Assessment of the alpha 1 beta 2 contact structure of valency hybrid hemoglobins by ultraviolet difference spectra

We have developed a rapid and useful method for purification of valency hybrid hemoglobins (alpha 2+ beta 2 and alpha 2 beta 2+: + denotes ferric heme) from a hemoglobin solution oxidized partially with ferricyanide by preparative high-performance liquid chromatography. This method does not involve the separation of hemoglobin subunits and the reconstitution of ferric and partner ferrous subunits. Using the valency hybrid hemoglobins thus prepared, the effect of the ferric spin state on the alpha 1 beta 2 subunit boundary structure was investigated by measuring the ultraviolet difference absorption spectra between the deoxy and the oxy valency hybrids associated with various ferric ligands (fluoride, aquo, azide and cyanide). All derivatives of both alpha 2+ beta 2 and alpha 2 beta 2+ showed the difference spectra characteristic of R-T quaternary structural transition. However, the magnitude of the difference spectral peak observed near 288 nm was larger for high-spin derivatives than for low-spin ones. The magnitude of the peak for the valency hybrid hemoglobin was closely correlated with the difference in the free energy of oxygen binding between the R and T states. Since the R state of high-spin hybrids is considered to be identical to that of low-spin hybrids, we concluded from these results that the alpha 1 beta 2 subunit boundary structure plays an important role in regulating the oxygen affinity of deoxy T state.     

Spectral changes upon subunit association in valency hybrid hemoglobins

The absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of valency hybrid hemoglobins and their constituents (alpha + and beta chains for alpha 2+beta 2, alpha and beta + chains for alpha 2 beta 2+: + denotes ferric heme) were measured in the Soret region for F-, H2O, N3- and CN- derivatives. Absorption and MCD spectra of valency hybrid hemoglobins were very similar to the arithmetic mean of respective spectra of their corresponding component chains in all derivatives. The Soret MCD intensity around 408 nm for various complexes of valency hybrid hemoglobins seems to reflect the spin state of ferric chains. Upon ferric and deoxy ferrous subunit association to make the deoxy valency hybrid hemoglobins, only the high-spin forms bound with F- and H2O of alpha 2+beta 2 displayed a blue shift in the peak position around 430 nm and those of alpha 2 beta 2+ an increase in intensity around 430 nm. The blue shift and the increase in intensity were considered to be caused by the structural changes in deoxy beta chains of alpha 2+beta 2 and deoxy alpha chains of alpha beta 2+, respectively. These spectral changes were interpreted on the basis of their oxygen-equilibrium properties. In contrast to absorption and MCD spectra, the CD spectra of valency hybrid hemoglobins were markedly different from the simple addition of those of their component chains in all derivatives examined. The large part of CD spectral changes upon subunit association were interpreted as changes in the heme vicinity accompanied by formation of the alpha 1 beta 1 subunit contact.     

Differential expression of VLA-alpha 4 and VLA-beta 1 discriminates multiple subsets of CD4+CD45R0+ "memory" T cells.

Given the importance of adhesion in T cell development, we have undertaken systematic flow cytometric analysis of CD4 T cells to determine relationships between the developmentally regulated marker CD45R0 and adhesion receptors (five VLA integrin chains). The most important findings are that: 1) expression of alpha 3, alpha 5, and alpha 6 are closely coregulated with beta 1 on CD4 cells, while regulation of VLA-alpha 4 is quite discordant. 2) CD45R0- cells, generally understood to be naive cells, have low homogeneous expression of VLA-alpha 3, VLA-alpha 4, VLA-alpha 5, VLA-alpha 6, and beta 1 integrin chains; studies of cord blood CD4 cells confirm the low homogeneous expression of alpha 4 and beta 1 on naive cells. 3) In marked contrast, CD45R0+ cells, generally understood to be memory cells, show not only an overall increase in expression of these integrins (relative to CD45R0- cells) but also heterogeneity. Dramatic heterogeneity is revealed when the markers VLA-alpha 4 and beta 1 are analyzed together. Many CD45R0+ cells show increased levels of both VLA-alpha 4 and VLA-beta 1; however, some have increased levels principally of either VLA-beta 1 or VLA-alpha 4. We hypothesize that T cells becoming memory cells in different microenvironments specialize their integrin phenotype, thereby acquiring distinctive functional and homing capacities; in this process, VLA-4 (CD49d) appears to play a unique role.     

