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This report offers some reflections for a better understanding of the realization of the European Open Science Cloud (EOSC) at a local level, describing the implementation of the newly established Austrian EOSC Mandated Organisation and the EOSC Support Office Austria.

Cerebral cortex 33(5), 2342-2360 (2023). doi:10.1093/cercor/bhac212, Published by Oxford Univ. Press, Oxford

Development of novel protein-based drugs such as antibodies or immunocytokines and initial validation of target binding typically occurs in 2D settings with e.g., surface plasmon resonance or reflection interferometry using antigen-coated planar sensors. However, a versatile tool to assess binding characteristics of tumour-targeting therapeutics where specific biochemical interactions can be monitored in real-time and in a more realistic 3D interaction setting is currently lacking. Here, we report on the development of versatile small optical 3D biosensors using protein G-coated spherical microparticles based on Whispering Gallery Modes (WGMs). These sensors allowed for an oriented immobilization of theoretically any immunoglobulin G (IgG) and IgG crystallizable fragment domain (Fc)-tagged protein and have been carefully optimized for specific detection of antigen-antibody interactions, as illustrated using the Epithelial Growth Factor Receptor (EGFR) antibody Cetuximab and Program Death Ligand 1 (PD-L1) antibody Atezolizumab. When both ligand and analyte contained an Fc-fragment, protein G binding capacity saturation followed by a “blocking” step with an irrelevant IgG enabled the label-free detection of Fc-tagged antigens with corresponding cognate ligands, as identified using EGFR-Fc, PD-L1-Fc and different members from the tumour necrosis factor receptor family, such as CD27-Fc and 4−1BB-Fc. Thus, WGMs and developed protein G-coated biosensors provide a widely applicable tool to evaluate binding of immunotherapeutics to their targets on the sensor surface, imitating cell surface properties.

Nanoparticles may limit off-tumor/on-target ubiquitous activation of signaling by protein-based drugs. However, many challenges still exist in the design of a nanoparticle for protein delivery. In this study, conditions to establish vaterite nanoparticles as a pH-sensitive drug delivery system (DDS) for encapsulated protein drugs are comprehensively evaluated. Low coprecipitation pH of vaterite and protein prevents protein denaturation and yields high loading efficiency. Unprotected vaterite recrystallizes in aqueous solutions within 3 h to calcite and releases the loaded protein completely, but surface-modified particles with carboxyl groups containing polymers prove stable for more than 5 months. Notably, modification of vaterite with sulfonated polymers increases the loading of cationic proteins by a multiple. A system is developed for vaterite exposure to (pH) conditions under body-like-flow rates, with the dissolution of vaterite and simultaneous release of active proteins at tumor microenvironmental pH reaching up to 80% and only 20% at physiological pH within 2 h. Importantly, the immunomodulatory protein tumor necrosis factor preserves its native structure and fully retains functional activity in vitro after release from the particles. In conclusion, the studies described here provide a framework for the development of vaterite-based DDS as a carrier for bioactive protein-based therapeutics.

Das Thema nachhaltige Einkaufstragetschen gewinnt zunehmend an Relevanz, nicht zuletzt aufgrund der 2015 eingefuhrten Richtlinie zur Reduzierung und ganzlichen Vermeidung von Kunststofftragetaschen. Dieses Thema wird im vorliegenden Beitrag anhand einer theoretischen Diskussion innerhalb der Wissenschaftsgebiete Nachhaltigkeit, Verpackungslogistik und Verhaltensokonomie und einer empirischen Studie anhand einer Kundenbefragung bei einer Filiale des dm-drogerie markts in Schwabisch Hall bearbeitet. Die Ergebnisse erlauben vielfaltige Ruckschlusse auf das Kundenverhalten der Studienteilnehmer sowie die Wirkungskraft der genannten Richtlinie.

