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G Schreiber

Publications and source records attributed to G Schreiber.

At least 19 recordsLinked to original sources
PubMed2004

The modular architecture of protein-protein binding interfaces.

Protein-protein interactions are essential for life. Yet, our understanding of the general principles governing binding is not complete. In the present study, we show that the interface between proteins is built in a modular fashion; each module is comprised of a number of closely interacting residues, with few interactions between the modules. The boundaries between modules are defined by clustering the contact map of the interface. We show that mutations in one module do not affect residues located in a neighboring module. As a result, the structural and energetic consequences of the deletion of entire modules are surprisingly small. To the contrary, within their module, mutations cause complex energetic and structural consequences. Experimentally, this phenomenon is shown on the interaction between TEM1-beta-lactamase and beta-lactamase inhibitor protein (BLIP) by using multiple-mutant analysis and x-ray crystallography. Replacing an entire module of five interface residues with Ala created a large cavity in the interface, with no effect on the detailed structure of the remaining interface. The modular architecture of binding sites, which resembles human engineering design, greatly simplifies the design of new protein interactions and provides a feasible view of how these interactions evolved.

Bacterial Proteins↗
PubMed2004

[Donor (heterologous) insemination in Germany--medical and legal aspects].

Donor insemination is a scientifically based reliable treatment for infertile couples--in Germany too. Reported results and long-term experience are uniformly good: neither the parents nor the children suffer any impairment of quality of life; stabilization of the family situation, low divorce rates and the frequently expressed desire for repeat treatments all militate in favour of this therapeutic option. Legal uncertainties, such as reservations on the part of the physician, documentation, anonymity and protection for donors, physicians and parents are points are points to be addressed by legislators and the relevant professional organizations.

Female↗
PubMed2004

Electrostatically optimized Ras-binding Ral guanine dissociation stimulator mutants increase the rate of association by stabilizing the encounter complex.

Association of two proteins can be described as a two-step process, with the formation of an encounter complex followed by desolvation and establishment of a tight complex. Here, by using the computer algorithm PARE, we designed a set of mutants of the Ras effector protein Ral guanine nucleotide dissociation stimulator (RalGDS) with optimized electrostatic steering. The fastest binding RalGDS mutant, M26K,D47K,E54K, binds Ras 14-fold faster and 25-fold tighter compared with WT. A linear correlation was found between the calculated and experimental data, with a correlation coefficient of 0.97 and a slope of 0.65 for the 24 mutants produced. The data suggest that increased electrostatic steering specifically stabilizes the encounter complex and transition state. This conclusion is backed up by Phi analysis of the encounter complex and transition state of the RalGDS(M26K,D47K,E54K)/Ras complex, with both values being close to 1. Upon further formation of the final complex, the increased Coulombic interactions are probably counterbalanced by the cost of desolvation of charges, keeping the dissociation rate constant almost unchanged. This mechanism is also reflected by the mutual compensation of enthalpy and entropy changes quantified by isothermal titration calorimetry. The binding constants of the faster binding RalGDS mutants toward Ras are similar to those of Raf, the most prominent Ras effector, suggesting that the design methodology may be used to switch between signal transduction pathways.

Binding Sites↗
PubMed2002

The evolutionary and integrative roles of transthyretin in thyroid hormone homeostasis.

