Biomedical subjects

J Desmet

Publications and source records attributed to J Desmet.

15 recordsLinked to original sources

PubMed1997

All in one: a highly detailed rotamer library improves both accuracy and speed in the modelling of sidechains by dead-end elimination.

BACKGROUND: About a decade ago, the concept of rotamer libraries was introduced to model sidechains given known mainchain coordinates. Since then, several groups have developed methods to handle the challenging combinatorial problem that is faced when searching rotamer libraries. To avoid a combinatorial explosion, the dead-end elimination method detects and eliminates rotamers that cannot be members of the global minimum energy conformation (GMEC). Several groups have applied and further developed this method in the fields of homology modelling and protein design. RESULTS: This work addresses at the same time increased prediction accuracy and calculation speed improvements. The proposed enhancements allow the elimination of more than one-third of the possible rotameric states before applying the dead-end elimination method. This is achieved by using a highly detailed rotamer library allowing the safe application of an energy-based rejection criterion without risking the elimination of a GMEC rotamer. As a result, we gain both in modelling accuracy and in computational speed. Being completely automated, the current implementation of the dead-end elimination prediction of protein sidechains can be applied to the modelling of sidechains of proteins of any size on the high-end computer systems currently used in molecular modelling. The improved accuracy is highlighted in a comparative study on a collection of proteins of varying size for which score results have previously been published by multiple groups. Furthermore, we propose a new validation method for the scoring of the modelled structure versus the experimental data based upon the volume overlap of the predicted and observed sidechains. This overlap criterion is discussed in relation to the classic RMSD and the frequently used +/- 40 degrees window in comparing chi 1 and chi 2 angles. CONCLUSIONS: We have shown that a very detailed library allows the introduction of a safe energy threshold rejection criterion, thereby increasing both the execution speed and the accuracy of the modelling program. We speculate that the current method will allow the sidechain prediction of medium-sized proteins and complex protein interfaces involving up to 150 residues on low-end desktop computers.

Algorithms↗

PubMed1997

Dead-end based modeling tools to explore the sequence space that is compatible with a given scaffold.

The dead-end elimination algorithm has proven to be a powerful tool in protein homology modeling since it allows one to determine rapidly the global minimum-energy conformation (GMEC) of an arbitrarily large collection of side chains, given fixed backbone coordinates. After introducing briefly the necessary background, we focus on logic arguments that increase the efficacy of the dead-end elimination process. Second, we present new theoretical considerations on the use of the dead-end elimination method as a tool to identify sequences that are compatible with a given scaffold structure. Third, we initiate a search for properties derived from the computed GMEC structure to predict whether a given sequence can be well packed in the core of a protein. Three properties will be considered: the nonbonded energy, the accessible surface area, and the extent by which the GMEC side-chain conformations deviate from a locally optimal conformation.

Amino Acid Sequence↗

PubMed1997

Computation of the binding of fully flexible peptides to proteins with flexible side chains.

Docking algorithms play an important role in the process of rational drug design and in understanding the mechanism of molecular recognition. An important determinant for successful docking is the extent to which the configurational space (including conformational changes) of the ligand/receptor system is searched. Here we describe a new, combinatorial method for flexible docking of peptides to proteins that allows full rotation around all single bonds of the peptide ligand and around those of a large set of receptor side chains. We have simulated the binding of several viral peptides to murine major histocompatibility complex class I H-2Kb. In addition, we have explored the limits of our method by simulating a complex between calmodulin and an 18-residue long helical peptide from calmodulin-dependent protein kinase IIalpha. The calculated peptide conformations generally matched well with the X-ray structures. Essential information about local flexibility and about residues that are responsible for strong binding was obtained. We have frequently observed considerable side-chain flexibility during the simulations, showing the need for a flexible treatment of the receptor. Our method may also be useful whenever the receptor side-chain conformation is not available or uncertain, as illustrated by the docking of an H-2Kb binding nonapeptide to the receptor structure taken from an octapeptide/H-2Kb complex.

Alanine↗

PubMed1997

Theoretical and algorithmical optimization of the dead-end elimination theorem.

The dead-end elimination theorem has proved to be a powerful method to reduce the theoretically accessible conformational space when modeling protein side chains by using a rotameric representation of possible conformations. In this work, theoretical details about variants to the original criterion are discussed. We also provide information on how the equations can be algorithmically implemented in such a way that both computational performance and structural accuracy are optimized. In addition, we discuss the theoretical and practical aspects of three new methods called the "bottom line theorem", dead-end elimination assisted by local modeling and a combinatorial search combined with conventional dead-end elimination. It is shown that the algorithm in its current from enables the determination of the global minimum energy side chain conformation of large proteins on a time scale of hours while for small proteins of up to 30 residues the calculations are done on a time scale of seconds. The latter opens a way to combine a main chain sampling algorithm with the dead-end elimination method to locally model entire fragments of a protein chain.