Due to their high secretion capacity, Gram-positive bacteria from the genus Bacillus are important expression hosts for the high-yield production of enzymes in industrial biotechnology; however, to date, strains from only few Bacillus species are used for enzyme production at industrial scale. Herein, we introduce Paenibacillus polymyxa DSM 292, a member of a different genus, as a novel host for secretory protein production. The model gene cel8A from Clostridium thermocellum was chosen as an easily detectable reporter gene with industrial relevance to demonstrate heterologous expression and secretion in P. polymyxa. The yield of the secreted cellulase Cel8A protein was increased by optimizing the expression medium and testing several promoter sequences in the expression plasmid pBACOV. Quantitative mass spectrometry was used to analyze the secretome in order to identify promising new promoter sequences from the P. polymyxa genome itself. The most abundantly secreted host proteins were identified, and the promoters regulating the expression of their corresponding genes were selected. Eleven promoter sequences were cloned and tested, including well-characterized promoters from Bacillus subtilis and Bacillus megaterium. The best result was achieved with the promoter for the hypothetical protein PPOLYM_03468 from P. polymyxa. In combination with the optimized expression medium, this promoter enabled the production of 5475 U/l of Cel8A, which represents a 6.2-fold increase compared to the reference promoter PaprE. The set of promoters described in this work covers a broad range of promoter strengths useful for heterologous expression in the new host P. polymyxa.

Interactions between lipid bilayers and the membrane-proximal regions of membrane-associated proteins play important roles in regulating membrane protein structure and function. The T-cell antigen receptor is an assembly of eight single-pass membrane-spanning subunits on the surface of T lymphocytes that initiates cytosolic signaling cascades upon binding antigens presented by MHC-family proteins on antigen-presenting cells. Its ζ-subunit contains multiple cytosolic immunoreceptor tyrosine-based activation motifs involved in signal transduction, and this subunit by itself is sufficient to couple extracellular stimuli to intracellular signaling events. Interactions of the cytosolic domain of ζ (ζcyt) with acidic lipids have been implicated in the initiation and regulation of transmembrane signaling. ζcyt is unstructured in solution. Interaction with acidic phospholipids induces structure, but its disposition when bound to lipid bilayers is controversial. Here, using surface plasmon resonance and neutron reflection, we characterized the interaction of ζcyt with planar lipid bilayers containing mixtures of acidic and neutral lipids. We observed two binding modes of ζcyt to the bilayers in dynamic equilibrium: one in which ζcyt is peripherally associated with lipid headgroups and one in which it penetrates deeply into the bilayer. Such an equilibrium between the peripherally bound and embedded forms of ζcyt apparently controls accessibility of the immunoreceptor tyrosine-based activation signal transduction pathway. Our results reconcile conflicting findings of the ζ structure reported in previous studies and provide a framework for understanding how lipid interactions regulate motifs to tyrosine kinases and may regulate the T-cell antigen receptor biological activities for this cell-surface receptor system.

Background: prior literature suggests that comorbidity with depression significantly worsens the health state of people with chronic diseases. Objective: the present study examines whether depressed mood increased medical care use for patients with a comorbid physical disease. Design, setting and subjects: the study was a population-based study (KORA-Age), with 3,938 participants aged 64–94. Methods: we investigated differences in health services use in participants with and without depressed mood (Geriatric Depression Scale). A further adjustment for disease was done and differences were examined with the Mann–Whitney U test. The incidence rate ratios (IRRs) for doctors’ appointments or the number of days in hospital were explored with (zero-inflated) negative binomial regression models. Results: there are increased self-neglecting behaviours and medical comorbidities in participants with depressed mood. Depressed mood increased participants’ use of medical services (P< 0.0001). Among participants who visited the doctor during the last 3 months, those with depressed mood had more visits than those without depressed mood, irrespective of somatic comorbidities (P< 0.0001 and P< 0.05 for ill and healthy, respectively). Additionally, patients with coexisting depressed mood and physical disease visited the doctor’s practice significantly more often. Having depressed mood significantly increases the likelihood for more doctor visits (IRR = 1.5, CI = 1.3–1.7) and longer hospital stays (IRR = 1.9, CI = 1.6–2.3). In participants with somatic comorbidities the risk is even greater (IRR = 1.6, CI = 1.3–2, for the number of doctors visits and IRR = 2, CI = 1.4–2.9, for the number of days in the hospital). Conclusions: results suggest that patients with depressed mood had increased use of health-care services overall, particularly those with somatic comorbidities.

Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex.