In larger mammals, thyroid hormone-binding plasma proteins are albumin, transthyretin (TTR) and thyroxine (T4)-binding globulin. They differ characteristically in affinities and release rates for T4 and triiodothyronine (T3). Together, they form a 'buffering' system counteracting thyroid hormone permeation from aqueous to lipid phases. Evolution led to important differences in the expression pattern of these three proteins in tissues. In adult liver, TTR is only made in eutherians and herbivorous marsupials. During development, it is also made in tadpole and fish liver. More intense TTR synthesis than in liver is found in the choroid plexus of reptilians, birds and mammals, but none in the choroid plexus of amphibians and fish, i.e. species without a neocortex. All brain-made TTR is secreted into the cerebrospinal fluid, where it becomes the major thyroid hormone-binding protein. During ontogeny, the maximum TTR synthesis in the choroid plexus precedes that of the growth rate of the brain and occurs during the period of maximum neuroblast replication. TTR is only one component in a network of factors determining thyroid hormone distribution. This explains why, under laboratory conditions, TTR-knockout mice show no major abnormalities. The ratio of TTR affinity for T4 over affinity for T3 is higher in eutherians than in reptiles and birds. This favors T4 transport from blood to brain providing more substrate for conversion of the biologically less active T4 into the biologically more active T3 by the tissue-specific brain deiodinases. The change in affinity of TTR during evolution involves a shortening and an increase in the hydrophilicity of the N-terminal regions of the TTR subunits. The molecular mechanism for this change is a stepwise shift of the splice site at the intron 1/exon 2 border of the TTR gene. The shift probably results from a sequence of single base mutations. Thus, TTR evolution provides an example for a molecular mechanism of positive Darwinian evolution. The amino acid sequences of fish and amphibian TTRs are very similar to those in mammals, suggesting that substantial TTR evolution occurred before the vertebrate stage. Open reading frames for TTR-like sequences already exist in Caenorhabditis elegans, yeast and Escherichia coli genomes.

Amino Acid Sequence↗
PubMed2001

New insights into the mechanism of protein-protein association.

Association of a protein complex follows a two-step mechanism, with the first step being the formation of an encounter complex that evolves into the final complex. Here, we analyze recent experimental data of the association of TEM1-beta-lactamase with BLIP using theoretical calculations and simulation. We show that the calculated Debye-Hückel energy of interaction for a pair of proteins during association resembles an energy funnel, with the final complex at the minima. All attraction is lost at inter-protein distances of 20 A, or rotation angles of >60 degrees from the orientation of the final complex. For faster-associating protein complexes, the energy funnel deepens and its volume increases. Mutations with the largest impact on association (hotspots for association) have the largest effect on the size and depth of the energy funnel. Analyzing existing evidence, we suggest that the transition state along the association pathway is the formation of the final complex from the encounter complex. Consequently, pairs of proteins forming an encounter complex will tend to dissociate more readily than to evolve into the final complex. Increasing directional diffusion by increasing favorable electrostatic attraction results in a faster forming and slower dissociating encounter complex. The possible applicability of electrostatic calculations for protein-protein docking is discussed.

Bacterial Proteins↗
PubMed2001

Structure of the interferon-receptor complex determined by distance constraints from double-mutant cycles and flexible docking.

The pleiotropic activity of type I interferons has been attributed to the specific interaction of IFN with the cell-surface receptor components ifnar1 and ifnar2. To date, the structure of IFN has been solved, but not that of the receptor or the complex. In this study, the structure of the IFN-alpha 2-ifnar2 complex was generated with a docking procedure, using nuclear Overhauser effect-like distance constraints obtained from double-mutant cycle experiments. The interaction free energy between 13 residues of the ligand and 11 of the receptor was measured by double-mutant cycles. Of the 100 pairwise interactions probed, five pairs of residues were found to interact. These five interactions were incorporated as distance constraints into the flexible docking program prodock by using fixed and movable energy-gradient grids attached to the receptor and ligand, respectively. Multistart minimization and Monte Carlo minimization docking of IFN-alpha 2 onto ifnar2 converged to a well-defined average structure, with the five distance constraints being satisfied. Furthermore, no structural artifacts or intraloop energy strain were observed. The mutual binding sites on IFN-alpha 2 and ifnar2 predicted from the model showed an almost complete superposition with the ones determined from mutagenesis studies. Based on this structure, differences in IFN-alpha 2 versus IFN-beta binding are discussed.

Interferon-alpha↗
PubMed2001

Experimental assignment of the structure of the transition state for the association of barnase and barstar.

