Analysis Of Conformational Changes Research Articles

Automatic Analysis of Protein Conformational Changes by Multiple Linkage Clustering

An automatic algorithm is presented for analyzing protein conformational changes such as those occurring upon substrate binding or in different crystal forms of the same protein. Using, as sole information, the atomic coordinates of a pair of protein structures, the procedure first generates structure alignments, which optimize the root-mean-square deviation of the backbone atoms. To this end, equivalent secondary stuctures and/or loops from both proteins are combined by a multiple linkage hierarchic clustering algorithm, which generates several intertwined clustering trees. Automatic analysis of these clustering trees is used to dissect the mechanism of the conformational change. It allows the identification of the static core, representing the collection of secondary structures which undergo no structural changes, as well as other entities which move like rigid bodies. It also permits the description of the movement of secondary structures or loops relative to this cre or entities. Using this information, it can be inferred, whether a particular conformational change involves shear or hinge motion, or components of both. The algorithm is applied to the analysis of the conformational changes of citrate synthase, lactate dehydrogenase, lactoferrin and β-glucosyltransferase, representing typical examples of shear- and hinge-type mechanisms, and a varied range in movement size. The results are shown to be in excellent agreement with previous analyses, and to provide additional information which gives a more complete and objective picture of the conformational change. Using our automatic algorithm, we find that any conformational change may be viewed as having components of both shear- and hinge-type motion. Determining which of these is most appropriate requires the combination of the information provided by our procedure with detailed knowledge of the protein tertiary structures.

Effect of modification of carbohydrate component on properties of glucoamylase

In this study, we investigated enzymatic deglycosylation of glucoamylase from Aspergillus awamori X 100/D27, a glycoprotein which has two N-linked and about forty short mannose-bearing O-linked sugars per molecule. O-Linked sugars were modified by treatment with α-mannosidase and N-linked sugars were removed using endo-β- N-acetylglucosaminidase F. Analysis ofconformational changes following deglycosylation suggests that O-linked sugars essentially contribute to the stabilization of glucoamylase domains. Modification of the carbohydrate component by adding 1-deoxymannojirimycin to the culture medium induced inhibition of α-mannosidases involved in the processing, leading to a more complete glycosylation and, consequently, to a higher stability of the enzyme.

Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed.

The cytochrome c oxidase-cytochrome c complex: spectroscopic analysis of conformational changes in the protein-protein interaction domain

Binding to cytochrome c oxidase induces a conformational change in the cytochrome c molecule. This conformational change has been characterized by comparing the binding of native cytochrome c and chemically modified cytochrome c derivatives to bovine cytochrome c oxidase by using absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy. The following derivatives were analyzed: (i) cytochrome c modified at all 19 lysine residues to yield the (N epsilon-acetimidyl)19 cytochrome c, (N epsilon-isopropyl)19 cytochrome c, and (N epsilon,N epsilon-dimethyl)19 cytochrome c; (ii) cytochrome c in which Met65 and Met80 are converted to the methionine sulfoxide; (iii) cytochrome c with a single break in the polypeptide chain at Arg38 or Gly37. The derivatives bind to cytochrome c oxidase at a ratio of one heme c per heme aa3. The association constants are similar to that of native cytochrome c except for (N epsilon-isopropyl)19 and (N epsilon,N epsilon-dimethyl)19 cytochromes c, which bind respectively four times and six times less strongly. The derivatives are good substrates for the cytochrome c oxidase reaction. The spectral changes accompanying the binding of the modified cytochromes c to cytochrome c oxidase are quite different from the spectral changes observed with native cytochrome c. The different optical absorption and MCD changes are explained by a polarity change around the exposed heme edge in the cytochrome c-cytochrome c oxidase complex. The CD changes indicate a conformational rearrangement restricted to the surface area surrounding the exposed heme edge. The rearrangement may involve a movement of the evolutionarily conserved Phe82 out of the vicinity of the heme.(ABSTRACT TRUNCATED AT 250 WORDS)

Complex of N-phosphonacetyl- l-aspartate with aspartate carbamoyltransferase : X-ray refinement, analysis of conformational changes and catalytic and allosteric mechanisms