Older adults often suffer from vitamin D deficiency and from the frailty syndrome charac-terized by different physical limitations, complicating independent everyday life. Previous studies have suggested a relationship between vitamin D status and the frailty syndrome, but results have been partly inconsistent, particularly regarding the shape of the association. Therefore, our aim was to further assess the association of 25-hydroxyvitamin D (25(OH)D) serum levels and frailty in older participants.Cross-sectional population-based study.The study population included 478 men and 462 women of the KORA (COoperative health research in the Region of Augsburg)-Age study born before 1944 examined in 2009.Classification of participants into different frailty states was performed according to the following criteria: weight loss, exhaustion, physical inactivity, slowness, and weakness. PARTICIPANTS who met 1-2 or ≥ 3 of the 5 criteria were classified as prefrail or frail, respectively. Total 25(OH)D was measured in non-fasting serum samples with an enhanced chemiluminescence immunoassay. Sequential logistic regression models adjusted for age, sex, season, lifestyle factors, diseases and biomarkers including parathyroid hormone (PTH) were calculated.High levels of 25(OH)D were inversely associated with being prefrail (N=351) or frail (N=38) in the model adjusted for age, sex, season and lifestyle factors. Compared to levels15 ng/ml, odds ratios (ORs) (95% confidence intervals (CIs) were 0.52 (0.34-0.78) for levels of 15-20 ng/ml, 0.55 (0.37-0.81) for levels of 20-30 ng/ml and 0.32 (0.21-0.51) for levels ≥ 30 ng/ml. Additional adjustment for potential mediators including PTH only slightly attenuated these associations. For single frailty-components, significantly decreased ORs were found for exhaustion, physical inactivity and slowness comparing 25(OH)D levels ≥ 30 ng/ml with levels15 ng/ml.Subjects with 25(OH)D serum levels ≥ 15 ng/ml were less frequently prefrail or frail.

Objective Physical activity is suggested to play a key role in the prevention of several chronic diseases. However, data on the association between physical activity and multimorbidity are lacking. Methods Using data from 1007 men and women aged 65–94 years who participated in the population-based KORA (Cooperative Health Research in the Region of Augsburg)-Age project conducted in Augsburg/Germany and two adjacent counties in 2008/09, 13 chronic conditions were identified, and physical activity scores were calculated based on the self-reported physical activity scale for the elderly (PASE). Multivariable sex-specific logistic regression was applied to determine the association of the continuous physical activity score with multimorbidity (≥ 2 out of 13 diseases). Results Physical activity (mean PASE score ± SD) was higher in men (125.1 ± 59.2) than in women (112.2 ± 49.2). Among men, the odds ratio (OR) for multimorbidity was 0.73 (95% CI: 0.60–0.90) for a 1 standard deviation increase of the PASE score. No significant results could be observed for women (OR: 1.05; 95% CI: 0.83–1.33). Conclusion We demonstrated an inverse association between physical activity and multimorbidity among men. Further prospective studies have to confirm the temporality of effects.

In the phototransduction pathway of rhodopsin, the metarhodopsin (Meta) III retinal storage form arises from the active G-protein binding Meta II by a slow spontaneous reaction through the Meta I precursor or by light absorption and photoisomerization, respectively. Meta III is a side product of the Meta II decay path and holds its retinal in the original binding site, with the Schiff base bond to the apoprotein reprotonated as in the dark ground state. It thus keeps the retinal away from the regeneration pathway in which the photolyzed all-trans-retinal is released. This study was motivated by our recent observation that Meta III remains stable for hours in membranes devoid of regulatory proteins, whereas it decays much more rapidly in situ. We have now explored the possibility of regulated formation and decay of Meta III, using intrinsic opsin tryptophan fluorescence and UV-visible and Fourier transform infrared spectroscopy. We find that a rapid return of Meta III into the regeneration pathway is triggered by the G-protein transducin (Gt). Depletion of the retinal storage is initiated by a novel direct bimolecular interaction of Gt with Meta III, which was previously considered inactive. Gt thereby induces the transition of Meta III into Meta II, so that the retinylidene bond to the apoprotein can be hydrolyzed, and the retinal can participate again in the normal retinoid cycle. Beyond the potential significance for retinoid metabolism, this may provide the first example of a G-protein-catalyzed conversion of a receptor.

The composition of the residential environment may have an independent influence on health, especially in older adults. In this cross-sectional study, we examined the associations between proximity to two features of the residential environment (green space and senior service centers) and three aspects of healthy aging (self-rated physical constitution, disability, and health-related quality of life). We included 1711 inhabitants from the city of Augsburg, Germany, aged 65years or older, who participated in the KORA-Age study conducted in 2008/2009. We calculated the Euclidian distances between each participant’s residential address and the nearest green space or senior service center, using a geographic information system. Multilevel logistic regression models were fitted to analyze the associations, controlling for demographic and socioeconomic factors. Contrary to expectations, we did not find clear associations between the distances to the nearest green space or senior service center and any of the examined aspects of healthy aging. The importance of living close to green space may largely depend on the study location. The city of Augsburg is relatively small (about 267,000 inhabitants) and has a high proportion of greenness. Thus, proximity to green space may not be as important as in a densely populated metropolitan area. Moreover, an objectively defined measure of access such as Euclidian distance may not reflect the actual use. Future studies should try to assess the importance of resources of the residential environment not only objectively, but also from the resident’s perspective.

