied polarizer angle φ _ij have been investigated (i = x, x, y; j = y, z, z). Results show that under our simulation model, SHG emission in the xy plane, such as I _2ω,x ,I _2ω,y ,γ _xy and I _2ω,xy varying as polarization angle (α) under collimated and focused light, presents no significant difference. The reverse of the fibril polarity has induced great impact on I _2ω,x ,γ _xy and I _2ω,xy in both collimated and focused light. I _2ω,x and γ _xy show similarity, but I _2ω,xy at α = 30° demonstrates a slight difference in focused light to that in collimated light. Under focused light, the reverse of fibril polarity causes obvious changes of the collected SHG intensity I _2ω,xz and I _2ω,yz at a special polarization angle α = 60° and γ _xz , γ _yz along α.     

Myogenic tone in mouse mesenteric arteries: evidence for P2Y receptor-mediated, Na+, K+, 2Cl− cotransport-dependent signaling

This study examines the action of agonists and antagonists of P2 receptors on mouse mesenteric artery contractions and the possible involvement of these signaling pathways in myogenic tone (MT) evoked by elevated intraluminal pressure. Both ATP and its non-hydrolyzed analog α,β-ATP triggered transient contractions that were sharply decreased in the presence of NF023, a potent antagonist of P2X₁ receptors. In contrast, UTP and UDP elicited sustained contractions which were suppressed by MRS2567, a selective antagonist of P2Y₆ receptors. Inhibition of Na⁺, K⁺, 2Cl⁻ cotransport (NKCC) with bumetanide led to attenuation of contractions in UTP- but not ATP-treated arteries. Both UTP-induced contractions and MT were suppressed by MRS2567 and bumetanide but were insensitive to NF023. These data implicate a P2Y₆-mediated, NKCC-dependent mechanism in MT of mesenteric arteries. The action of heightened intraluminal pressure on UTP release from mesenteric arteries and its role in the triggering of P2Y₆-mediated signaling should be examined further.     

Investigation of the functional expression of purine and pyrimidine receptors in porcine isolated pancreatic arteries

Receptors for purines and pyrimidines are expressed throughout the cardiovascular system. This study investigated their functional expression in porcine isolated pancreatic arteries. Pancreatic arteries (endothelium intact or denuded) were prepared for isometric tension recording and preconstricted with U46619, a thromboxane A₂ mimetic; adenosine-5′-diphosphate (ADP), uridine-5′-triphosphate (UTP) and MRS2768, a selective P2Y₂ agonist, were applied cumulatively, while adenosine-5′-triphosphate (ATP) and αβ-methylene-ATP (αβ-meATP) response curves were generated from single concentrations per tissue segment. Antagonists/enzyme inhibitors were applied prior to U46619 addition. ATP, αβ-meATP, UTP and MRS2768 induced vasoconstriction, with a potency order of αβ-meATP > MRS2768 > ATP ≥ UTP. Contractions to ATP and αβ-meATP were blocked by NF449, a selective P2X1 receptor antagonist. The contraction induced by ATP, but not UTP, was followed by vasorelaxation. Endothelium removal and DUP 697, a cyclooxygenase-2 inhibitor, had no significant effect on contraction to ATP but attenuated that to UTP, indicating actions at distinct receptors. MRS2578, a selective P2Y₆ receptor antagonist, had no effect on contractions to UTP. ADP induced endothelium-dependent vasorelaxation which was inhibited by MRS2179, a selective P2Y₁ receptor antagonist, or SCH58261, a selective adenosine A_2A receptor antagonist. The contractions to ATP and αβ-meATP were attributed to actions at P2X1 receptors on the vascular smooth muscle, whereas it was shown for the first time that UTP induced an endothelium-dependent vasoconstriction which may involve P2Y₂ and/or P2Y₄ receptors. The relaxation induced by ADP is mediated by P2Y₁ and A_2A adenosine receptors. Porcine pancreatic arteries appear to lack vasorelaxant P2Y₂ and P2Y₄ receptors.     