Changes of near-UV CD spectrum of human hemoglobin upon oxygen binding: a study of mutants at alpha 42, alpha 140, beta 145 tyrosine or beta 37 tryptophan

The deoxy-form of human adult hemoglobin (Hb A) exhibits a distinct negative CD band at 287 nm that disappears in the oxy-form. It has been suggested that the negative CD band is due to the environmental alteration of Tyr-alpha 42 or Trp-beta 37 at the alpha(1)beta(2) contact upon deoxygenation. To evaluate the contributions of the aromatic residues at the alpha(1)beta(2) contact and the penultimate tyrosine residues of the alpha and beta subunits (alpha 140 and beta 145) to the negative CD band, three recombinant (r) Hbs (rHb Ser-alpha 42, rHb His-beta 37, and rHb Thr-beta 145) were produced in Escherichia coli, and we compared the near-uv CD spectra of these three rHbs and Hb Rouen (Tyr-alpha 140-->His) with the spectra of Hb A under the condition in which all mutant Hbs were able to undergo the T-->R transition (Hill's n > 2.0). The contributions of Tyr-alpha 42, Trp-beta 37, Tyr-alpha 140, and Tyr-beta 145 to the negative CD band were estimated from changes in the ellipticity of the negative CD band at 287 nm to be 4, 18, 32, and 27%, respectively. These results indicate that environmental alteration of the penultimate tyrosine residues caused by the formation of salt bridges upon the R-->T transition is primarily responsible for the negative CD band.     

Characterization of a dissimilatory-type sulfite reductase, desulfoviridin, from Desulfovibrio africanus Benghazi

A desulfoviridin-type sulfite reductase having the alpha band at 638 nm was purified from Desulfovibrio africanus Benghazi (NCIB 8401) by chromatography on DEAE-cellulose, Sephadex G-200, and DEAE-Sepharose columns and by disc gel electrophoresis. The content of desulfoviridin in the soluble protein was estimated to be about 6% from the purification indexes. Like the typical desulfoviridin from D. vulgaris Miyazaki K, it formed mainly trithionate besides thiosulfate and sulfide in sulfite reduction coupled to hydrogenase and methyl viologen. No significant differences in the amino acid compositions, CD patterns in the UV (205-250 nm) region, and subunit structures were found, except for a pI value about 1 unit larger (pI 5.3). The split Soret (410 +/- 2 nm, less intense peak at 391 +/- 2 nm with a shoulder around 380 nm) and beta (584 +/- 2 nm) band maxima of the enzyme as isolated, and the visible absorption and fluorescence spectra of the acidic acetone-extracted chromophore were almost identical to those ascribed to sirohydrochlorin in spite of the reported difference in the native enzyme (alpha band maxima at 638 nm as against 628 +/- 2 nm in a typical desulfoviridin). Iron was the only significant chelatable metal contained in the chromophore. Some differences between africanus and vulgaris desulfoviridins were observed in the CD patterns in the UV to near UV region (250-340 nm) and also in the visible absorption spectra in the presence of dithionite.     