Algorithms↗

PubMed1995

A Ca(2+)-binding chimera of human lysozyme and bovine alpha-lactalbumin that can form a molten globule.

In contrast to lysozymes, which undergo two-state thermal denaturation, the Ca(2+)-free form of the homologous alpha-lactalbumins forms an intermediate "molten globule" state. To understand this difference, we have produced a chimera of human lysozyme and bovine alpha-lactalbumin. In the synthetic gene of the former the sequence coding for amino acid residues 76-102 was replaced by that for bovine alpha-lactalbumin 72-97, which represents the Ca(2+)-binding loop and the central helix C. The chimeric protein, LYLA1, expressed in Saccharomyces cerevisiae was homogeneous on electrophoresis and mass spectrometry. Its Ca2+ binding constant was 2.50 (+/- 0.04) x 10(8) M-1, and its muramidase activity 10% of that of human lysozyme. One-dimensional NMR spectroscopy indicated the presence of a compact, well structured protein. From two-dimensional NMR spectra, main chain resonances for 118 of a total of 129 residues could be readily assigned. Nuclear Overhauser effect analysis and hydrogen-deuterium exchange measurements indicated the presence and persistence of all expected secondary structure elements. Thermal denaturation, measured by circular dichroism, showed a single transition temperature for the Ca2+ form at 90 degrees C, whereas unfolding of the apo form occurred at 73 degrees C in the near-UV and 81 degrees C in the far-UV range. These observations illustrate that by transplanting the central part of bovine alpha-lactalbumin, we have introduced into human lysozyme two important properties of alpha-lactalbumins, i.e. stabilization through Ca2+ binding and molten globule behavior.

Amino Acid Sequence↗

PubMed1995

Enhanced dead-end elimination in the search for the global minimum energy conformation of a collection of protein side chains.

Although the conformational states of protein side chains can be described using a library of rotamers, the determination of the global minimum energy conformation (GMEC) of a large collection of side chains, given fixed backbone coordinates, represents a challenging combinatorial problem with important applications in the field of homology modelling. Recently, we have developed a theoretical framework, called the dead-end elimination method, which allows us to identify efficiently rotamers that cannot be members of the GMEC. Such dead-ending rotamers can be iteratively removed from the system under study thereby tracking down the size of the combinatorial problem. Here we present new developments to the dead-end elimination method that allow us to handle larger proteins and more extensive rotamer libraries. These developments encompass (i) a procedure to determine weight factors in the generalized dead-end elimination theorem thereby enhancing the elimination of dead-ending rotamers and (ii) a novel strategy, mainly based on logical arguments derived from the logic pairs theorem, to use dead-ending rotamer pairs in the efficient elimination of single rotamers. These developments are illustrated for proteins of various sizes and the flow of the current method is discussed in detail. The effectiveness of dead-end elimination is increased by two orders of magnitude as compared with previous work. In addition, it now becomes feasible to use extremely detailed libraries. We also provide an appendix in which the validity of the generalized dead-end criterion is shown. Finally, perspectives for further applications which may now become within reach are discussed.

Aprotinin↗

PubMed1993

Complexes of the polyamines spermine, spermidine and putrescine with alpha-lactalbumins.

The effects of polyamines on the spectral properties and thermal stability of different alpha-lactalbumins were measured. Addition of millimolar concentrations of spermine to the Ca(2+)-free (apo) form of bovine or goat alpha-lactalbumin resulted in spectral shifts, in both the far- and near-ultraviolet ranges, similar to those induced by Ca2+ binding. Fluorescence emission spectra of tryptophan residues underwent a pronounced blue shift, concomitant with a decrease in quantum yield. Also, in the presence of spermine, thermal stability was increased. In contrast, in the case of human and to some extent also equine alpha-lactalbumin, the shifts of the CD and fluorescence spectra and the thermal denaturation curves were in the opposite direction. Direct competition of spermine with Mn2+ could be observed for binding to the Ca2+ site of bovine alpha-lactalbumin. As the main binding mode of spermine showed a 1:1 stoichiometry [K1 = (2 +/- 0.5) x 10(4) M-1], we have assumed that binding occurs primarily at this site with one of the distal ammonium groups. In order to accommodate the remaining positively charged ammonium groups of spermine, a binding model was constructed by computer modeling, based on the atomic coordinates of both interacting species. This model makes use of the cluster of negatively charged glutamate residues present in the N-terminal sequence of bovine alpha-lactalbumin, sterically close to the Ca2+ site. The spermine molecule could be nicely fitted in a space delineated by the Ca2+ site, on the one hand, and Glu1, Glu7 and Glu11, on the other. Our model, which has been extended to the shorter polyamines spermidine and putrescine, is able to fully account for the observed stabilization of bovine (and goat) alpha-lactalbumins against thermal denaturation, and for the observed CD and fluorescence spectral shifts. It also explains why human (and equine) alpha-lactalbumin behave differently.