Effects of micromolar concentrations of Mn, Mo, and Si on α1adrenoceptor-mediated contraction in porcine coronary artery

We studied the effects of trace elements, Mn, Mo, and Si, on vasoconstriction induced by norepinephrine (NE) or electrical field stimulation in isolated porcine right coronary arteries. α₁-Adrenoceptor (AR) antagonist prazosin dose-despondently suppressed vasoconstriction in response to NE or field stimulation indicating an α₁-AR mediated response. Mn, Mo, and Si at 0.3-3 μmol/L dosedespondently inhibited NE mediated contraction (allp < 0.05). In contrast, Mn, Mo, and Si at the same concentrations (0.3-3 μmol/L) enhanced the maximal contractile response to field stimulation in a dose-dependent manner (allp < 0.05), but these elements at 10 μmol/L suppressed the vasoconstrictive response. The results indicate that in porcine right coronary arteries, the α₁-AR-mediated vasoconstriction by NE or electrical field stimulation was affected differently by micromolar concentrations of Mn, Mo, and Si and that the elements might facilitate NE release presynaptically but inhibit the contractile response postsynaptically.     

Transforming growth factor-β1 modulates the effect of 1α,25-dihydroxyvitamin D3 on leukemic cells

Summary The human leukemic cells HL-60, U937, KG-1 and THP-1 incubated with transforming growth factor-β1 (TGF-β1) were studied by examining cell surface antigens and macrophage-specific activities. The addition of 0.5 ng/ml (20 pM) of TGF-β1 with 1α,25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] induced more Leu-M3 (CD14)-positive cells (approximately 80%) than 5×10⁻⁸ M 1α,25(OH)₂D₃ alone did (30 to 50%), although original HL-60 cells did not express any Leu-M3 antigen at all. Tumor necrosis factor-α (TNF-α) with TGF-β1 and 1α,25(OH)₂D₃ was found to potentiate the expression of these surface antigens. Furthermore, the phagocytic activity was also induced strongly. The expression of CR3 (CD11b) antigen was also increased, and all Leu-M3-positive cells were found CR3-positive when HL-60, U937, and THP-1 cells were treated with these stimulants. In contrast, CR3 but not Leu-M3 was induced in KG-1 cells after the same treatment. This may indicate that the responsiveness of leukemic cells to TGF-β1 and 1α,25(OH)₂D₃ might vary depending on a differentiation stage of the target cells. Furthermore, K562 cells originated from a more undifferentiated precursor, were not able to respond to these two inducers. These results suggested that some of TGF-β superfamily proteins might represent potent modulators in hematopoiesis, especially in the development of monocytes-macrophages or their precursors.     

Relationship between tolerance and accumulation characteristics of cadmium in higher plants

Ten plant species belonging to 5 families,i.e., Cruciferae, Cucurbitaceae, Gramineae, Leguminosae, and Solanaceae, were grown in a sand soil at two pH levels. The soil was subjected to an application of CdCl₂ at rates of 0 to 700 mgCd·kg⁻¹ soil. The relationship between Cd concentration in the shoots (tc) and soil (sc; NH₄NO₃ extractable) was expressed by the equation: log (tc)=α+βlog (sc). The coefficients α and β were estimated for each species at each level of soil pH. Plottings of the scores on α and β axes showed that the Cd accumulation characteristics in the plants appeared to depend on the families irrespective of soil pH. Based on theupper critical concentration of Cd in the tops (C_t), the Cruciferae and Leguminosae species were found to be the most and the least tolerant to Cd, respectively. The C_t values correlated exceedingly well with the values of α.     

Rho GTPases mediated integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> activation in sphingosine-1-phosphate stimulated chemotaxis of endothelial cells

Integrins, a family of transmembrane heterodimeric polypeptides, mediate various biological responses including cell adhesion and migration. In this report, we show that sphingosine-1-phosphate (S1P) activates integrin α_vβ₃ in endothelial cells (ECs) via the sphingosine-1-phosphate receptor subtype 1 (S1P1)-mediated signaling pathway. S1P treatment results in the activation of integrin α_vβ₃ in the lamellipodia region of ECs, suggesting that integrin α_vβ₃ plays a critical role in the S1P-stimulated chemotactic response of ECs. Indeed, S1P treatment induces the association of focal adhesion kinase (FAK) and cytoskeletal proteins with integrin α_vβ₃, the ligation of α_v and β₃ subunits, as well as enhances endothelial migration on vitronectin-coated substrata. Knockdown endothelial S1P1 receptor, treatments with pertussis toxin or dominant-negative-Rho family GTPases abrogates the S1P-induced integrin α_vβ₃ activation in ECs. Consequently, these treatments markedly inhibit the S1P-induced endothelial migratory response on vitronectin-coated substrata. Collectively, these data indicate that the S1P-mediated signaling via the S1P1/G_i/Rho GTPases pathway activates integrin α_vβ₃, which is indispensable for S1P-stimulated chemotactic response of ECs.     