Objective We aimed to examine the association between various sleep disturbances and falls among older individuals from the general population while considering the influence of age and dizziness. Methods Data were derived from the population-based cross-sectional KORA (Cooperative Health Research in the Region of Augsburg)-Age study, whereby information was conducted in standardized telephone interviews with 4127 men and women aged ⩾65 years in 2008 and 2009. Unstratified and stratified (by age and dizziness) multivariable logistic regression model analyses were performed. Results The multivariable analysis showed a marginally significant association between trouble staying asleep and ⩾1 fall in the previous year (odds ratio [OR], 1.23 [95% confidence interval (CI), 1.01–1.50]). This association was more pronounced in participants older than the age of 75 years (OR, 1.58 [95% CI, 1.16–2.16]) and in individuals without dizziness (OR, 1.35 [95% CI, 1.04–1.76]). There was no association between daytime sleepiness and falls in the fully-adjusted models, but the odds of falls in the previous year in individuals older than the age of 75 years were significantly higher for individuals with difficulty falling asleep. Although sleep duration was not associated with falls in multivariable analyses when stratified by dizziness, sleep duration of 9 h daily was significantly associated with higher odds of experiencing at least one fall in the previous year. Conclusions Our study suggested that the positive relationship between a trend towards longer sleep duration, trouble falling and staying asleep, and falls is strongest in older individuals and in individuals who did not experience dizziness in the previous year.

Nef is the viral gene product employed by the majority of primate lentiviruses to overcome restriction by tetherin (BST-2 or CD317), an interferon-inducible transmembrane protein that inhibits the detachment of enveloped viruses from infected cells. Although the mechanisms of tetherin antagonism by HIV-1 Vpu and HIV-2 Env have been investigated in detail, comparatively little is known about tetherin antagonism by SIV Nef. Here we demonstrate a direct physical interaction between SIV Nef and rhesus macaque tetherin, define the residues in Nef required for tetherin antagonism, and show that the anti-tetherin activity of Nef is dependent on clathrin-mediated endocytosis. SIV Nef co-immunoprecipitated with rhesus macaque tetherin and the Nef core domain bound directly to a peptide corresponding to the cytoplasmic domain of rhesus tetherin by surface plasmon resonance. An analysis of alanine-scanning substitutions identified residues throughout the N-terminal, globular core and flexible loop regions of Nef that were required for tetherin antagonism. Although there was significant overlap with sequences required for CD4 downregulation, tetherin antagonism was genetically separable from this activity, as well as from other Nef functions, including MHC class I-downregulation and infectivity enhancement. Consistent with a role for clathrin and dynamin 2 in the endocytosis of tetherin, dominant-negative mutants of AP180 and dynamin 2 impaired the ability of Nef to downmodulate tetherin and to counteract restriction. Taken together, these results reveal that the mechanism of tetherin antagonism by Nef depends on a physical interaction between Nef and tetherin, requires sequences throughout Nef, but is genetically separable from other Nef functions, and leads to the removal of tetherin from sites of virus release at the plasma membrane by clathrin-mediated endocytosis., Author Summary Tetherin (BST-2, CD317 or HM1.24) is an interferon-inducible cellular restriction factor that prevents the release of enveloped viruses from infected cells. Human and simian immunodeficiency viruses have evolved to use different viral proteins to overcome the anti-viral effects of tetherin. Whereas HIV-1 Vpu and HIV-2 Env counteract human tetherin, most SIVs use the accessory protein Nef to counteract tetherin in their non-human primate hosts. Here we show that the mechanism of tetherin antagonism by SIV Nef involves a direct physical interaction between the core domain of Nef and the cytoplasmic domain of tetherin, which results in the removal of tetherin from sites of virus assembly and release on the cell surface by a mechanism that depends on clathrin and dynamin 2. The Nef-mediated internalization of tetherin leads to the accumulation of tetherin within lysosomal compartments, suggesting that, similar to CD4− and MHC I-downregulation, Nef promotes the lysosomal degradation of tetherin.