Association of a protein complex follows a two step reaction mechanism, with the first step being the formation of an encounter complex which evolves into the final complex. Here we present new experimental data for the association of the bacterial ribonuclease barnase and its polypeptide inhibitor barstar which shed light on the thermodynamics and structure of the transition state and preceding encounter complex of association at diminishing electrostatic attraction. We show that the activation entropy at the transition state is close to zero, with the activation enthalpy being equal to the free energy of binding. This observation was independent of the magnitude of the mutual electrostatic attraction, which were altered by mutagenesis or by addition of salt. The low activation entropy implies that the transition state is mostly solvated at all ionic strengths. The structure of the transition state was probed by measuring pairwise interaction energies using double-mutant-cycles. While at low ionic strength all proximal charge-pairs form contacts, at high salt only a subset of these interactions are maintained. More specifically, charge-charge interactions between partially buried residues are lost, while exposed charged residues maintain their ability to form specific interactions even at the highest salt concentration. Uncharged residues do not interact at any ionic strength. The results presented here suggest that the barnase-barstar binding sites are correctly aligned during the transition state even at diminishing electrostatic attraction, although specific short range interactions of uncharged residues are not yet formed. Furthermore, most of the interface desolvation (which contributes to the entropy of the system) has not yet occurred. This picture seems to be valid at low and high salt. However, at high salt, interactions of the activated complex are limited to a more restricted set of residues which are easier approached during diffusion, prior to final docking. This suggest that the steering region at high salt is more limited, albeit maintaining its specificity.

Bacillus↗
PubMed2001

Fast transient cytokine-receptor interactions monitored in real time by reflectometric interference spectroscopy.

Investigating protein-protein interactions by mutational analysis requires practical techniques for quantifying rate constants and equilibrium constants over several orders of magnitude with reasonably high sample throughput. We have employed spectroscopic interferometry for label-free monitoring of the interaction between the cytokine interferon alpha2 (IFNalpha2) and the extracellular domain of its receptor ifnar2 (ifnar2-EC). We implemented a versatile surface chemistry for the glass substrate of this transducer for covalent immobilization of proteins. Affinity capturing with a monoclonal anti-ifnar2-EC antibody (mAb) followed by crosslinking with a second, noncompetitive mAb provided stable, but still reversible, immobilization of ifnar2-EC. We measured kinetics and affinity of numerous of mutants of IFNalpha2 and ifnar2-EC. Dissociation rate constants up to 0.3 s(-1) and association rate constants up to 3 x 10(6) M(-)1 s(-1) were resolved by the system. Dissociation constants down to 200 microM were measured with protein concentrations up to 50 microM without no background signal or nonspecific binding. The instrument detection limit is approximately 10 pm without the need for temperature stabilization or referencing channels. The system proved effective for large-scale mutational analysis involving alanine scanning mutagenesis and double mutant cycles.

Antibodies, Monoclonal↗
PubMed2001

Prolonging the half-life of human interferon-alpha 2 in circulation: Design, preparation, and analysis of (2-sulfo-9-fluorenylmethoxycarbonyl)7- interferon-alpha 2.

Polypeptide drugs are generally short-lived species in circulation. In this study, we have covalently linked seven moieties of 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) to the amino groups of human interferon-alpha2. The derivative thus obtained (FMS(7)-IFN-alpha2) has approximately 4% the biological potency and 33 +/- 4% the receptor binding capacity of the native cytokine. Upon incubation, FMS(7)-IFN-alpha2 undergoes time-dependent spontaneous hydrolysis, generating active interferon with t(1/2) values of 24 +/- 2 h at pH 8.5 and 98 +/- 10 h at pH 7.4. When native IFN-alpha2 is intravenously administered to mice, circulating antiviral activity is maintained for a short duration and then declines with t(1/2) = 4 +/- 0.5 h, reaching undetectable values at approximately 18 h after administration. With intravenously administered FMS(7)-IFN-alpha2, there is a lag period of 2 h, followed by a progressive elevation in circulating antiviral-active protein, which peaked at 20 h and declined with t(1/2) = 35 +/- 4 h. FMS(7)-IFN-alpha2 is resistant to alpha-chymotrypsin digest and to proteolytic inactivation by human serum proteases in vitro. We have thus introduced here an inactive IFN-alpha2 derivative, which is resistant to in situ inactivation and has the capability of slowly reverting to the native active protein at physiological conditions in vivo and in vitro. Having these attributes, FMS(7)-IFN-alpha2 maintains prolonged circulating antiviral activity in mice, exceeding 7-8 times the activity of intravenously administered native cytokine.

