Improving the prediction of secondary structure of -TIM-barrel— enzymes

The information contained in aligned sets of homologous protein sequences should improve the score of secondary structure prediction. Seven different enzymes having the (β/α)8 or TIM-barrel fold were used to optimize the prediction with regard to this class of enzymes. The α-helix, β-strand and loop propensities of the Garnier—Osguthorpe—Robson method were averaged at aligned residue positions, leading to a significant improvement over the average score obtained from single sequences. The increased accuracy correlates with the average sequence variability of the aligned set. Further improvements were obtained by using the following averaged properties as weights for the averaged state propensities: amphipathic moment and α-helix; hydropathy and β-strand; chain flexibility and loop. The clustering of conserved residues at the C-terminal ends of the 13-strands was used as an additional positive weight for β-strand propensity and increased the prediction of otherwise unpredicted β-strands decisively. The automatic weighted prediction method identifies >95% of the secondary structure elements of the set of seven TIM-barrel enzyme

The predicted secondary structure of the G-type glutamineamidotransferase is compatible with TEM-barrel topology

Glutamine amidotransferase (GAT) subunits or domains catalyze an important partial reaction in many complex biosynthetic reactions. The structure of one member of the F-type GATs is known, but the structure of the unrelated G-type is still unknown. Because many protein sequences are available for anthranilate synthase component II (product of the trpG gene), we have predicted its average secondary structure by a joint prediction method [Niermann and Kirschner (1991a) Protein Engng, 4, 359-370]. The predicted eight β-strands and seven α-helices follow an 8-fold cyclic repetition of a β-strand-loop-α-helix-loop module with helix α7 missing. This pattern of secondary structure suggests that the G-type GAT domain has an 8-fold βα-barrel topology, as found first in triose phosphate isomerase (TIM-barrel). This model is supported by the location of known catalytically essential residues in loops between (β-strands and α-helices. Evidence from published sequencing and mutational studies on selected members of the GAT superfamily (carbamoyl phosphate, imidazoleglycerol phosphate, GMP and CTP synthases) support both the secondary structure prediction and the TIM-barrel topolog

An 8-fold βα barrel protein with redundant folding possibilities

Protein sequences containing redundant segments of secondary structure at both termini have the choice a priori of folding into several possible circularly permuted variants of the wild-type tertiary structure. To test this hypothesis the gene of phosphoribosyl anthranilate isomerase from yeast, which is a single-domain 8-fold βα barrel protein, was modified to produce a 10-fold βα homologue in Escherichia coli. It contained a duplicate of the two C-terminal βα units of supersecondary structure fused to its N-terminus. Most of the protein was recovered from the insoluble fraction of disrupted cells by dissolution in guanidinium chloride solutions and refolding. Pristine protein was purified from the soluble fraction. The purified (βα)10 proteins were enzymically almost fully active. Absorbance, fluorescence and circular dichroism spectra as well as the reversible unfolding behaviour of both proteins were also very similar to the properties of the original (βα)8 protein. Digestion with endopeptidases converted both the pristine and the refolded (βα)10 variant to the same large fragment that had the N-terminal sequence and mol. wt of the wild-type βα)8 protein. The data suggest that the folding of the (βα)10 variant is controlled thermodynamically both in vivo and in vitr

2.0 å structure of indole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus solfataricus: possible determinants of protein stability

AbstractBackground: Recent efforts to understand the basis of protein stability have focussed attention on comparative studies of proteins from hyperthermophilic and mesophilic organisms. Most work to date has been on either oligomeric enzymes or monomers comprising more than one domain. Such studies are hampered by the need to distinguish between stabilizing interactions acting between subunits or domains from those acting within domains. In order to simplify the search for determinants of protein stability we have chosen to study the monomeric enzyme indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus (sIGPS), which grows optimally at 90°C.Results The 2.0 å crystal structure of sIGPS was determined and compared with the known 2.0 å structure of the IGPS domain of the bifunctional enzyme from the mesophilic bacterium Escherichia coli (eIGPS). sIGPS and eIGPS have only 30% sequence identity, but share high structural similarity. Both are single-domain (β/α)8 barrel proteins, with one (eIGPS) or two (sIGPS) additional helices inserted before the first β strand. The thermostable sIGPS has many more salt bridges than eIGPS. Several salt bridges crosslink adjacent α helices or participate in triple or quadruple salt-bridge clusters. The number of helix capping, dipole stabilizing and hydrophobic interactions is also increased in sIGPS.Conclusion The higher stability of sIGPS compared with eIGPS seems to be the result of several improved interactions. These include a larger number of salt bridges, stabilization of α helices and strengthening of both polypeptide chain termini and solvent-exposed loops

