Effect of lipid-protein interaction on the color of bacteriorhodopsin

Detergent solubilization and subsequent delipidation of bacteriorhodopsin (bR) results in the formation of a new species absorbing maximally at 480 nm (bR480). Upon lowering the pH, its absorption shifts to 540 nm (bR540). The pK of this equilibrium is 2.6, with the higher pH favoring bR480 (Baribeau, J. and Boucher, F. (1987) Biochim. Biophysica Acta, 890, 275-278). Resonance Raman spectroscopy shows that bR480, like the native bR, contains a protonated Schiff base (PSB) linkage between the chromophore and the protein. However, the Schiff base vibrational frequency in bR480, and its shift upon deuteration, are quite different from these in the native bR, suggesting changes in the Schiff base environment upon delipidation. Infrared absorption and circular-dichroism (CD) spectral studies do not show any net change in the protein secondary structure upon formation of bR480. It is shown that deprotonation of the Schiff base is not the only mechanism of producing hypsochromic shift in the absorption maximum of bR-derived pigments, subtle changes in the protein tertiary structure, affecting the Schiff base environment of the chromophore, may play an equally significant role in the color regulation of bR-derived pigments.     

Deep-breath frequency in bronchoconstricted monkeys (Macaca fascicularis).

Deep-breath frequency has been shown to increase in spontaneously obstructed asthmatic subjects. Furthermore, deep breaths are known to be regulated by lung rapidly adapting receptors, yet the mechanism by which these receptors are stimulated is unclear. This study tested the hypothesis that deep-breath frequency increases during experimentally induced bronchoconstriction, and the magnitude of the increased deep-breath frequency is dependent on the method by which bronchoconstriction is induced. Nine cynomolgus monkeys (Macaca fascicularis) were challenged with methacholine (MCh), Ascaris suum (AS), histamine, or an external mechanical resistance. Baseline (BL) and challenge deep-breath frequency were calculated from the number of deep breaths per trial period. Airway resistance (Raw) and tissue compliance (Cti), as well as tidal volume, respiratory rate, and minute ventilation, were analyzed for BL and challenged conditions. Transfer impedance measurements were fit with the DuBois model to determine the respiratory parameters (Raw and Cti). The flow at the airway opening was measured and analyzed on a breath-by-breath basis to obtain the ventilatory parameters (tidal volume, respiratory rate, and minute ventilation). Deep-breath frequency resulting from AS and histamine challenges [0.370 (SD 0.186) and 0.467 breaths/min (SD 0.216), respectively] was significantly increased compared with BL, MCh, or external resistance challenges [0.61 (SD 0.046), 0.156 (SD 0.173), and 0.117 breaths/min (SD 0.082), respectively]. MCh and external resistance challenges resulted in insignificant changes in deep-breath frequency compared with BL. All four modalities produced similar levels of bronchoconstriction, as assessed through changes in Raw and Cti, and had similar effects on the ventilatory parameters except that non-deep-breath tidal volume was decreased in AS and histamine. We propose that increased deep-breath frequency during AS and histamine challenge is the result of increased vascular permeability, which acts to increase rapidly adapting receptor activity.     

Probing protein dynamics using temperature jump relaxation spectroscopy

There have been recent advances in initiating and perturbing chemical reactions on very fast timescales, as short as picoseconds, thus making it feasible to study a vast range of chemical kinetics problems that heretofore could not be studied. One such approach is the rapid heating of water solutions using laser excitation. Laser-induced temperature jump relaxation spectroscopy can be used to determine the dynamics of protein motion, an area largely unstudied for want of suitable experimental and theoretical probes, despite the obvious importance of dynamics to protein function. Coupled with suitable spectroscopic probes of structure, relaxation spectroscopy can follow the motion of protein atoms over an enormous time range, from picoseconds to minutes (or longer), and with substantial structural specificity.     

An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21

We have measured the Raman spectrum of GDP bound to the elongation factor protein, EF-Tu, and the c-Harvey-ras protein, p21, two proteins of the guanine nucleotide binding family. In order to separate the Raman spectrum of the nucleotide from the much more intense protein spectrum, we investigate the feasibility of "tagging" the normal modes of the nucleotide by isotopic substitution, here by incoporating deuterium-labeled guanine at the C8 position into the active site. A difference spectrum between the labeled and unlabeled protein-nucleotide complex shows the changes in the Raman spectrum of the bound nucleotide that arise from the isotopic exchange. We find that surprisingly good Raman spectra of bound ligands can be obtained with this method and that the method can be easily generalized to other systems. The data show that the guanine amino group of the nucleotide interacts differently with both EF-Tu and p21 than it does with water, showing a change in hydrogen-bonding properties upon binding. On the other hand, no change in hydrogen bonding is observed at guanine's N7. The data strongly suggest that the conformation of the nucleotide when bound to EF-Tu and that p21 is the C2' endo pucker of the ribose ring and anti about the glycosidic bond. These results are compared to previous structural and chemical studies.     