NMR studies on puerarin and its interaction with beta-cyclodextrin

The interaction between puerarin and β-cyclodextrin (CD) has been studied in D₂O, H₂O/acetone-d ₆, acetone-d ₆ and DMSO-d ₆ solutions by ¹H NMR spectroscopy. The NMR data obtained from hydroxy protons indicate that the formation of the inclusion complex between the two molecules is not stabilized by strong hydrogen bond interactions. The sugar part of puerarin as well as the A ring are outside the β-CD cavity while the B and C rings are located inside the cavity and the interaction is mainly stabilized by hydrophobic interactions. In DMSO at 30°C and in acetone-d ₆/H₂O at temperature below −5°C, doubling of some signals indicated that, in these solvent systems, free rotation of the C-glycosyl bond was restricted due to the steric hindrance between the phenolic hydroxy group at C-7 and the bulky sugar moiety at C-8. In acetone, fast exchange of phenolic protons on the NMR timescale was observed, showing the effect of the solvent on the hindered rotation.     

Rice <Emphasis Type="Italic">Importin β1</Emphasis> gene affects pollen tube elongation

Importin β1 interacts with nuclear transport factors and mediates the import of nuclear proteins. We isolated a pollen-expressed gene, rice Importin β1 (OsImpβ1), from a T-DNA insertional population that was trapped by a promoterless β-glucuronidase (GUS) gene. The GUS reporter was expressed in the anthers and ovaries from early through mature developmental stages. Its expression was also observed in all floral organs. However, these patterns changed as the spikelet developed. T-DNA was inserted into the OsImpβ1 gene at 339 bp downstream from the translation initiation site. We obtained another T-DNA insertional allele by searching the flanking sequence tag database. In both lines, the wild-type and T-DNA-carrying progeny segregated at a ratio close to 1:1. The latter genotype was heterozygous (OsImpβ1/osimpβ1). Reciprocal crosses between WT and heterozygous plants demonstrated that the mutant alleles could not be transmitted through the male gametophyte. Close examination of the heterozygous anthers revealed that the mutant pollen matured normally. However, in vitro assays showed that tube elongation was hampered in the mutant grains. These results indicate that OsImpβ1 is specifically required for pollen tube elongation.     

GABA Analogues Derived from 4-Aminocyclopent-1-enecarboxylic Acid

The incorporation of extra binding groups onto known ligands is a powerful tool for the development of more potent and selective agents at target sites such as the GABA receptors. In the present work we have attempted to build on the activity of the know potent GABA_A agonist 4-ACP-3-CA and its cis and trans saturated analogues CACP and TACP. We have investigated reactions to add thiol substituents to the α,β-unsaturated system of 4-ACP-3-CA. The reaction was successful with a limited number of thiols but gave products of mixed stereochemistry. The resultant thioether amino acids were screened for activity at human recombinant α₁β₂ γ_2L GABA_A receptors. The most interesting derivative was the benzylthioether which acted as an antagonist with an IC₅₀ of 42 μM for the inhibition of a GABA EC₅₀ dose (50 μM). This study has shown that GABA analogues derived by thiol addition to 4-aminocyclopent-1-enecarboxylic acid display interesting antagonist activity at the α₁β₂γ_2L GABA_A receptor. Special issue article in honour of Dr. Graham Johnston.     

Stereoelectronic Properties of N-acetyl-α,β-dehydroamino acid N′-methylamides

Summary α,β-Dehydroamino acids are useful peptide modifiers. However, their stereoelectronic properties still remain insufficiently recognized. Based on FTIR experiments in the range ofv _s(N-H), AI, AII andv _s(C^α=C^β) and ab initio calculations with B3LYP/6–31G*, we studied the solution conformational preferences and the amide electron density perturbation of Ac-ΔXaa-NHMe, where ΔXaa=ΔAla, (E)-ΔAbu, (Z)-ΔAbu, (Z)-ΔLeu, (Z)-ΔPhe and ΔVal. Each of these dehydroamides adopts a C₅ structure, which in Ac-ΔAla-NHMe is fully extended and accompanied by the strong C₅ hydrogen bond. Interaction with bond C^α=C^β lessens the amidic resonance within the flanking amide groups. TheN-terminal C=O bond is noticeably shorter, both amide bonds are longer than the corresponding bonds in the saturated entities and the N-terminal amide system is distorted. Ac-ΔAla-NHMe constitutes an exception. ItsC-terminal amide bond is shorter than the standard one and both amide systems are ideally planar. Ac-(E)-ΔAbu-NHMe shares stereoelectronic features with both Ac-ΔAla-NHMe and (Z)-dehydroamides.     