Contribution of alpha140Tyr and beta37Trp to the near-UV CD spectra on quaternary structure transition of human hemoglobin A

The CD band of human adult hemoglobin (Hb A) at 280 approximately 290 nm shows a pronounced change from a small positive band to a definite negative band on the oxy (R) to deoxy (T) structural transition. This change has been suggested to be due to environmental alteration of Tyrs (alpha42, alpha140, and beta145) or beta37 Trp residues located at the alpha1beta2 subunit interface by deoxygenation. In order to evaluate contributions of alpha140Tyr and beta37Trp to this change of CD band, we compared the CD spectra of two mutant Hbs, Hb Rouen (alpha140Tyr-->His) and Hb Hirose (beta37Trp-->Ser) with those of Hb A. Both mutant Hbs gave a distinct, but smaller negative CD band at 287nm in the deoxy form than that of deoxyHb A. Contributions of alpha140Tyr and beta37Trp to the negative band at the 280 approximately 290 nm region were estimated from difference spectra to be 30% and 26%, respectively. These results indicate that the other aromatic amino acid residues, alpha42Tyr and beta145Tyr, at the alpha1beta2 interface, are also responsible for the change of the CD band upon the R-->T transition of Hb A.     

The domain structure of tryptophan synthase. A neutron scattering study

Tryptophan synthase from Escherichia coli is a complex of two alpha subunits and two beta subunits. Small-angle neutron scattering involving deuterium-labelled isomers revealed the quaternary structure of the enzyme at the level of the beta 2 subunit and the two structural domains P1 and P2 which constitute the alpha subunits. Within the alpha 2 beta 2 complex, the two alpha subunits are completely separated. They are situated on opposite sides of the beta 2 subunit. The most probable distance between the two alpha protomers is 10.5 +/- 1 nm; the nearest distance is 5.8 +/- 0.5 nm, and the largest distance is 13.5 +/- 0.5 nm. The two domains of the same alpha subunit are intimately juxtaposed. The distances between two like or unlike domains belonging to opposite alpha subunits are roughly equal. All domains exhibit about equal distances to the beta 2 subunit which is situated in the centre of the complex. Thus the cleft between P1 and P2, which probably contains the active site of the alpha subunit, makes intimate contact with the beta 2 subunit. Neutron scattering allows us to determine the shape of the beta 2 subunit within the complex. Comparison with the free dimer suggests a conformational change, upon assembly, from an elongated into a more compact form.     

Effects of T3R alpha 1 and T3R alpha 2 gene deletion on T and B lymphocyte development

Thyroid hormones bind to several nuclear receptors encoded by T3R alpha and T3R beta genes. There is now accumulating evidence that thyroid hormones act on the immune system. Indeed, mice deficient for thyroid hormones show a reduction in lymphocyte production. However, the mechanisms involved and, in particular, the role of the different thyroid hormone receptors in lymphocyte development have not been investigated. To address that question, we have studied lymphocyte development in mice deficient for the T3R alpha 1 and T3R alpha 2 gene products. A strong decrease in spleen cell numbers was found compared with wild-type littermates, B lymphocytes being more severely affected than T lymphocytes. A significant decrease in splenic macrophage and granulocyte numbers was also found. In bone marrow, a reduction in CD45+/IgM- pro/pre-B cell numbers was found in these mice compared with wild-type littermates. This decrease seems to result from a proliferation defect, as CD45+/IgM- cells incorporate less 5-bromo-2'-deoxyuridine in vivo. To define the origin of the bone marrow development defect, chimeric animals between T3R alpha-/- and Rag1-/- mice were generated. Results indicate that for B cells the control of the population size by T3R alpha 1 and T3R alpha 2 is intrinsic. Altogether, these results show that T3R alpha 1 or T3R alpha 2 gene products are implicated in the control of the B cell pool size.     

Allosteric kinetics and equilibria of triligated, cross-linked hemoglobin.

