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Biomedical subjects

M Buehner

Publications and source records attributed to M Buehner.

17 recordsLinked to original sources
PubMed1995

Human interleukin-4 and variant R88Q: phasing X-ray diffraction data by molecular replacement using X-ray and nuclear magnetic resonance models.

The structure of recombinant human interleukin-4 (hIL-4) has been determined by both NMR and X-ray diffraction methods in several laboratories, including ours. The X-ray and NMR structures were successfully applied for solving the X-ray crystal structure by molecular replacement. Due to the small size of the hIL-4 molecule (129 residues) and its lack of structural diversity (4-helix bundle), this task was especially difficult and required special care with rotation function applications. The crucial point was that proper removal of the Patterson origin peaks was indispensable in all cases. All available structures of hIL-4 were checked, in a standardized procedure, for their suitability as templates for molecular replacement. The models derived from the various structures are close to, but not in all loop details identical with, the genuine X-ray structures. The deviations of the X-ray structure-derived models are of the same magnitude as the differences between the original X-ray structures, while the deviations of the NMR structure-derived models are two to three times as large. The hIL-4 variant R88Q is a binding mutant, its affinity to the receptor is decreased by a factor of about 200. Its X-ray structure was determined by molecular replacement using the wild-type X-ray structure determined in our laboratory as a model. The structure of R88Q is virtually identical with that of the wild-type protein. All differences besides the shortened side-chain of residue 88 occur at surface residues with high temperature factors, i.e. at spots where the structure is not well defined. Since the structure is not perturbed, the biological effect of decreased receptor affinity has to be attributed to the loss of a single positive charge in the surface area of the main receptor contact.

Computer Simulation↗
PubMed1992

The wandering spleen.

Wandering spleen is an unusual entity, occurring in both sexes and at any age, but is more frequent in women of reproductive age and in children. Wandering spleen is probably most often a result of congenital anomalies of development of the dorsal mesogastrium, but acquired factors may have a role in certain instances. Patients present most commonly with an asymptomatic mass, mass and subacute abdominal or gastrointestinal complaints or with acute abdominal findings. Clinical diagnosis can be difficult, but noninvasive imaging procedures, such as sonography, nuclear scintigraphy, computed tomography and magnetic resonance imaging are usually diagnostic. Laboratory tests are usually nonspecific, but may occasionally reveal evidence of hypersplenism or functional splenia. Symptoms may remain limited or absent for long periods of time, but complications related to torsion or compression of abdominal organs by the spleen or the pedicle are quite common. Splenomegaly is usually a result of torsion of the pedicle and splenic sequestration. Significant morbidity and mortality rates seem to be considerably less than described in 1933 and limited primarily to patients presenting initially with acute abdominal findings. Management recommendations have varied, but recognition of a significant risk of postsplenectomy sepsis supports a conservative approach. Patients with limited symptomatology may be medically managed until they exhibit worsening symptoms indicating progressive splenic torsion or gastrointestinal compression. Detorsion and splenopexy may be considered a reasonable surgical option even in patients presenting with acute abdomen, if there is no evidence of infarction, thrombosis or hypersplenism. Splenic preservation is especially recommended in extremely young patients who are at particular risk for postsplenectomy sepsis. However, it should be noted that follow-up evaluation data on splenopexy patients are notably lacking. Splenectomy is ideally reserved for patients presenting with acute abdomen and splenic infarction or thrombosis or with hypersplenism and patients in whom splenopexy is technically unfeasible. Subtotal splenectomy and splenic autotransplantation may be of limited value. Pneumococcal, Hemophilus and meningococcal vaccines are indicated before elective splenectomy and shortly after nonelective splenectomy. Antibiotic prophylaxis is recommended for those at particular risk. Prospective studies are unlikely, but extended follow-up information on patients already reported, particularly those managed expectantly or with conservative surgical measures, is needed.

Congenital Abnormalities↗
PubMed1990

Crystallization of prostaglandin-H synthase for X-ray structure analysis.

Prostaglandin-H (PGH) synthase from ram seminal vesicles is a dimeric integral membrane protein of molecular weight 140 kDa. PGH synthase is a key enzyme in the biosynthesis of prostaglandins, has cyclooxygenase and peroxidase activities, and contains heme as a coenzyme. In the peroxidation step of its reaction. PGH synthase can use xenobiotics as co-substrates and can catalyze the metabolic activation of carcinogens such as diethylstilbestrol. To gain a detailed understanding of the inner workings of PGH synthase, we are investigating its three-dimensional structure by X-ray crystallography. A purification procedure was established that yields stable homogeneous PGH synthase that is at least 80% holoenzyme. The crucial aspect is the proper choice of type and concentration of detergent in all steps of the procedure. Single crystals can be obtained from concentrated solutions of PGH synthase in the presence of polyethylene glycol 4000 as a precipitant. Crystallization occurs during gas phase equilibration with a concentrated salt solution. The enzyme solution becomes turbid and forms a second liquid phase in which PGH synthase crystals grow up to 0.2 mm in length in the course of days. Manipulation of these crystals is very difficult due to the small volume of the growth phase. The crystals dissolved rapidly in all aqueous media into which they were transferred for mounting in X-ray capillaries. Therefore, we have not yet been able to demonstrate their true X-ray scattering power. A crystal provisionally dry mounted diffracted to about 8 A resolution.