Extraction,Purification and Characterization of Thermostable,Alkaline Tolerant α-Amylase from <Emphasis Type="Italic">Bacillus cereus</Emphasis>

Thermostable alkaline α-amylase producing bacterium Bacillus cereus strain isolated from Cuddalore harbour waters grew maximally in both shake flask and fermentor, and produced α-amylase at 35°C, pH 7.5 and 1.0% of substrate concentrations. α-Amylase activity was maximum at 65°C, pH 8.0, 89% of its activity was sustained even at pH 11.0. Added with MnCl_2, α-amylase activity showed 4% increase but it was inhibited by EDTA. The molecular weight of the purified α-amylase is 42 kDa.     

Hemoglobin types in saanen goats and Barbary sheep: Genetic and comparative aspects

By the use of the Immobiline technique at pH ranges 7.0–7.6 and 6.9–7.9, 16 different hemoglobin (Hb) phenotypes were observed in 61 English Saanen goats. They are explained in this breed by a genetic theory of five β-globin genes (A ₄,A ₆,A ₈,E, andD) and two closely linked α-globin loci (′α and ″α) of which the ″α has a variant allele, provisionally called ″α ^X . Family data together with observed and expected Hb frequencies were in agreement with the genetic theory. Among six Barbary sheep there were three Hb phenotypes explained by the occurrence of the β-chain allelesB andC ^na.     

Structure and Mechanism of Vacuolar Na<Superscript>+</Superscript>-Translocating ATPase From <Emphasis Type="Italic">Enterococcus hirae</Emphasis>

V-type Na⁺-ATPase from Entercoccus hirae consists of nine kinds of subunits (NtpA₃, B₃, C₁, D₁, E₁₋₃, F₁₋₃, G₁, I₁, and K₁₀) which are encoded by the ntp operon. The amino acid sequences of the major subunits, A, B, and K (proteolipid), were highly similar to those of A, B, and c subunits of eukaryotic V-ATPases, and those of β, α, and c subunits of F-ATPases. We modeled the A and B subunits by homology modeling using the structure of β and α subunits of F-ATPase, and obtained an atomic structure of NtpK ring by X-ray crystallography. Here we briefly summarize our current models of the whole structure and mechanism of the E. hirae V-ATPase.     

Expression of Inhibin/activin Subunits alpha (-α), beta A (-β A) and beta B (-β B) in Placental Tissue of Normal and Intrauterine Growth Restricted (IUGR) Pregnancies

During human pregnancy the placenta produces a variety of proteins like steroid hormones and their receptors that are responsible for the establishment and ongoing of the feto-placental unit. Inhibins are dimeric glycoproteins, composed of an α-subunit and one of two possible β-subunits (β _A or β _B). Aims of the present study were the determination of the frequency and tissue distribution patterns of the inhibin/activin subunits in human placental tissue of normal pregnancies and pregnancies complicated with fetal growth restriction (IUGR). Slides of paraffin embedded placental tissue were obtained after delivery from patients diagnosed with IUGR (n = 6) and normal term placentas (n = 8). Tissue samples were fixed and incubated with monoclonal antibodies inhibin/activin-subunits -α, -β _A, -β _B. Intensity of immunohistochemical reaction on the slides was analysed using a semi-quantitative score and statistical analysis was performed (P<0.05). A significant lower expression of the inhibin-α subunit in IUGR extravillous trophoblast compared to normal pregnancies was observed, while the inhibin-α immunostaining was significantly upregulated in syncytiotrophoblast. Additionally, a significant down-regulation of inhibin-β _B subunit in extravillous trophoblast cells in IUGR syncytiotrophoblast cells was demonstrated. A co-localisation of inhibin-α and the β-subunits was also observed, suggesting a production and secretion of intact inhibin A and inhibin B. Although the precise role of these inhibin/activin subunits in human placenta and IUGR pregnancies is still unclear, they could be involved in autocrine/paracrine signalling, contributing to several aspects like angiogenesis and tissue remodelling.     