Antiviral Agents↗
PubMed2001

Elevated dopamine receptor-coupled G(s) protein measures in mononuclear leukocytes of patients with schizophrenia.

Heterotrimeric G proteins play a pivotal role in post-receptor information transduction and were previously implicated in the pathophysiology and treatment of mood disorders. Changes previously detected in G protein levels in post-mortem brain of patients with schizophrenia could reflect effects of antipsychotic medication. The present study aims at quantitatively and functionally evaluating receptor-coupled G proteins in mononuclear leukocytes obtained from 23 untreated patients with schizophrenia and 30 healthy subjects in an attempt to unravel a pattern of G protein measures in schizophrenia distinctive from patterns previously obtained in mood disorders. Dopamine-enhanced guanine nucleotide binding capacity to G(s) protein through D1/D5 receptor in mononuclear leukocytes of untreated patients with schizophrenia was significantly increased in comparison with healthy subjects, and positively correlated with both the total PANSS score and the positive subscale. beta-Adrenergic and muscarinic receptor-coupled G protein functions, as well as G(s)alpha, G(i)alpha and Gbeta immunoreactivities, were similar to healthy subjects. These findings, distinctive for schizophrenia, unrelated to drug treatment, and differential from previous findings in mania and depression, may potentially help to differentially diagnose, after the first psychotic episode, between the major psychoses: schizophrenia and manic-depressive illness.

Acute Disease↗
PubMed2001

Structure and expression of the transthyretin gene in the choroid plexus: a model for the study of the mechanism of evolution.

Thyroid hormones are key regulators of brain differentiation and function. They permeate strongly into lipid membranes. However, a substantial portion of thyroid hormone is retained in the intravascular/extracellular compartments by binding to plasma proteins. In the brain, transthyretin is the most important of these proteins. This transthyretin is synthesized in the epithelial cells of the choroid plexus and exclusively secreted towards the brain. A net movement of thyroid hormones from the blood to the brain ensues. During evolution, transthyretin synthesis in the choroid plexus and the beginnings of a neocortex first appeared at the stage of the stem reptiles. The affinity of transthyretin for thyroxine increased and that for triiodothyronine decreased during evolution. This could augment the importance of deiodination for regulation of metabolism and gene expression by thyroid hormones in the brain. Successive shifts of the splice site at the 5' end of exon 2 of transthyretin precursor mRNA in the 3' direction led to a shortening of the N-terminal sections and to an increase in hydrophilicity of the N-terminal regions of transthyretin. This shift can be explained by a sequence of single base mutations. It could be an example for a molecular mechanism of positive Darwinian evolution. The selection pressure, which led to the expression of the transthyretin gene in the choroid plexus during evolution, might have been the maintenance of thyroid hormone homeostasis in the extracellular compartment of the brain in the presence of the greatly increasing volume of the lipid phase.

Amino Acid Sequence↗
PubMed2001

Exploring the relationship between timing of menarche and eating disorder symptoms in Black and White adolescent girls.

OBJECTIVE: This study examined the relationship between timing of sexual maturation and eating disorders symptoms in adolescent girls. METHOD: Data were collected over 10 years for a cohort of 1,213 Black girls and 1,166 White girls who were either 9 or 10 years old at study entry. Annually, girls' height and weight were measured and, biannually, girls completed self-report measures of eating disorders symptoms. RESULTS: Early-onset menarche is a risk factor for the development of body image and dieting concerns, but the effect of timing is due to the impact of early and late maturation on body weight. DISCUSSION: Findings underscore the importance of adiposity as a risk factor for poor mental health.

Adolescent↗
PubMed2001

Retinal anatomy and function of the transthyretin null mouse.

