Circular permutation within the coenzyme binding domain of the tetrameric glyceraldehyde‐3‐phosphate dehydrogenase from <i>Bacillus stearothermophilus</i>

Vignais, Marie-Luce; Corbier, Catherine; Mulliert, Guillermo; Branlant, Christiane; Branlant, Guy

doi:10.1002/pro.5560040519

Cited by 18 publications

(1 citation statement)

References 27 publications

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“…Circular permutation, in which the natural termini are connected by a linker and new termini are introduced at desired positions within the protein, makes it possible to maintain the structure while altering the connectivity of the chain. For most proteins, circular permutation results in only minor structural changes and minimal loss of stability (Zhang et al, 1993;Hahn et al, 1994;Ritco-Vonsovici et al, 1995;Vignais et al, 1995;Viguera et al, 1995;Aÿ et al, Ó The Author 2006. Published by Oxford University Press.…”

Section: Introductionmentioning

confidence: 99%

The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of dihydrofolate reductase from E.coli

Svensson¹,

Zitzewitz²,

Matthews³

et al. 2006

Protein Engineering, Design and Selection

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The role of domains in defining the equilibrium and kinetic folding properties of dihydrofolate reductase (DHFR) from Escherichia coli was probed by examining the thermodynamic and kinetic properties of a set of variants in which the chain connectivity in the discontinuous loop domain (DLD) and the adenosine-binding domain (ABD) was altered by permutation. To test the concept that chain cleavage can selectively destabilize the domain in which the N- and C-termini are resident, permutations were introduced at one position within the ABD, one within the DLD and one at a boundary between the domains. The results demonstrated that a continuous ABD is required for a stable thermal intermediate and a continuous DLD is required for a stable urea intermediate. The permutation at the domain interface had both a thermal and urea intermediate. Strikingly, the observable kinetic folding responses of all three permuted proteins were very similar to the wild-type protein. These results demonstrate a crucial role for stable domains in defining the energy surface for the equilibrium folding reaction of DHFR. If domain connectivity affects the kinetic mechanism, the effects must occur in the sub-millisecond time range.

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Section: Introductionmentioning

confidence: 99%

The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of dihydrofolate reductase from E.coli

Svensson¹,

Zitzewitz²,

Matthews³

et al. 2006

Protein Engineering, Design and Selection

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Crystal structures and properties of de novo circularly permuted 1,3-1,4-β-glucanases

et al. 1998

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The 1,3-1,4-beta-glucanases from Bacillus macerans and Bacillus licheniformis, as well as related hybrid enzymes, are stable proteins comprised of one compact jellyroll domain. Their structures are studied in an effort to reveal the degree of redundancy to which the three-dimensional structure of protein domains is encoded by the amino acid sequence. For the hybrid 1,3-1,4-beta-glucanase H(A16-M), it could be shown recently that a circular permutation of the sequence giving rise to the variant cpA16M-59 is compatible with wildtype-like enzymatic activity and tertiary structure (Hahn et al., Proc. Natl. Acad. Sci. USA 91:10417-10421, 1994). Since the circular permutation yielding cpA16M-59 mimicks that found in the homologous enzyme from Fibrobacter succinogenes, the question arose whether de novo circular permutations, not guided by molecular evolution of the 1,3-1,4-beta-glucanases, could also produce proteins with native-like fold. The circularly permuted variants cpA16M-84, cpA16M-127, and cpA16M-154 were generated by PCR mutagenesis of the gene encoding H(A16-M), synthesized in Escherichia coli and shown to be active in beta-glucan hydrolysis. CpA16M-84 and cpA16M-127 were crystallized in space groups P2(1) and P1, respectively, and their crystal structures were determined at 1.80 and 2.07 A resolution. In both proteins the main parts of the beta-sheet structure remain unaffected by the circular permutation as is evident from a root-mean-square deviation of main chain atoms from the reference structure within the experimental error. The only major structural perturbation occurs near the novel chain termini in a surface loop of cpA16M-84, which becomes destabilized and rearranged. The results of this study are interpreted to show that: (1) several circular permutations in the compact jellyroll domain of the 1,3-1,4-beta-glucanases are tolerated without radical change of enzymatic activity or tertiary structure, (2) the three-dimensional structures of simple domains are encoded by the amino acid sequence with sufficient redundancy to tolerate a change in the sequential order of secondary structure elements along the sequence, and (3) the native N-terminal region is not needed to guide the folding polypeptide chain toward its native conformation.

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Are turns required for the folding of ribonuclease T1?

1996

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Ribonuclease T1 (RNase T1) is a small, globular protein of 104 amino acids for which extensive thermodynamic and structural information is known. To assess the specific influence of variations in amino acid sequence on the mechanism for protein folding, circularly permuted variants of RNase T1 were constructed and characterized in terms of catalytic activity and thermodynamic stability. The disulfide bond connecting Cys-2 and Cys-10 was removed by mutation of these residues to alanine (C2,lOA) to avoid potential steric problems imposed by the circular permutations. The original amino-terminus and carboxyl-terminus of the mutant (C2,lOA) were subsequently joined with a tripeptide linker to accommodate a reverse turn and new termini were introduced throughout the primary sequence in regions of solvent-exposed loops at Ser-35 (cp35S1), Asp-49 (cp49D1), Gly-70 (cp70G1), and Ser-96 (cp96S1). These circularly permuted RNase T1 mutants retained 35-100% of the original catalytic activity for the hydrolysis of guanylyl(3' + 5')cytidine, suggesting that the overall tertiary fold of these mutants is very similar to that of wild-type protein. Chemical denaturation curves indicated thermodynamic stabilities at pH 5.0 of 5.7, 2.9, 2.6, and 4.6 kcal/mol for cp35S1, cp49D1, cp70G1, and cp96S1, respectively, compared to a value of 10.1 kcal/mol for wild-type RNase TI and 6.4 kcal/mol for (C2,lOA) T1. A fifth set of circularly permuted variants was attempted with new termini positioned in a tight &turn between Glu-82 and (3111-85. New termini were inserted at Asn-83 (cp83N1), Asn-84 (cp84N1), and Gln-85 (cp85Q1). No detectable amount of protein was ever produced for any of the mutations in this region, suggesting that this turn may be critical for the proper folding and/or thermodynamic stability of RNase T1. Keywords: circular permuted proteins; protein folding; ribonuclease T1The mechanism by which proteins fold from a linear chain of amino acids to a specific tertiary structure has been of interest to biological chemists for some time. Even though there are several overlapping theories to explain the protein-folding process, current experimental data from a number of laboratories indicate that many proteins fold along a small number of sequential pathways and form a finite number of transient intermediates (Jennings &Wright, 1993;Bai et al., 1995). It appears evident that protein folding begins with the initial formation of discreet secondary structural units that subsequently undergo reordering or further interaction to form the framework for the final tertiary structure. If the initial steps along the folding pathway involve the nucleation of secondary structural units, then it is important to address what role the linear organization of the primary amino acid sequence has on these steps. This feature of the proteinfolding process can be examined by circularly permuting the priReprint requests to: Frank M. Raushel,

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Circular permutation within the coenzyme binding domain of the tetrameric glyceraldehyde‐3‐phosphate dehydrogenase from Bacillus stearothermophilus

Cited by 18 publications

References 27 publications

The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of dihydrofolate reductase from E.coli

The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of dihydrofolate reductase from E.coli

Crystal structures and properties of de novo circularly permuted 1,3-1,4-β-glucanases

Are turns required for the folding of ribonuclease T1?

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