Microbial transformation of ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">Acremonium strictum</Emphasis>

Preparative-scale fermentation of ginsenoside Rb₁ (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12β-hydroxydammar-3-one-20 (S)-O-β-d-glucopyranoside (7), 12β, 25-dihydroxydammar-(E)-20(22)-ene-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8), and 12β, 20 (R), 25-trihydroxydammar-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg₃ (4), ginsenoside F₂ (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2–6 are also the metabolites of ginsenoside Rb₁ in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.     

Subunit Regulation of the Human Brain α1E Calcium Channel

The α₁ subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary α₂δ and β subunits. α_1E channels directed with the expression of Ba²⁺ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of α_1E with α_2bδ shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of α_1E with β_2a shifted the peak current relationship by −10 mV, and strongly reduced Ba²⁺ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 ± 10%, n= 8) of the Ba²⁺ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain β_2a and the neuronal α_2bδ subunits increased by ≈10-fold whole-cell Ba²⁺ currents although coinjection with either β_2a or α_2bδ alone failed to significantly increase α_1E peak currents. Coexpression with β_2a and α_2bδ yielded Ba²⁺ currents with inactivation kinetics similar to the β_2a induced currents, indicating that the neuronal α_2bδ subunit has little effect on α_1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four β subunit genes. The slower inactivation was unique to α_1E/β_2a currents. Coexpression with β_1a, β_1b, β₃, and β₄, yielded faster-inactivating Ba²⁺ currents than currents recorded from the α_1E subunit alone. Furthermore, α_1E/α_2bδ/β_1a; α_1E/α_2bδ/β_1b; α_1E/α_2bδ/β₃; α_1E/α_2bδ/β₄ channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The β subunit-induced changes in the properties of α_1E channel were comparable to modulation effects reported for α_1C and α_1A channels with β₃≈β_1b > β_1a≈β₄≫β_2a inducing fastest to slowest rate of whole-cell inactivation. Received: 27 March 1997/Revised: 10 July 1997     

Domain <Emphasis Type="Italic">a</Emphasis>’ of protein disulfide isomerase plays key role in inhibiting α-synuclein fibril formation

α-Synuclein (αSyn) is the main component of Lewy bodies formed in midbrain dopaminergic neurons which is a pathological characteristic of Parkinson's disease. It has been recently showed to induce endoplasmic reticulum (ER) stress and impair ER functions. However, the mechanism of how ER responds to αSyn toxicity is poorly understood. In the present study, we found that protein disulfide isomerase (PDI), a stress protein abundant in ER, effectively inhibits αSyn fibril formation in vitro. In PDI molecule with a structure of abb’xa’c, domain a’ was found to be essential and sufficient for PDI to inhibit αSyn fibril formation. PDI was further found to be more avid for binding with intermediate species formed during αSyn fibril formation, and the binding was more intensive in the later lag phase. Our results provide new insight into the role of PDI in protecting ER from the deleterious effects of misfolded protein accumulation in many neurodegenerative diseases.     

GFT projection NMR based resonance assignment of membrane proteins: application to subunit c of <Emphasis Type="Italic">E. coli</Emphasis> F<Subscript>1</Subscript>F<Subscript>0</Subscript> ATP synthase in LPPG micelles

G-matrix FT projection NMR spectroscopy was employed for resonance assignment of the 79-residue subunit c of the Escherichia coli F₁F₀ ATP synthase embedded in micelles formed by lyso palmitoyl phosphatidyl glycerol (LPPG). Five GFT NMR experiments, that is, (3,2)D HNNCO, L-(4,3)D HNNC ^αβ C ^α, L-(4,3)D HNN(CO)C ^αβ C ^α, (4,2)D HACA(CO)NHN and (4,3)D HCCH, were acquired along with simultaneous 3D ¹⁵N, ¹³C^aliphatic, ¹³C^aromatic-resolved [¹H,¹H]-NOESY with a total measurement time of ∼43 h. Data analysis resulted in sequence specific assignments for all routinely measured backbone and ¹³C^β shifts, and for 97% of the side chain shifts. Moreover, the use of two G²FT NMR experiments, that is, (5,3)D HN{N,CO}{C ^αβ C ^α} and (5,3)D {C ^αβ C ^α}{CON}HN, was explored to break the very high chemical shift degeneracy typically encountered for membrane proteins. It is shown that the 4D and 5D spectral information obtained rapidly from GFT and G²FT NMR experiments enables one to efficiently obtain (nearly) complete resonance assignments of membrane proteins. Qi Zhang, Hanudatta S. Atreya, Douglas E. Kamen, Mark E. Girvin and Thomas Szyperski—New York Consortium on Membrane Protein Structure.