Using modulated excitation, we have measured the forward and reverse rates of the allosteric transition between relaxed (R) and tense (T) quaternary structures for triply ligated hemoglobin (Hb), cross-linked between the alpha chains at Lys 99. Oxygen, carbon monoxide, and water were used as ligands and were studied in phosphate and low Cl- bis-Tris buffers at neutral pH. Since the cross-link prohibits disproportionation, triply ligated aquomet Hb species with ferrous beta chains were specifically isolated by isoelectric focusing. Modulated excitation provides rate pairs and therefore gives equilibrium constants between quaternary structures. To coordinate with that information, oxygen binding curves of fully ferrous and tri-aquomet Hb were also measured. L3, the equilibrium constant between three liganded R and T structures, is determined by modulated excitation to be of order unity for O2 or CO (1.1 to 1.5 for 3O2 and 0.7 for 3CO bound), while with three aquomet subunits it is much greater (> or = 23). R-->T conversion rates are similar to those found for HbA, with weak sensitivity to changes in L3. The L3 values from HbXL O2 were used to obtain a unique allosteric decomposition of the ferrous O2 binding curve in terms of KT, KR, and L3. From these values and the O2 binding curve of tri-aquomet HbXL, L3 was calculated to be 2.7 for the tri-aquomet derivative. Consistency in L3 values between equilibrium and modulated excitation data for tri-aquomet-HbXL can be achieved if the equilibrium constant for O2 binding to the alpha chains is six times lower than that for binding to the beta chains in the R state, while the cooperative properties remain homogeneous. The results are in quantitative agreement with other studies, and suggest that the principal effect of the cross-link is to decrease the R state and T state affinity of the alpha subunits with almost no change in the affinity of the beta subunits, leaving the allosteric parameters L and c unchanged.     

CD8alphabeta has two distinct binding modes of interaction with peptide-major histocompatibility complex class I

Interaction of CD8 (CD8alphaalpha or CD8alphabeta) with the peptide-major histocompatibility complex (MHC) class I (pMHCI) is critical for the development and function of cytolytic T cells. Although the crystal structure of CD8alphaalpha.pMHCI complex revealed that two symmetric CD8alpha subunits interact with pMHCI asymmetrically, with one subunit engaged in more extensive interaction than the other, the details of the interaction between the CD8alphabeta heterodimer and pMHCI remained unknown. The Ig-like domains of mouse CD8alphabeta and CD8alphaalpha are similar in the size, shape, and surface electrostatic potential of their pMHCI-binding regions, suggesting that their interactions with pMHCI could be very similar. Indeed, we found that the CD8alpha variants CD8alpha(R8A) and CD8alpha(E27A), which were functionally inactive as homodimers, could form an active co-receptor with wild-type (WT) CD8beta as a CD8alpha(R8A)beta or CD8alpha(E27A)beta heterodimer. We also identified CD8beta variants that could form active receptors with WT CD8alpha but not with CD8alpha(R8A). This observation is consistent with the notion that the CD8beta subunit may replace either CD8alpha subunit in CD8alphaalpha.pMHCI complex. In addition, we showed that both anti-CD8alpha and anti-CD8beta antibodies were unable to completely block the co-receptor activity of WT CD8alphabeta. We propose that CD8alphabeta binds to pMHCI in at least two distinguishable orientations.     

Ruthenium-iron hybrid hemoglobins as a model for partially liganded hemoglobin: NMR studies of their tertiary and quaternary structures

Diruthenium-substituted Ru-Fe hybrid hemoglobins (Hb) were synthesized by heme substitution from protoheme to ruthenium (II) carbonyldeuteroporphyrin in the alpha or beta subunits. As the carbon monoxide coordinated to ruthenium (II) is not released under physiological conditions, deoxygenated Ru-Fe hybrid derivatives [alpha(Fe)2 beta(Ru-CO)2 and alpha(Ru-CO)2 beta(Fe)2] can serve as models for half-liganded Hbs. On the basis of proton NMR spectra of hyperfine-shifted proton resonances, these Ru-Fe hybrid Hbs have only small structural changes in the heme environment of the partner subunits at low pH. The proton NMR spectra of the intersubunit hydrogen-bonded protons also showed that the quaternary structures of the two complementary hybrids both remain in the "T-like state" at low pH, suggesting that the T to R structural conversion is induced by ligation of the third ligand molecule. Marked conformational changes in the heme vicinity are observed at high pH only for alpha(Ru-CO)2 beta(Fe)2, and its quaternary structure is converted into the "R state"; the alpha(Fe)2 beta(Ru-CO)2 hybrid does not undergo this change. This implies that the free-energy difference between the two quaternary states is smaller in the alpha-liganded hybrid than in the beta-liganded one.     