Immunoprophylaxis of respiratory syncytial virus: global experience

In sarcoidosis, host genetic factors are discussed as contributing to disease susceptibility and course. Since tumor necrosis factor (TNF)-α is a central mediator of granuloma formation and since elevated TNF-α levels are found during active phases of sarcoidosis, genetic polymorphisms correlating with influences on TNF-α levels are of special interest. The complete sequencing of the MHC region and the increase in the number of identified gene polymorphisms in this locus associated with TNF-α production offer the opportunity of detecting new genes associated with sarcoidosis and perhaps of defining disease-associated haplotypes that bear the potential of serving as predictive markers for this disease.     

Is photobleaching necessary for Raman imaging of bone tissue using a green laser?

Raman microspectroscopy is widely used for musculoskeletal tissues studies. But the fluorescence background obscures prominent Raman bands of mineral and matrix components of bone tissue. A 532-nm laser irradiation has been used efficiently to remove the fluorescence background from Raman spectra of cortical bone. Photochemical bleaching reduces over 80% of the fluorescence background after 2 h and is found to be nondestructive within 40 min. The use of electron multiplying couple charge detector (EMCCD) enables to acquire Raman spectra of bone tissues within 1-5 s range and to obtain Raman images less than in 10 min.     

Determination of the ionization state and catalytic function of Glu-133 in peptide deformylase by difference FTIR spectroscopy

Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria and the organelles of certain eukaryotes. PDF is a novel class of amide hydrolase, which utilizes an Fe2+ ion to effect the hydrolysis of an amide bond. The ferrous ion is tetrahedrally coordinated by two histidines from a conserved HEXXH motif, a cysteine, and a water molecule. In this work, the function of the conserved glutamate (Glu-133 in Escherichia coli PDF) is evaluated by difference FTIR spectroscopic analysis of a Co(II)-substituted E. coli wild-type and E133D mutant PDF. At pH <6, the wild-type enzyme exhibited a relatively sharp C=O stretch band at 1742 cm(-1), which is assigned to the COOH group of Glu-133. The pH titration study and curve fitting to the data revealed a pK(a) of 6.0 for Glu-133 (in the presence of 500 mM NaCl). For the E133D mutant, which is only approximately 10-fold less active than the wild-type enzyme, a similar pH titration study of the Asp-133 C=O stretch band at 1740 cm(-1) revealed a pK(a) of 10.1. This unusually high pK(a) for a carboxyl group is likely due to its hydrophobic environment and electrostatic repulsion from the metal-bound hydroxide. These results argue that in the active form of E133D PDF, Asp-133 is protonated and therefore acts as a general acid during the decomposition of the tetrahedral intermediate by donating a proton to the leaving amide ion perhaps through a water molecule in the cavity created by the E133D mutation. In contrast, Glu-133 is deprotonated in the active form of wild-type PDF. We propose that Glu-133 acts as a proton shuttle accepting a proton from the metal-bound water and subsequently acts as a general acid during the decomposition of the tetrahedral intermediate.     

Mek1 Suppression of Meiotic Double-Strand Break Repair Is Specific to Sister Chromatids,Chromosome Autonomous and Independent of Rec8 Cohesin Complexes