Multimorbidity is a common problem in aged populations with a wide range of individual and societal consequences. The objective of the study was to explore patterns of comorbidity and multimorbidity in an elderly population using different analytical approaches. Data were gathered from the population-based KORA-Age project, which included 4,127 persons aged 65-94 years living in the city of Augsburg and its two surrounding counties in Southern Germany. Information on the presence of 13 chronic conditions was collected in a standardized telephone interview and a self-administered questionnaire. Patterns of comorbidity and multimorbidity were analyzed using prevalence figures, logistic regression models and exploratory tetrachoric factor analysis. The prevalence of multimorbidity (≥2 diseases) was 58.6% in the total sample. Hypertension and diabetes (Odds Ratio [OR] 2.95, 99.58% confidence interval [CI] [2.19-3.96]), as well as hypertension and stroke (OR 2.00, 99.58% CI [1.26-3.16]) most often occurred in combination. This association was independent of age, sex and the presence of other conditions. Using factor analysis, we identified four patterns of multimorbidity: the first pattern includes cardiovascular and metabolic diseases, the second includes joint, liver, lung and eye diseases, the third covers mental and neurologic diseases and the fourth pattern includes gastrointestinal diseases and cancer. 44% of the persons were assigned to at least one of the four multimorbidity patterns; 14% could be assigned to both the cardiovascular/metabolic and the joint/liver/lung/eye pattern. Further common pairs were the mental/neurologic pattern combined with the cardiovascular/metabolic pattern (7.2%) or the joint/liver/lung/eye pattern (5.3%), respectively. Our results confirmed the existence of co-occurrence of certain diseases in elderly persons, which is not caused by chance. Some of the identified patterns of multimorbidity and their overlap may indicate common underlying pathological mechanisms.

Hintergrund. Das KORA-Age-Verbundprojekt hat zum Ziel, die Determinanten und Folgen von Multimorbidität im Alter zu ermitteln und nach Faktoren des erfolgreichen Alterns in der Allgemeinbevölkerung zu suchen. Material und Methoden. Die KORA-Age-Kohorte besteht aus 9197 Personen, die 1943 oder früher geboren wurden und Teilnehmer der KORA-Kohorte (KORA: Kooperative Gesundheitsforschung in der Region Augsburg) zwischen 1984 und 2001 waren. In der randomisierten Interventionsstudie KORINNA (Koronarinfarktnachbehandlung im Alter) wurde ein von Krankenschwestern durchgeführtes Case-Management-Programm mit 338 Herzinfarktpatienten getestet und gesundheitsökonomisch bewertet. Ergebnisse. In der KORA-Age-Kohorte wurden 2734 Todesfälle registriert, 4565 Personen nahmen an einer schriftlichen Befragung und 4127 Personen an einem Telefoninterview teil (Teilnahme: 76,2% bzw. 68,9%). Zusätzlich wurde eine alters- und geschlechtsstratifizierte Stichprobe von 1079 Personen untersucht (Teilnahme: 53,8%). Schlussfolgerung. Das KORA-Age-Verbundprojekt untersuchte eine große bevölkerungsbezogene Stichprobe älterer Menschen, die Aufschluss über die Verteilung und Determinanten von Multimorbidität und erfolgreichem Altern gibt.

In the phototransduction pathway of rhodopsin, the metarhodopsin (Meta) III retinal storage form arises from the active G-protein binding Meta II by a slow spontaneous reaction through the Meta I precursor or by light absorption and photoisomerization, respectively. Meta III is a side product of the Meta II decay path and holds its retinal in the original binding site, with the Schiff base bond to the apoprotein reprotonated as in the dark ground state. It thus keeps the retinal away from the regeneration pathway in which the photolyzed all-trans-retinal is released. This study was motivated by our recent observation that Meta III remains stable for hours in membranes devoid of regulatory proteins, whereas it decays much more rapidly in situ. We have now explored the possibility of regulated formation and decay of Meta III, using intrinsic opsin tryptophan fluorescence and UV-visible and Fourier transform infrared spectroscopy. We find that a rapid return of Meta III into the regeneration pathway is triggered by the G-protein transducin (G(t)). Depletion of the retinal storage is initiated by a novel direct bimolecular interaction of G(t) with Meta III, which was previously considered inactive. G(t) thereby induces the transition of Meta III into Meta II, so that the retinylidene bond to the apoprotein can be hydrolyzed, and the retinal can participate again in the normal retinoid cycle. Beyond the potential significance for retinoid metabolism, this may provide the first example of a G-protein-catalyzed conversion of a receptor.