Structure of tubulin C-terminal domain obtained by subtilisin treatment. The major alpha and beta tubulin isotypes from pig brain are glutamylated.

Limited subtilisin digestion of the tubulin alpha, beta heterodimer has been used in this work to reduce the total number of tubulin isotypes from 20 for native to 9 for subtilisin-cleaved tubulin. This indicates that the major part of tubulin heterogeneity is located at the C-terminus of the molecule. The C-terminal peptides of both alpha and beta subunits of tubulin were purified by anion-exchange HPLC. Combined use of Edman degradation chemistry and mass spectrometry on the isolated peptides shows that subtilisin cleavage occurs at position Asp-438 and His-406 of alpha and Gln-433 and His-396 of beta tubulin chains. Quantitative analysis of our data show that cleavage at positions His-406 (alpha) and His-396 (beta) occurs with a low efficiency and indicates that the major isotypes of pig brain tubulin are modified by sequential attachment of 1 to 5 glutamic acid residues at positions Glu-445 or -435 of alpha and beta tubulin, respectively.     

Chemical cross-linking of bovine retinal transducin and cGMP phosphodiesterase

The bifunctional reagents para-phenyldimaleimide and maleimidobenzoyl-N-hydroxysuccinimide ester were used to chemically cross-link the subunits of the transducin and cGMP phosphodiesterase (PDE) complexes of bovine rod photoreceptor cells. The cross-linked products were identified by Western immunoblotting using antisera against purified subunits of transducin (T alpha and T beta gamma) and PDE. Oligomeric cross-linked products of transducin subunits as large as (T alpha beta gamma)3 were observed in the latent form of transducin with bound GDP. In addition to the expected T alpha beta and T beta gamma cross-linked products, a (T alpha gamma)2 structure was detected. The close proximity of T alpha and T gamma suggests that T gamma may play a role in conferring the specificity of the interaction between T alpha and rhodopsin. Most of the oligomeric cross-linked structures between T alpha and T beta gamma were diminished in the activated form of transducin, with guanosine 5'-(beta, gamma-imidotriphosphate) (Gpp(NH)p) bound. However, cross-linking between T beta and T gamma was not altered. These results suggest that transducin exists as an oligomer in solution which dissociates upon the binding of Gpp(NH)p. To identify the possible interacting domains between the T alpha, T beta, and T gamma subunits, the cross-linked products were subjected to limited tryptic proteolysis. Several cross-linked tryptic peptides of transducin subunits were found and include the cross-linked products of the N terminus 15-kDa fragment of T beta and the C terminus 5-kDa fragment of T alpha, T gamma and the 12-kDa fragment of T alpha, T gamma and the 15-kDa as well as the 23-kDa fragments of T beta, and an intra-T alpha cross-linked product of the 2- and 21-kDa fragments. These results have allowed the construction of a topographical model for the transducin subunits. The organization of the subunits of PDE (P alpha, P beta, and P gamma) was also studied. The formation of the high molecular size cross-linked products of PDE resulted in the concurrent loss of the P beta and P gamma subunits, suggesting that they are in close proximity. Finally, the interaction between transducin and PDE was examined by chemical cross-linking of transducin-Gpp(NH)p and PDE. Two additional cross-linked products of 180 and 210 kDa were obtained which could be due to the cross-linking of T alpha or T beta with P alpha beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of the T cell antigen receptor (TCR) complex by UDP-glucose:glycoprotein glucosyltransferase. TCR folding is finalized convergent with formation of alpha beta delta epsilon gamma epsilon complexes.