Amino Acid Sequence↗

PubMed1993

The fuzzy-end elimination theorem: correctly implementing the side chain placement algorithm based on the dead-end elimination theorem.

Recently it has been shown that the dead-end elimination theorem is a powerful tool in the search for the global minimum energy conformation (GMEC) of a large collection of protein side chains given known backbone coordinates and a library of allowed side chain conformational states, also known as rotamers. A side chain placement algorithm based on this theorem iteratively applies this theorem to single as well as to pairs of rotamers leading to the identification of rotamers, single or pairs, that are incompatible with the GMEC and that can thus be qualified as 'dead-ending'. Here we formulate a theorem which proves that contrary to intuition dead-end rotamer pairs cannot simply be discarded from consideration in the iterative process leading to the further elimination of dead-end rotamers. We refer to this theorem as the fuzzy-end elimination theorem. We also describe how the obtained dead-end rotamer pairs can contribute to the search for the GMEC in the protein side chain placement problem. Hence the present work forms a theoretical basis for the correct implementation of a side chain placement algorithm based on the dead-end elimination theorem. In addition, possible future perspectives are presented.

Algorithms↗

PubMed1991

Thermodynamic data on the binding of six M2(+)-ions to bovine, goat, and human alpha-lactalbumin.

By batch microcalorimetry we titrated the apo-forms of bovine, goat, and human alpha-lactalbumin with Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, and Cd2+ ions at pH 7.5 and 25 degrees C. The titration curves enabled us to calculate the apparent enthalpy changes and binding constants and thus, also the free energy and the entropy changes of the binding. CD-spectra showed that all cations induce the same conformational change to the native form of the protein. The calorimetric and spectroscopic results, as well as sequence comparisons confirm the hypothesis that all these ions occupy the very same site on the molecule. The thermodynamic parameters, plotted vs the ionic radii, run parallel for the three proteins, which illustrates the earlier proposed "rigid site" model.

Animals↗

PubMed1991

Thermodynamics of Mn(2+)-binding to goat alpha-lactalbumin.

By means of reaction calorimetry we measured the apparent enthalpy change, delta Happ, of the binding of Mn(2+)-ions to goat alpha-lactalbumin as a function of temperature. The observed delta Happ can be written as the sum of contributions resulting from a conformational and a binding process. In combination with the thermal unfolding curve of goat alpha-lactalbumin, we succeeded in separating the complete set of thermodynamic parameters (delta H, delta G, delta S, delta Cp) into the binding and conformational contributions. By circular dichroism we showed that NH+4-ions, upon binding to bovine alpha-lactalbumin, induce the same conformational change as do Na+ and K+: the binding constant KappNH+4 equals 98 +/- 9 M-1.

Animals↗

PubMed1989

High performance liquid chromatography stability study of malonyl-coenzyme A, using statistical experimental designs.

Malonyl-CoA is a biochemically important compound, formed by an acetyl-coenzyme A carboxylase catalysed reaction. The stability of this short-chain coenzyme A derivative under various experimental conditions is discussed in this article. High-performance liquid chromatography was used for the analysis of the reaction mixture because of its excellent selectivity and sufficient sensitivity. Several variables were investigated as possible stability-influencing factors: pH, magnesium and buffer concentration, reaction temperature and time. The Plackett-Burman screening design was first used for selecting the most important variables, with which a central composite design was constructed. In this way, a response surface was obtained with the percentage remaining malonyl-CoA as a function of magnesium concentration, reaction temperature and time. The usefulness of this approach is demonstrated by obtaining kinetic data from the mathematical function and by the evaluation of the stopping of reaction procedure in the activity assay of acetyl-coenzyme A carboxylase.

Acyl Coenzyme A↗

PubMed1989

Comparison of the binding of Ca2+ and Mn2+ to bovine alpha-lactalbumin and equine lysozyme.

The enthalpy change of the binding of Ca2+ and Mn2+ to equine lysozyme was measured at 25 degrees C and pH 7.5 by batch microcalorimetry: delta H degrees Ca2+ = -76 +/- 5 kJ mol-1, delta H degrees Mn2+ = -21 +/- 10 kJ mol-1. Binding constants, log KCa2+ = 6.5 +/- 0.2 and log KMn2+ = 4.1 +/- 0.5, were calculated from the calorimetric data. Therefore, delta S degrees Ca2+ = -131 +/- 20 JK-1 mol-1 and delta S degrees Mn2+ = 8 +/- 44 JK-1 mol-1. Removal of Ca2+ induces small but significant changes in the circular dichroism spectrum, indicating the existence of a partially unfolded apo-conformation, comparable with, but different from, the apo-conformation of bovine alpha-lactalbumin.