Rhodopsin bears 11-cis-retinal covalently bound by a protonated Schiff base linkage. 11-cis/all-trans isomerization, induced by absorption of green light, leads to active metarhodopsin II, in which the Schiff base is intact but deprotonated. The subsequent metabolic retinoid cycle starts with Schiff base hydrolysis and release of photolyzed all-trans-retinal from the active site and ends with the uptake of fresh 11-cis-retinal. To probe chromophore-protein interaction in the active state, we have studied the effects of blue light absorption on metarhodopsin II using infrared and time-resolved UV-visible spectroscopy. A light-induced shortcut of the retinoid cycle, as it occurs in other retinal proteins, is not observed. The predominantly formed illumination product contains all-trans-retinal, although the spectra reflect Schiff base reprotonation and protein deactivation. By its kinetics of formation and decay, its low temperature photointermediates, and its interaction with transducin, this illumination product is identified as metarhodopsin III. This species is known to bind all-trans-retinal via a reprotonated Schiff base and forms normally in parallel to retinal release. We find that its generation by light absorption is only achieved when starting from active metarhodopsin II and is not found with any of its precursors, including metarhodopsin I. Based on the finding of others that metarhodopsin III binds retinal in all-trans-C(15)-syn configuration, we can now conclude that light-induced formation of metarhodopsin III operates by Schiff base isomerization ("second switch"). Our reaction model assumes steric hindrance of the retinal polyene chain in the active conformation, thus preventing central double bond isomerization.

Binding of herbicides to photosystem II inhibits the electron transfer from QA to QB due to competition of herbicides with plastoquinone bound at the QB site. We investigated herbicide binding to monomeric and dimeric photosystem II core complexes (PSIIcc) isolated from Thermosynechococcus elongatus by a combination of different methods (isothermal titration and differential scanning calorimetry, CD spectroscopy and measurements of the oxygen evolution) yielding binding constants, enthalpies and stoichiometries for various herbicides as well as information regarding stabilization/destabilization of the complex. Herbicide binding to detergent-solubilized PSIIcc can be described by a model of single independent binding sites present on this important membrane protein. Interestingly, binding stoichiometries herbicide:PSIIcc are lower than 1:1 and vary depending on the herbicide under study. Strong binding herbicides such as terbutryn stabilize PSIIcc in thermal unfolding experiments and endothermically binding herbicides like ioxynil probably cause large structural changes accompanied with the binding process as shown by differential scanning calorimetry experiments of the unfolding reaction of PSIIcc monomer in the presence of ioxynil. In addition we studied the occupancy of the QB sites with plastoquinone (PQ9) by measuring flash induced fluorescence relaxation yielding a possible explanation for the deviations of herbicide binding from a 1:1 herbicide/binding site model. © 2005 Elsevier B.V. All rights reserved.

The T cell antigen receptor (TCR) is a cell surface protein complex that binds peptide antigen fragments presented by the Major Histocompatibility Complex (MHC). The interaction of TCR with MHC leads to T cell activation and initiation of an immune response,1 but the molecular details of the transmembrane signaling process remain unknown. TCRζ is the 115 amino acid long cytosolic signaling domain of the CD3-T cell receptor complex that plays a central role in this process. TCRζ is predominantly unstructured2 and carries several copies of the immunoreceptor tyrosine-based activation motif (ITAM). ITAMs become phosphorylated upon receptor engagement, constituting an early and obligatory event in the signaling cascade that blocks lipid association and lipid-associated conformational rearrangements3 of TCRζ. We present a characterization of the affinity of TCRζ to tethered lipid bilayers by SPR and a structural study of its association with such membranes using neutron reflectometry (NR). We studied the binding of unphosphorylated TCRζ to highly charged stBLMs rich in anionic phosphatidylserine (PS) or phosphatidylglycerol (PG) and observed affinity constants of Kd ≈ 10 μM. Minute amounts of phosphoinositides (PI(4,5)P2) increase the membrane affinity of the protein by more than 10-fold. An NR characterization of TCRζ in the membrane-associated state shows the major portion of the protein interfacially associated with the bilayer surface and a minor protein penetrating the bilayer deeply. This is consistent with a model in which an α-helical structure is aligned on the membrane surface that may be connected via a hinge with another α-helical segment inserted into the bilayer.[1] Call, M.E., and Wucherpfennig, K.W., Annu. Rev. Immunol. 23:101 (2005).[2] Sigalov, A.B., et al., Biochemistry 45:15731 (2006).[3] Aivazian, D.A., et al., Nat. Struct. Biol. 7:1023 (2000).