Most T lymphocytes express on their surfaces a multisubunit receptor complex, the T cell antigen receptor (TCR) containing alpha, beta, gamma, delta, epsilon, and zeta molecules, that has been widely studied as a model system for protein quality control. Although the parameters of TCR assembly are relatively well established, little information exists regarding the stage(s) of TCR oligomerization where folding of TCR proteins is completed. Here we evaluated the modification of TCR glycoproteins by the endoplasmic reticulum folding sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (GT) as a unique and sensitive indicator of how TCR subunits assembled into multisubunit complexes are perceived by the endoplasmic reticulum quality control system. These results demonstrate that all TCR subunits containing N-glycans were modified by GT and that TCR proteins were differentially reglucosylated during their assembly with partner TCR chains. Importantly, these data show that GT modification of most TCR subunits persisted until assembly of CD3alpha beta chains and formation of CD3-associated, disulfide-linked alpha beta heterodimers. These studies provide a novel evaluation of the folding status of TCR glycoproteins during their assembly into multisubunit complexes and are consistent with the concept that TCR folding is finalized convergent with formation of alpha beta delta epsilon gamma epsilon complexes.     

A circular dichroism study of sheath contraction in pyocin R1

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, is a protein particle shaped like a bacteriophage tail composed of a contractile sheath, core, baseplate and tail fibers. Alkaline treatment with sodium carbonate caused sheath contraction without considerable disassembly of other components. Circular dichroism (CD) spectra of pyocin R1 before and after the treatment, and of isolated sheath, were measured in wavelength regions around 220 and 290 nm at neutral pH. The alkaline treatment caused a red shift of the minimum from 208 nm to 212 nm. A marked difference in the CD spectrum was found in the near-ultraviolet region. THe difference is considered to be mainly due to a CD spectra change of tryptophan residues in the sheath subunits.     

The gamma and epsilon subunits of the CD3 complex inhibit pre-Golgi degradation of newly synthesized T cell antigen receptors

The T cell receptor for antigen (TCR) is composed of six different transmembrane proteins. T cells carefully control the intracellular transport of the receptor and allow only complete receptors to reach the plasma membrane. In an attempt to understand how T cells regulate this process, we used c-DNA transfection and subunit-specific antibodies to follow the intracellular transport of five subunits (alpha beta gamma delta epsilon) of the receptor. In particular, we assessed the intracellular stability of each chain. Our results showed that the chains were markedly different in their susceptibility to intracellular degradation. TCR alpha and beta and CD3 delta were degraded rapidly, whereas CD3 gamma and epsilon were stable. An analysis of the N-linked oligosaccharides of the glycoprotein subunits suggested that the chains were unable to reach the medial Golgi during the metabolic chase. This was supported by immunofluorescence micrographs that showed both the stable CD3 gamma and unstable CD3 delta chain localized in the endoplasmic reticulum. To study the effects of subunit associations on intracellular transport we used cotransfection to reconstitute precise combinations of subunits. Associations between stable and unstable subunits expressed in the same cell led to the formation of stable complexes. These complexes were retained in or close to the endoplasmic reticulum. The results suggested that the intracellular transport of the T cell receptor could be regulated by two mechanisms. The TCR alpha and beta and CD3 delta subunits were degraded rapidly and as a consequence failed to reach the plasma membrane. CD3 gamma or epsilon were stable but were retained inside the cell. The results also demonstrated that there was an interplay between the two pathways such that the CD3 gamma and epsilon subunits were able to protect labile chains from rapid intracellular degradation. In this way, they could seed subunit assembly in or close to the endoplasmic reticulum and allow a stable receptor to form before its transport to the plasma membrane.     

The three-dimensional structure of guanine-specific ribonuclease F1 in solution determined by NMR spectroscopy and distance geometry.