Animals↗

PubMed1988

Calorimetric experiments of Mn2+ -binding to alpha-lactalbumin.

We measured by batch microcalorimetry the standard enthalpy change delta H degrees of the binding of Mn2+ to apo-bovine alpha-lactalbumin; delta H degrees = -90 +/- k J.mol-1. The binding constants, KMn2+, calculated from the calorimetric and circular dichroism titration curves, are (4.6 +/- 1).10(5) M-1 and (2.1 +/- 0.4).10(5) M-1, respectively. Batch calorimetry confirms the competitive binding Ca2+, Mn2+ and Na+ to the same site. The relatively small enthalpy change for Mn2+ binding compared to Ca2+ binding favours a model of a rigid and almost ideal Ca2+-complexating site, different from the well-known EF-hand structures. Cation binding to the high-affinity site most probably triggers the movement of an alpha-helix which is directly connected to the complexating loop.

Binding Sites↗

PubMed1987

Comparison of the binding of Na+ and Ca2+ to bovine alpha-lactalbumin.

alpha-Lactalbumin is a metal-binding protein which binds Ca2+- and Na+-ions competitively to one specific site, giving rise to a large conformational change of the protein. For this reason, the enthalpy change of binding Ca2+ to apo-alpha-lactalbumin (delta Ho) is strongly dependent on the concentration of Na+ ions in the medium. From that relationship a molar enthalpy of -145 +/- 3 kJ X mol-1 is calculated for the Ca2+-binding at pH 7.4 and 25 degrees C, while a delta Ho of -5 +/- 3 kJ X mol-1 is found to substitute a complexed Na+ by a Ca2+-ion. These measurements also allowed us to calculate a binding constant for Na+ of 195 +/- 18 M-1. The molar enthalpy of Na+-loading was found to be -142 +/- 3 kJ X mol-1, a value very close to delta Ho of the binding of Ca2+ to alpha-lactalbumin. Both enthalpy changes in binding Ca2+ and Na+ are independent of the protein concentration. These exothermic values are in agreement with the hypothesis that both Na+- and Ca2+-ions are able to induce the same conformational change in alpha-lactalbumin upon which hydrophobic regions are removed from the solvent, yielding a less hydrophobic protein. The latter is confirmed by means of affinity measurements of the hydrophobic fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulphonate](bis-ANS) to alpha-lactalbumin. The association constant (Ka) decreased from (6.6 +/- 0.5) X 10(4) M-1 in the absence of NaCl to (2.7 +/- 0.2) X 10(4) M-1 in 75 mM NaCl, while the maximum intensity (Imax) of the binary bis-ANS-alpha-lactalbumin complex remained constant at 0.44 +/- 0.02 (arbitrary units). The Ka value of bis-ANS for Ca2+-alpha-lactalbumin was determined at (1.7 +/- 0.2) X 10(4) M-1 and Imax was 0.43 +/- 0.02 (arbitrary units). The difference in hydrophobicity between the two conformational states of the protein was further demonstrated by adsorption experiments of both conformers to phenyl-Sepharose. Apo-alpha-lactalbumin, hydrophobically bound to phenyl-Sepharose, can be eluted by adding Ca2- or Na+-solutions.

Animals↗

PubMed1985

Coexistence of hypocalciuric hypercalcaemia and interstitial lung disease in a family: a cross-sectional study.

In a prospective investigation, a large kindred (twenty-one subjects) with unexplained association of familial hypocalciuric hypercalcaemia and idiopathic interstitial lung disease was studied. Serum calcium was increased in fifteen patients (the youngest being 7 years old) and was associated with hypo- or normocalciuria. The abnormalities were not age-dependent. The serum concentrations of parathyroid hormone, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and calcitonin were normal. In twelve patients the diffusing capacity (DLCO) and/or DLCO per unit lung volume was less than 75% predicted. This was often accompanied by a vital capacity of less than 80% predicted, and increased Tiffeneau index, and a reticulo-micronodular pattern with high diaphragm on chest X-ray. The decrease in DLCO was more pronounced in older non-smoking as well as smoking subjects (P less than 0.02) suggesting a progressing interstitial disease with age. The fibrosing alveolitis, which had been confirmed by open lung biopsy in three subjects, could not be attributed to sarcoidosis, collagen-vascular disease, or exogenous causes. The disturbances in the calcium homeostasis and in the diffusing capacity of the lung coexisted in seven of the twenty-one patients. Apparently, both abnormalities were inherited following an autosomal-dominant pattern but with a different penetration in each person, and seemed not be causally related to each other.

Adolescent↗