Structure and function of mutationally generated monomers of dimeric phosphoribosylanthranilate isomerase from Thermotoga maritima.

BACKGROUND: Oligomeric proteins may have been selected for in hyperthermophiles because subunit association provides extra stabilization. Phosphoribosylanthranilate isomerase (PRAI) is monomeric and labile in most mesophilic microorganisms, but dimeric and stable in the hyperthermophile Thermotoga maritima (tPRAI). The two subunits of tPRAI are associated back-to-back and are locked together by a hydrophobic loop. The hypothesis that dimerization is important for thermostability has been tested by rationally designing monomeric variants of tPRAI. RESULTS: The comparison of tPRAI and PRAI from Escherichia coli (ePRAI) suggested that levelling the nonplanar dimer interface would weaken the association. The deletion of two residues in the loop loosened the dimer. Subsequent filling of the adjacent pocket and the exchange of polar for apolar residues yielded a weakly associating and a nonassociating monomeric variant. Both variants are as active as the parental dimer but far more thermolabile. The thermostability of the weakly associating monomer increased significantly with increasing protein concentration. The X-ray structure of the nonassociating monomer differed from that of the parental subunit only in the restructured interface. The orientation of the original subunits was maintained in a crystal contact between two monomers. CONCLUSIONS: tPRAI is dimeric for reasons of stability. The clearly separated responsibilities of the betaalpha loops, which are involved in activity, and the alphabeta loops, which are involved in protein stability, has permitted the evolution of dimers without compromising their activity. The preserved interaction in the crystal contacts suggests the most likely model for dimer evolution

Crystal structure at 2.0 A resolution of phosphoribosyl anthranilate isomerase from the hyperthermophile Thermotoga maritima: possible determinants of protein stability.

The structural basis of thermostability of proteins is of great scientific and biotechnological interest. Differences in the X-ray structues of orthologous proteins from hyperthermophilic and mesophilic organisms can indicate crucial stabilizing interactions. To this end the crystal structure of dimeric phosphoribosyl anthranilate isomerase from the hyperthermophile Thermotoga maritima (tPRAI) was determined using phases derived from the isomorphous replacement method and was refined at 2.0 A resolution. The comparison to the known 2.0 A structure of PRAI from Escherichia coli (ePRAI) shows that tPRAI has the complete TIM- or (beta alpha)8-barrel fold, whereas helix alpha5 in ePRAI is replaced by a loop. The subunits of tPRAI associate via the N-terminal faces of their central beta-barrels. Two long, symmetry-related loops that protrude reciprocally into cavities of the other subunit provide for multiple hydrophobic interactions. Moreover, the side chains of the N-terminal methionines and the C-terminal leucines of both subunits are immobilized in a hydrophobic cluster, and the number of salt bridges is increased in tPRAI. These features appear to be mainly responsible for the high thermostability of tPRAI. In contrast to other hyperthermostable enzymes, tPRAI at 25 degrees C is catalytically more efficient than ePRAI, mainly due to its small K(M) value for the substrate [Sterner, R., Kleemann, G. R., Szadkowski, H., Lustig, A., Hennig, M., & Kirschner, K. (1996) Protein Sci. 5, 2000-2008]. The increased number of hydrogen bonds between the phosphate ion and tPRAI compared to ePRAI could be responsible for this effect

Purification, characterization and crystallization of thermostable anthranilate phosphoribosyltransferase from Sulfolobus solfataricus

DkrColorVolume 65, Page 1