During meiosis, recombination is directed to occur between homologous chromosomes to create connections necessary for proper segregation at meiosis I. Partner choice is determined at the time of strand invasion and is mediated by two recombinases: Rad51 and the meiosis-specific Dmc1. In budding yeast, interhomolog bias is created in part by the activity of a meiosis-specific kinase, Mek1, which is localized to the protein cores of condensed sister chromatids. Analysis of meiotic double-strand break (DSB) repair in haploid and disomic haploid strains reveals that Mek1 suppresses meiotic intersister DSB repair by working directly on sister chromatids. Rec8 cohesin complexes are not required, however, either for suppression of intersister DSB repair or for the repair itself. Regulation of DSB repair in meiosis is chromosome autonomous such that unrepaired breaks on haploid chromosomes do not prevent interhomolog repair between disomic homologs. The pattern of DSB repair in haploids containing Dmc1 and/or Rad51 indicates that Mek1 acts on Rad51-specific recombination processes.IN eukaryotes, meiosis is a specialized type of cell division that produces the gametes required for sexual reproduction. In meiosis, one round of DNA replication is followed by two rounds of chromosome segregation, termed meiosis I and II. As a result of the two divisions, four haploid cells are produced, each containing half the number of chromosomes as the diploid parent. Proper segregation at meiosis I requires connections between homologous chromosomes that are created by a combination of sister chromatid cohesion and recombination (Petronczki et al. 2003). In vegetative cells, cohesion is mediated by multisubunit ring-shaped complexes that are removed by proteolysis of the kleisin subunit, Mcd1/Scc1 (Onn et al. 2008). In meiotic cells, introduction of a meiosis-specific kleisin subunit, Rec8, allows for a two-step removal of cohesion with loss of arm cohesion at anaphase I and centromere cohesion at anaphase II (Klein et al. 1999). Missegregation of chromosomes during meiosis causes abnormal chromosome numbers in gametes that may lead to infertility and genetic disorders such as trisomy 21 or Down''s syndrome.In mitotically dividing budding yeast cells, recombination is mediated by an evolutionarily conserved RecA-like recombinase, Rad51, and occurs preferentially between sister chromatids (Kadyk and Hartwell 1992). In contrast, recombination during meiosis is initiated by the deliberate formation of double-strand breaks (DSBs) by an evolutionarily conserved, topoisomerase-like protein, Spo11, and occurs preferentially between homologous chromosomes (Jackson and Fink 1985; Schwacha and Kleckner 1997; Keeney 2001). After DSB formation, the 5′ ends on either side of the breaks are resected, resulting in 3′ single stranded (ss) tails. Rad51, and the meiosis-specific recombinase Dmc1, bind to the 3′ ssDNA tails to form protein/DNA filaments that promote strand invasion of homologous chromosomes. DNA synthesis and ligation result in the formation of double Holliday junctions, which are then preferentially resolved into crossovers (Allers and Lichten 2001; Hunter 2007).The precise roles that the Rad51 and Dmc1 recombinase activities play in meiotic recombination have been unclear because experiments have indicated both overlapping and distinct functions for the two proteins (Sheridan and Bishop 2006; Hunter 2007). While both rad51Δ and dmc1Δ mutants reduce interhomolog recombination, other studies suggest that Rad51, in complex with the accessory protein Rad54, is involved primarily in intersister DSB repair. In contrast, Dmc1, in conjunction with the accessory protein Rdh54/Tid1 (a paralog of Rad54), effects DSB repair in meiotic cells by invasion of nonsister chromatids (Dresser et al. 1997; Schwacha and Kleckner 1997; Shinohara et al. 1997a,b; Arbel et al. 1999; Bishop et al. 1999; Hayase et al. 2004; Sheridan and Bishop 2006).The preference for recombination to occur between homologous chromosomes during meiosis is created in part by Dmc1. DSBs accumulate in dmc1Δ diploids due to a failure in strand invasion (Bishop et al. 1992; Hunter and Kleckner 2001). In the efficiently sporulating SK1 strain background, these unrepaired breaks trigger the meiotic recombination checkpoint, resulting in prophase arrest (Lydall et al. 1996; Roeder and Bailis 2000). In dmc1Δ mutants, Rad51 is present at DSBs, yet there is no strand invasion of sister chromatids (Bishop 1994; Shinohara et al. 1997a). These results suggest that in addition to Dmc1 promoting interhomolog strand invasion, Rad51 activity must also be suppressed.Recent studies have shown that during meiosis Rad51 recombinase activity is inhibited by two different mechanisms that decrease the formation of Rad51/Rad54 complexes: (1) binding of the meiosis-specific Hed1 protein to Rad51, thereby excluding interaction with Rad54, and (2) reduction in the affinity of Rad54 for Rad51 due to phosphorylation of Rad54 by Mek1 (Tsubouchi and Roeder 2006; Busygina et al. 2008; Niu et al. 2009). Mek1 is a meiosis-specific kinase that is activated in response to DSBs (Niu et al. 2005, 2007; Carballo et al. 2008). In addition to phosphorylating Rad54, Mek1 phosphorylation of an as yet undetermined substrate is required to suppress Rad51/Rad54-mediated strand invasion of sister chromatids (Niu et al. 2009).To dissect the mechanism by which Mek1 suppresses meiotic intersister DSB repair, we took advantage of the ability of yeast cells to undergo haploid meiosis. The lack of homologous chromosomes in haploid cells makes it possible to examine sister-chromatid-specific events in the absence of interhomolog recombination. De Massy et al. (1994) previously observed a delay in DSB repair in haploid cells and proposed that this delay was due to a constraint in using sister chromatids. We have shown that this delay is dependent on MEK1 and utilized the haploid system to determine various biological parameters required to suppress meiotic intersister DSB repair. Our results indicate that Rad51 and Dmc1 recombinase activities have distinct roles during meiosis and that interhomolog bias is established specifically on sister chromatids through regulation of Rad51, not Dmc1. rec8Δ diploids exhibit defects in meiotic DSB repair (Klein et al. 1999; Brar et al. 2009). Given that cohesin complexes are specific for sister chromatids, we investigated the role of REC8 in intersister DSB repair and found it is required neither for suppressing intersister DSB repair during meiosis nor for the repair itself.