Biochemische und biophysikalische Charakterisierung des GARB2-Proteins aus Sehstäbchen der Rindernetzhaut

The GARP proteins of rods have been localized exclusively at the disc incisures and rims that are in close proximity to the plasma membrane. GARP2 is the most abundant GARP species, and a major protein in ROS. The localization and the amount of GARP2 indicate that it may have an important role in rod phototransduction. Studies focusing on the protein/protein interactions involving GARP proteins have revealed contradictory results (Körschen et al., 1999 ; Poetsch et al., 2001). The present work describes the purification of native GARP2 from bovine ROS. Furthermore this work describes the cloning, expression and purification of two different recombinant GARP2 proteins. A detailed analysis of the structural and biochemical characteristics of the purified proteins shows that the unusual running behavior of GARP2 is not due to dimerisation. The results also strongly suggest that GARP2 is not a globular protein. Moreover the GARP2 secondary structure was analyzed by CD. The interaction of GARP2 with proteins was studied by gel filtration, affinity chromatography, and crosslinking. These experiment show that GARP2 interacts with RDS/peripherin and possibly also with RGS9. Besides, an interaction to unknown proteins with an apparent M$_{w}$ of $\sim$18 kDa, $\sim$ 30 kDa and $\sim$ 70 kDa could be detected. The interactions with the PDE, the GC, and the ABCR could not be proven. Using the purified GARP2 proteins, the effect on the activated PDE was examined. The previously shown powerful inhibition of PDE by the recombinant HT-GARP2 could be reproduced, but using native GARP2 no inhibition was observed. These results suggest that the fusion part of the HT-GARP2 is responsible for the inhibitory effect. These findings and the monoclonal antibodies against the different GARP proteins, provide a solid basis to perform further biochemical, immunohistochemical and cristallographic studies

Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel

MthK is a Ca2+-gated K+ channel whose activity is inhibited by cytoplasmic H+. To determine possible mechanisms underlying the channel’s proton sensitivity and the relation between H+ inhibition and Ca2+-dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different [Ca2+] (ranging from nominally 0 to 30 mM) at a given [H+], in which the solutions bathing the cytoplasmic side of the channels were changed via a perfusion system to ensure complete solution exchanges. We observed a steep relation between [Ca2+] and open probability (Po), with a mean Hill coefficient (nH) of 9.9 ± 0.9. Neither the maximal Po (0.93 ± 0.005) nor nH changed significantly as a function of [H+] over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca2+ was not H+ sensitive over pH ranging from 7.3 to 9.0. However, increasing [H+] raised the EC50 for Ca2+ activation by ∼4.7-fold per tenfold increase in [H+], displaying a linear relation between log(EC50) and log([H+]) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H+ binding does not directly modulate either the channel’s closed–open equilibrium or the allosteric coupling between Ca2+ binding and channel opening. We can account for the Ca2+ activation and proton sensitivity of MthK gating quantitatively by assuming that Ca2+ allosterically activates MthK, whereas H+ opposes activation by destabilizing the binding of Ca2+

Biochemische und biophysikalische Charakterisierung des GARP2-Proteins aus Sehstäbchen der Rindernetzhaut

The GARP proteins of rods have been localized exclusively at the disc incisures and rims that are in close proximity to the plasma membrane. GARP2 is the most abundant GARP species, and a major protein in ROS. The localization and the amount of GARP2 indicate that it may have an important role in rod phototransduction. Studies focusing on the protein/protein interactions involving GARP proteins have revealed contradictory results (Körschen et al., 1999 ; Poetsch et al., 2001). The present work describes the purification of native GARP2 from bovine ROS. Furthermore this work describes the cloning, expression and purification of two different recombinant GARP2 proteins. A detailed analysis of the structural and biochemical characteristics of the purified proteins shows that the unusual running behavior of GARP2 is not due to dimerisation. The results also strongly suggest that GARP2 is not a globular protein. Moreover the GARP2 secondary structure was analyzed by CD. The interaction of GARP2 with proteins was studied by gel filtration, affinity chromatography, and crosslinking. These experiment show that GARP2 interacts with RDS/peripherin and possibly also with RGS9. Besides, an interaction to unknown proteins with an apparent M$_{w}$ of $\sim$18 kDa, $\sim$ 30 kDa and $\sim$ 70 kDa could be detected. The interactions with the PDE, the GC, and the ABCR could not be proven. Using the purified GARP2 proteins, the effect on the activated PDE was examined. The previously shown powerful inhibition of PDE by the recombinant HT-GARP2 could be reproduced, but using native GARP2 no inhibition was observed. These results suggest that the fusion part of the HT-GARP2 is responsible for the inhibitory effect. These findings and the monoclonal antibodies against the different GARP proteins, provide a solid basis to perform further biochemical, immunohistochemical and cristallographic studies

El cuerpo y el movimiento en la educación como una propuesta para potenciar las habilidades comunicativas y expresivas en personas con alteración del habla

Pensar, Vivir y Hacer educación: visiones compartidas Vol. 2 nos introduce en el examen del hecho educativo a la luz de tres ejes inseparables: pensar, vivir y hacer. Por ello, desea recordarnos la importancia de “hacer” la educación de forma metódica y sistemática, pero sobre todo es un llamado a “vivirla” con pasión y a “reflexionar” sobre ella en sosiego. De la adecuada articulación de esos tres centros complementarios depende el futuro de la educación.15-7

Glutamic acid-rich proteins of rod photoreceptors are natively unfolded

Broadly neutralizing HIV antibodies (bNAbs) can recognize carbohydrate-dependent epitopes on gp120. In contrast to previously characterized glycan-dependent bNAbs that recognize high-mannose N-glycans, PGT121 binds complex-type N-glycans in glycan microarrays. We isolated the B-cell clone encoding PGT121, which segregates into PGT121-like and 10-1074-like groups distinguished by sequence, binding affinity, carbohydrate recognition, and neutralizing activity. Group 10-1074 exhibits remarkable potency and breadth but no detectable binding to protein-free glycans. Crystal structures of unliganded PGT121, 10-1074, and their likely germ-line precursor reveal that differential carbohydrate recognition maps to a cleft between complementarity determining region (CDR)H2 and CDRH3. This cleft was occupied by a complex-type N-glycan in a "liganded" PGT121 structure. Swapping glycan contact residues between PGT121 and 10-1074 confirmed their importance for neutralization. Although PGT121 binds complex-type N-glycans, PGT121 recognized high-mannose-only HIV envelopes in isolation and on virions. As HIV envelopes exhibit varying proportions of high-mannose- and complex-type N-glycans, these results suggest promiscuous carbohydrate interactions, an advantageous adaptation ensuring neutralization of all viruses within a given strain