Vitamin A (retinol) is vital for the normal development and function of many tissues in the body including the eye. The purpose of this project was to characterize the retinal anatomy and function of the transthyretin (TTR) null mouse. Mice lacking TTR have been constructed by homologous recombination. Immunocytochemistry was performed to localize short and mid-long wavelength cone opsins as well as morphological examination of the entire retina in wild-type and TTR null mice. Visual function was assessed using the electroretinogram (ERG) and resulting waveforms were analysed in terms of receptoral and postreceptoral components. Retinal morphology of the TTR null mouse was normal. In addition, short and mid-long wavelength cone opsins were localized normally in both TTR null and wild-type retinae. Consistent with these findings, TTR null mice show no anomalies of receptoral (P3) nor post-receptoral (b-wave) ERG components compared with wild-type mice. The results suggest that although circulating plasma levels of retinol and retinol binding protein (RBP) are extremely low, this reduction has little effect on the retinal structure or function of the TTR null mouse. These data are consistent with the existence of mechanisms for the transport of retinol to the retina independent of the classical retinol-RBP-TTR complex.

Analysis of Variance↗
PubMed2001

Differential effects of the antipsychotics haloperidol and clozapine on G protein measures in mononuclear leukocytes of patients with schizophrenia.

AIMS: Heterotrimeric G proteins play a pivotal role in postreceptor information transduction. These proteins were previously implicated in the pathophysiology and treatment of mood and other neuropsychiatric disorders. Recently we showed that untreated patients with schizophrenia have a significantly elevated dopamine-induced Gs protein function which is correlated with the severity of the psychotic symptoms. In contrast, an inverse picture with reduction in the function and the immunoreactivity of Gs protein was detected in patients with Parkinson's disease. The present study aims at investigating the effect of antipsychotic medications on dopamine-induced Gs protein hyperfunction in schizophrenia comparing the classical antipsychotic haloperidol and the newer antipsychotic clozapine, which is devoid of extrapyramidal side effects, on G protein measures. METHODS: G protein functional measurements coupled to beta-adrenergic, muscarinic, and dopamine receptors were undertaken through bacterial toxin sensitive, agonist enhanced [3H]-Gpp(NH)p binding capacity, substantiated by quantitative measures of Gs alpha, Gi alpha, and G beta subunit proteins through immunoblot analysis in mononuclear leukocytes obtained from patients with schizophrenia under haloperidol, or clozapine treatments in comparison with untreated patients with schizophrenia and healthy volunteers. RESULTS: Dopamine-induced Gs hyperfunction characteristic of untreated patients with schizophrenia was not detected under antipsychotic treatment with either haloperidol or clozapine. Haloperidol caused a significant decrease in Gs function and immunoreactivity below normal levels. The extend of reduction in Gs function was found to be correlated with the intensity of extrapyramidal side effects. The pattern of G protein subunits levels in patients with schizophrenia under haloperidol treatment resembles the one obtained in patients with Parkinson's disease. CONCLUSIONS: In the present study it is shown that G protein measurements in patients with schizophrenia under antipsychotic treatments can be used to biochemically monitor effects of antipsychotic medications in living patients. Moreover, these measurements may be used also for monitoring parkinsonian side effects induced by antipsychotic medications.

Adult↗
PubMed2001

Dynamic analysis of inter-words time intervals: a method to analyze the structure of communicative signals.

Speech analyses are usually focused on words as signifiers ignoring inter-words time intervals (IWIs), which are related to the 'form' of speech, rather than to its 'content'. Applying the method of power spectrum analysis to inter-vocalizations time intervals of bird singing, underlying periodic processes were detected. In contrast, human IWIs revealed non-periodicity, which may be random or chaotic. To differentiate between these two possibilities, the non-linear dynamic methods of unstable periodic orbits and correlation dimension were applied to show that IWIs are characterized by a low dimensional chaotic attractor. Its correlation dimension of 3.2 +/- 1.1 suggests a minimum number of four variables underlying the system. The methods developed in the present communication can be further applied: (a) for the measurement of specific alterations in the processes underlying the form of speech in human disorders, i.e., schizophrenia, (b) for the assessment of normal and pathological developmental aspects of speech processes in children; (c) for comparing communicative signals between humans and other species.

Adult↗