An efficient general-purpose least-squares refinement program for macromolecular structures

Tronrud, Dale E.; Eyck, Lynn F. Ten; Matthews, B.W.

doi:10.1107/s0108767387099124

Cited by 925 publications

(606 citation statements)

References 12 publications

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“…The structural changes associated with the various mutants were first visualized using difference electron density maps and then refined (Tronrud et al, 1987) using procedures essentially as described by . The refined model of wild-type lysozyme (Bell et al, 1991) was used as the starting point for refinement.…”

Section: Resultsmentioning

confidence: 99%

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

1992

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In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within a-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the a-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions.All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest ( 5 0 . 4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133 +Ala made a-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino-and carboxyterminal domains changed by 3.6". This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.Keywords: alanine; lysozyme; protein folding; protein structure; thermostability In a systematic attempt to identify residues important in the folding and stability of phage T4 lysozyme we previously substituted four alanines within the a-helix that includes residues 126-134 . The helix is amphipathic, located on the surface of the carboxy-terminal domain, and is remote from the active site ( Fig. 1; Kinemage 1). In wild-type lysozyme this a-helix already includes three alanines. It was found that substitution of three solvent-exposed residues, Glu 128, Val 131, and Asn

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Section: Resultsmentioning

confidence: 99%

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

1992

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“…Subsequent refinement (Tronrud et al, 1987) of the structure has decreased R to 0.190 for all data (37,621 reflections) between 10.0 and 1.7 A for the native structure and to 0.185 (36,903 reflections) for FNR bound to 2'-phospho AMP (Bruns & Karplus, in prep.). Comparisons in this paper are based on the refined coordinates.…”

Section: Analyses Of Fnr and Pdrmentioning

confidence: 99%

Structural prototypes for an extended family of flavoprotein reductases: Comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin

et al. 1993

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The structure of phthalate dioxygenase reductase (PDR), a monomeric iron-sulfur flavoprotein that delivers electrons from NADH to phthalate dioxygenase, is compared to ferredoxin-NADP+ reductase (FNR) and ferredoxin, the proteins that reduce NADP+ in the final reaction of photosystem I. The folding patterns of the domains that bind flavin, NAD(P), and [2Fe-2S] are very similar in the two systems. Alignment of the X-ray structures of PDR and FNR substantiates the assignment of features that characterize a family of flavoprotein reductases whose members include cytochrome P-450 reductase, sulfite and nitrate reductases, and nitric oxide synthase. Hallmarks of this subfamily of flavoproteins, here termed the FNR family, are an antiparallel 0-barrel that binds the flavin prosthetic group, and a characteristic variant of the classic pyridine nucleotide-binding fold. Despite the similarities between FNR and PDR, attempts to model the structure of a dissociable FNR:ferredoxin complex by analogy with PDR reveal features that are at odds with chemical crosslinking studies (Zanetti, G., Morelli, D., Ronchi, S . , Negri, A., Aliverti, A., & Curti, B., 1988, Biochemistry 27, 3753-3759).Differences in the binding sites for flavin and pyridine nucleotides determine the nucleotide specificities of FNR and PDR. The specificity of FNR for NADP' arises primarily from substitutions in FNR that favor interactions with the 2' phosphate of NADP'. Variations in the conformation and sequences of the loop adjoining the flavin phosphate affect the selectivity for FAD versus FMN.The midpoint potentials for reduction of the flavin and [2Fe-2S] groups in PDR are higher than their counterparts in FNR and spinach ferredoxin, by about 120 mV and 260 mV, respectively. Comparisons of the structure of PDR with spinach FNR and with ferredoxin from Anabaena 7120, along with calculations of electrostatic potentials, suggest that local interactions, including hydrogen bonds, are the dominant contributors to these differences in potential.

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“…The resulting maps were then used to construct models for the new side chains using the FRODO package (Jones, 1982). The mutant structures were refined using the TNT package (Tronrud et al, 1987;Tronrud, 1992) as described by Wilson et al (1992). Bank (Brookhaven National Laboratory) with codes lLSN and 1 LSM, respectively.…”

Section: Preparation and Crystallization Of Mutant Lysozymesmentioning

confidence: 99%

Thermal stability determinants of chicken egg‐white lysozyme core mutants: Hydrophobicity, packing volume, and conserved buried water molecules

1995

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A series of 24 mutants was made in the buried core of chicken lysozyme at positions 40, 5 5 , and 91. The midpoint temperature of thermal denaturation transition (T,) values of these core constructs range from 60.9 to 77.3 "C, extending an earlier, more limited investigation on thermostability. The T,,, values of variants containing conservative replacements for the wild type (WT) (Thr 40-Ile 55-Ser 91) triplet are linearly correlated with hydrophobicity (r = 0.81) and, to a lesser degree, with combined side-chain volume (r = 0.75). The X-ray structures of the S91A (1.9A) and 155L/S91T/DlOlS (1.7 A) mutants are presented. The former amino acid change is found in duck and mammalian lysozymes, and the latter contains the most thermostable core triplet. A network of four conserved, buried water molecules is associated with the core. It is postulated that these water molecules significantly influence the mutational tolerance at the individual triplet positions. The pH dependence of T, for the S91D mutant was compared with that of WT enzyme. The pK, of S91D is 1.2 units higher in the native than in the denatured state, corresponding to AAG298 = 1.7 kcal/mol. This is a low value for charge burial and likely reflects the moderating influence of the buried water molecules or a conformational change. Thermal and chemical denaturation and far UV CD spectroscopy were used to characterize the in vitro properties of I55T. This variant, which buries a hydroxyl group, has similar properties to those of the human amyloidogenic variant I56T.

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An efficient general-purpose least-squares refinement program for macromolecular structures

Cited by 925 publications

References 12 publications

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

Multiple alanine replacements within α‐helix 126–134 of T4 lysozyme have independent, additive effects on both structure and stability

Structural prototypes for an extended family of flavoprotein reductases: Comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin

Thermal stability determinants of chicken egg‐white lysozyme core mutants: Hydrophobicity, packing volume, and conserved buried water molecules

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