Animals↗
PubMed1987

Refinement of the C222(1) crystal form of oxidized uteroglobin at 1.34 A resolution.

The structure of uteroglobin, a progesterone binding protein from rabbit uterine fluid, was determined and refined at 1.34 A resolution to a conventional R-factor of 0.229. The accuracy of the co-ordinates is estimated to be 0.15 A. The isotropic temperature factor of individual atoms was refined and its average value is 11.9 A2 for the 548 non-hydrogen atoms of the protein monomer. A total of 83 water molecules was located in difference electron density maps and refined, first using a constant occupancy factor of 1 and then variable occupancy, the final (Q) being 0.63. The mean temperature factor of the water oxygen atoms is 26.4 A2. Uteroglobin is a dimer and its secondary structure consists of four alpha-helices per monomer that align in an anti-parallel fashion. There is one beta-turn between helix 2 and helix 3 (Lys26 to Glu29); 76% of the residues are part of the alpha-helices. In the core of the dimeric protein molecule, between the two monomers that are held together by two disulfide bridges, we have observed a closed cavity. Its length is 15.6 A and its width is 9 A; 14 water molecules could be positioned inside. In the "bottom" part of the protein, near the C terminus, we have observed a smaller cavity, occupied by two water molecules. The calculation of the molecular surface revealed four surface pockets whose possible functional implications are discussed below.

Amino Acid Sequence↗
PubMed1975

Studies of asymmetry in the three-dimensional structure of lobster D-glyceraldehyde-3-phosphate dehydrogenase.

An improved electron density map of lobster holo-D-glyceraldehyde-3-phosphate dehydrogenase has been computed to 2.9 A resolution based on two heavy atom isomorphous derivatives. This has been averaged only over the Q molecular 2-fold axis, which is known to be exact in the human holoenzyme. The map showed possible asymmetry between the subunits in which the active centers are closely related across the R axis (that is, between the red and green or between the yellow and blue subunits). A difference map between the electron density of citrate and sulfate-soaked crystals gave further evidence for possible asymmetry. The major differences of electron density between R axis-related subunits appear around the active center and suggest the following interpretations. 1. The conformation of the adenine about the glycosidic bond is the more frequently observed anti with a C-2' endo conformation for the ribose ring in the red and yellow subunits, but is probably syn with a C-3' endo conformation in the green and blue subunits.2. The adenine ribose has its 3'-hydroxyl group hydrogen-bonded to a main chain carbonyl group in the red and yellow subunits but not in the green and blue subunits, as a consequence of the differing ribose conformations. 3. Cysteine-149 is more closely associated with histidine-176 in the green and blue subunits, and appears nearer the nicotinamide in the red and yellow subunits.

Amino Acid Sequence↗
PubMed1973

D-glyceraldehyde-3-phosphate dehydrogenase: three-dimensional structure and evolutionary significance.

A 3.0-A resolution electron density map of lobster glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) was computed. The essentially single isomorphous replacement map was very substantially improved by averaging subunits. NAD binds in an open conformation at sites close to subunit interfaces. The coenzyme binding portion of the enzyme has almost the same fold as the corresponding portion of lactate dehydrogenase (EC 1.1.1.27). The presence of this structure in the five enzymes, analyzed so far, that use nucleotide coenzymes might indicate a fundamental primordial structural element.

Animals↗
PubMed1973

Structure-function relationships in lactate dehydrogenase.

The binding of coenzyme and substrate are considered in relation to the known primary and tertiary structure of lactate dehydrogenase (EC 1.1.1.27). The adenine binds in a hydrophobic crevice, and the two coenzyme phosphates are oriented by interactions with the protein. The positively charged guanidinium group of arginine 101 then folds over the negatively charged phosphates, collapsing the loop region over the active center and positioning the unreactive B side of the nicotinamide in a hydrophobic protein environment. Collapse of the loop also introduces various charged groups into the vicinity of the substrate binding site. The substrate is situated between histidine 195 and the C4 position on the nicotinamide ring, and is partially oriented by interactions between its carboxyl group and arginine 171. The spatial arrangements of these groups may provide the specificity for the L-isomer of lactate.

Amino Acid Sequence↗