The allosteric enzyme aspartate Carbamoyltransferase of Escherichia coli consists of six regulatory chains (R) and six catalytic chains (C) in D 3 symmetry. The less active T conformation, complexed to the allosteric inhibitor CTP has been refined to 2.6 Å ( R-factor of 0.155). We now report refinement of the more active R conformation, complexed to the bisubstrate analog N-phosphonacetyl- l-aspartate (PALA) to 2.4 Å ( R-factor of 0.165, rootmean-square deviations from ideal bond distances and angles of 0.013 Å and 2.2 °, respectively). The antiparallel β-sheet in the revised segment 8–65 of the regulatory chain of the T conformation is confirmed in the R conformation, as is also the interchange of alanine 1 with the side-chain of asparagine 2 in the catalytic chain. The crystallographic asymmetric unit containing one-third of the molecule (C 2R 2) includes 925 sites for water molecules, and seven side-chains in alternative conformations. The gross conformational changes of the T to R transition are confirmed, including the elongation of the molecule along its threefold axis by 12 Å, the relative reorientation of the catalytic trimers C 3 by 10 °, and the rotation of the regulatory dimers R 2 about the molecular twofold axis by 15 °. No changes occur in secondary structure. Essentially rigid-body transformations account for the movement of the four domains of each catalytic-regulatory unit; these include the allosteric effector domain, the equatorial (aspartate) domain, and the combination of the polar (carbamyl phosphate) and zinc domain, which moves as a rigid unit. However, interfaces change, for example the interface between the zinc domain of the R chain and the equatorial domain of the C chain, is nearly absent in the T state, but becomes extensive in the R state of the enzyme; also, one catalytic-regulatory interface (C1-R4) of the T state disappears in the more active R state of the enzyme. Segments 50–55, 77–86 and 231–246 of the catalytic chain and segments 51–55, 67–72 and 150–153 of the regulatory chain show conformational changes that go beyond the rigid-body movement of their corresponding domains. The localized conformational changes in the catalytic chain all derive from the interactions of the enzyme with the inhibitor PALA; these changes may be important for the catalytic mechanism. The conformation changes in segments 67–72 and 150–153 of the regulatory chain may be important for the allosteric control of substrate binding. On the basis of the conformational differences of the T and R states of the enzyme, we present a plausible scheme for catalysis that assumes the ordered binding of substrates and the ordered release of products. We present as well those interactions in the hexamer that may be responsible for positive co-operativity in catalysis and the modulation of catalytic rates by allosteric effectors.

A general formulation of the “quantum chemical le Chatelier principle”

AbstractRegularities observed in the variations of calculated energy components, for example, those of electronic and nuclear repulsion energies in the analysis of conformational changes and in studies of the propagation of basis set errors in ab initio calculations, are found to be related to the variational principle and to the boundedness of energy expectation value functionals. These relations are analogous to the le Chatelier principle of equilibrium thermodynamics, and may be formulated as a general “compensation principle” for two sets of general parameters of the molecular total energy functional.

Lower and upper bounds for the number of critical points on energy hypersurfaces

Lower and upper bounds are derived for the number of critical points of all indices λ(λ=0, ⋯ n), for potential energy hypersurfaces defined over a subset S of the nuclear configuration space n R. The number of minima (λ = 0) and saddle points of transition states (λ = 1) are important in analysis of conformational changes and chemical reactions.

Denaturation of proteins and nucleic acids by thermal-gradient electrophoresis.

A polyacrylamide-gel-electrophoresis method has been developed that permits the analysis of conformational changes that occur during the thermal denaturation of macromolecules. A stable transverse temperature gradient was produced in an aluminium heating jacket clamped around a vertical polyacrylamide slab gel. After temperature equilibration, gels were loaded with either a layer of protein solution (20-200 micrograms/gel) or a solution of double-stranded DNA (20 micrograms/gel) and electrophoresis begun. At the end of the run the gels were stained and the effect of temperature on mobility observed. The technique proved informative both for the irreversible unfolding of proteins (Drosophila alcohol dehydrogenase and lactic acid dehydrogenase) and for a protein that was reversibly denatured by heat (beta-lactamase). In the latter case a clear transition between the native enzyme and a slower-migrating denatured state was observed. The patterns obtained were analogous to the type produced by the transverse-urea-gradient-electrophoretic method of Creighton [(1979) J. Mol. Biol. 129, 253-264]. The method also resolved a complex mixture of double-stranded-DNA restriction-digest fragments.

Analysis of conformational changes in polystyrene and its derivatives in dilute solution by using the ultraviolet absorption behavior of model compounds

AbstractStudy of the variation as a function of temperature of the molar extinction coefficients of dilute solutions of various alkylbenzenes, the configurational isomers of 2, 4‐diphenylpentane and 2, 4, 6‐triphenylheptane, and polystyrene and certain of its halogenated and methylated derivatives, has revealed behavioral anomalies. These have been attributed to changes in the position of the benzene rings whether accompanied or not by rotations of the backbone bonds leading to an overall conformational change.

Sedimentation analysis of conformation changes of circular PM2 DNA in relation to the ionic strength

Sedimentation coefficients of both circular DNA components and the number of superhelix turns of Component I of PM2 DNA were measured as a function of the ionic strength. The resulting conformation changes of both circular DNA components as revealed by the sedimentation measurements are discussed in relation to the twisting and the polyelectrolyte expansion of the DNA molecule.