Two-dimensional 1H-NMR studies have been performed on ribonuclease F1 (RNase F1), which contains 106 amino acid residues. Sequence-specific resonance assignments were accomplished for the backbone protons of 99 amino acid residues and for most of their side-chain protons. The three-dimensional structures were constructed on the basis of 820 interproton-distance restraints derived from NOE, 64 distance restraints for 32 hydrogen bonds and 33 phi torsion-angle restraints. A total of 40 structures were obtained by distance geometry and simulated-annealing calculations. The average root-mean-square deviation (residues 1-106) between the 40 converged structures and the mean structure obtained by averaging their coordinates was 0.116 +/- 0.018 nm for the backbone atoms and 0.182 +/- 0.015 nm for all atoms including the hydrogen atoms. RNase F1 was determined to be an alpha/beta-type protein. A well-defined structure constitutes the core region, which consists of a small N-terminal beta-sheet (beta 1, beta 2) and a central five-stranded beta-sheet (beta 3-beta 7) packed on a long helix. The structure of RNase F1 has been compared with that of RNase T1, which was determined by X-ray crystallography. Both belong to the same family of microbial ribonucleases. The polypeptide backbone fold of RNase F1 is basically identical to that of RNase T1. The conformation-dependent chemical shifts of the C alpha protons are well conserved between RNase F1 and RNase T1. The residues implicated in catalysis are all located on the central beta-sheet in a geometry similar to that of RNase T1.     

Closed-state cross-linking of adjacent beta1 subunits in alpha1beta1 GABAa receptors via introduced 6' cysteines

The pore structural changes associated with Cys-loop receptor gating are currently the subject of considerable interest. Several functional approaches have shown that surface exposure of pore-lining side chains does not change significantly during activation. However, a disulfide trapping study on alpha1(T6'C)beta1(T6'C) gamma-aminobutyric acid type A (GABA(A)) receptors (GABA(A)Rs), which showed that adjacent beta subunits cross-link in the open state only, concluded that channel gating is mediated by beta subunits contra-rotating through a summed angle of approximately 120 degrees. Such a large rotation is not easily reconciled with other evidence. The present study initially sought to investigate an observation that appeared inconsistent with the rotation model: namely that alpha1(T6'C)beta1(T6'C) GABA(A)Rs expressed in HEK293 cells form 6' cysteine-mediated disulfide bonds in the closed state. On the basis of electrophysiological and Western blotting experiments, we conclude that adjacent beta(T6'C) subunits dimerise efficiently in the closed state via cross-links between their respective 6' cysteines and that this locks the channels closed. This questions the beta subunit contra-rotation model of activation and raises the question of how the closed state cross-links form. We propose that beta subunit 6' cysteines move into sufficiently close proximity for disulfide formation via relatively large amplitude random thermal motions that appear to be a unique feature of beta subunits. Because dimerized channels are locked closed, we conclude either that the spontaneous beta subunit movements or asymmetries in the movements of adjacent beta subunits during activation are essential for pore opening. Our results identify a novel feature of GABA(A)R gating that may be important for understanding its activation mechanism.     

Interaction of pirprofen enantiomers with human serum albumin.

The interaction of pirprofen enantiomers with human serum albumin (HSA) was investigated by means of high-performance liquid chromatography (HPLC), circular dichroism (CD), and 1H NMR spectroscopy. HPLC experiments indicated that both pirprofen enantiomers were bound to one class of high-affinity binding sites (n(+) = 1.91 +/- 0.13, K(+) = (4.09 +/- 0.64) x 10(5) M-1, n(-) = 2.07 +/- 0.13, K(-) = (6.56 +/- 1.35) x 10(5) M-1) together with nonspecific binding (n'K'(+) = (1.51 +/- 0.21) x 10(4) M-1, n'K'(-) = (0.88 +/- 0.13) x 10(-4) M-1). Slight stereoselectivity in specific binding was demonstrated by the difference in product n(+)K(+) = (0.77 +/- 0.08) x 10(6) M-1 vs. n(-)K(-) = (1.30 +/- 0.21) x 10(6) M-1, i.e., the ratio n(-)K(-)/n(+)K(+) = 1.7. CD measurements showed changes in the binding sites located on the aromatic amino acid side chains (a small positive band at 315 nm and a pronounced negative extrinsic Cotton effect in the region 250-280 nm). The protein remains, however, in its predominantly alpha-helical conformation. The 1H NMR difference spectra confirmed that both pirprofen enantiomers interacted with HSA specifically, most probably with site II on the albumin molecule.