Circularly permuted dihydrofolate reductase of <i>E.coli</i> has functional activity and a destabilized tertiary structure

Protasova, N Y; Kireeva, Maria L.; Murzina, Natalia V.; Murzin, Alexey G.; Uversky, Vladimir N.; Gryaznova, O.I.; Gudkov, A.T.

doi:10.1093/protein/7.11.1373

Cited by 44 publications

(31 citation statements)

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“…Several other monomeric proteins have since been circularly permuted through genetic manipulations, all of which maintain a nativelike function. [3][4][5][6][7][8][9][10][11] Single subunits of multimeric proteins can also withstand circularly permuted sequences. For example, circularly permuted catalytic chains of aspartate transcarbamoylase combined in vitro with regulatory chains produced a folded and functional multimeric protein.…”

Section: Introductionmentioning

confidence: 99%

Intersubunit circular permutation of human hemoglobin

Sanders

Sligar

2002

Blood

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For many years, human hemoglobin (Hb) isolated from erythrocytes has been investigated as a potential oxygen delivery therapeutic. Advantages with respect to the need for blood typing were balanced with various undesirable properties of cell-free Hb, including cost, overall oxygen affinity, alterations in cooperativity, and ready dissociation into toxic dimeric species. The use of total gene synthesis has resulted in very high levels of functional human Hb expression in Escherichia coli, but there remains a desire for effecting the crosslinking of the hemoglobin tetramer and providing for ready means for increasing the globular molecular weight. In this communication, we report a novel method for linking alpha chains. By circularly permuting one alpha sequence, the second alpha chain in the Hb tetramer can be linked with glycine residues to form 2 bridges across the central cavity. The second alpha chain thus presents its amino and carboxyl termini on a solvent exposed surface, providing for additional polymerization of oxygen-carrying subunits or attachment of any other peptide-based therapeutic. IntroductionA mechanistic and predictive understanding of how a primary sequence can fold to a single tertiary structure remains undefined. Since a thorough understanding of this process will enable the rational design of unique protein structures, this remains an area of active research. One theory suggests that short continuous regions within a protein develop local interactions early in the folding process to minimize the number of accessible structures and catalyze the formation of a functional protein. Radical perturbations of protein structure by circular permutation is one technique being used in an attempt to explore this issue. In addition, utilization of circular permutation itself can be exploited in the design of new protein structures.A circularly permuted protein is created in 2 steps. First, the original termini are linked to form a circular polypeptide. New termini are then created by cleavage of a peptide bond at a location distant from the original termini. Goldenberg and Creighton engineered the first circularly permuted sequence by chemically condensing the termini of bovine pancreatic trypsin inhibitor (BPTI) and generating new termini by limited proteolysis. 1 This circularly permuted variant was shown to refold, in vitro, to a native functional conformation. This seminal experiment suggested that the location of the termini has little effect on the final 3-dimensional structure of the protein. Luger and coworkers genetically circularly permuted phosphoribosyl anthranilate isomerase. 2 The Escherichia coli translated variant was structurally and functionally similar to the wild-type protein. Several other monomeric proteins have since been circularly permuted through genetic manipulations, all of which maintain a nativelike function. [3][4][5][6][7][8][9][10][11] Single subunits of multimeric proteins can also withstand circularly permuted sequences. For example, circularly permuted catalytic cha...

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Section: Introductionmentioning

confidence: 99%

Intersubunit circular permutation of human hemoglobin

Sanders

Sligar

2002

Blood

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“…However, circular permutation of E. coli as well as mDHFR has shown that its primary structure can be radically altered and yet yield active enzyme with native-like structure (16). We have designed two fragments of mDHFR consisting of the sequences coding for F [1,2], and for F [3], each of which is covalently linked to an interacting domain (GCN4 leucine zipper).…”

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confidence: 99%

Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments

Pelletier

Campbell-Valois

Michnick

1998

Proc. Natl. Acad. Sci. U.S.A.

331

266

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Reassembly of enzymes from peptide fragments has been used as a strategy for understanding the evolution, folding, and role of individual subdomains in catalysis and regulation of activity. We demonstrate an oligomerization-assisted enzyme reassembly strategy whereby fragments are covalently linked to independently folding and interacting domains whose interactions serve to promote efficient refolding and complementation of fragments, forming active enzyme. We show that active murine dihydrofolate reductase (E.C. 1.5.1.3) can be reassembled from complementary N-and C-terminal fragments when fused to homodimerizing GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners. Reassembly is detected by an in vivo selection assay in Escherichia coli and in vitro. The effects of mutations that disrupt fragment affinity or enzyme activity were assessed. The steady-state kinetic parameters for the reassembled mutant (Phe-31 3 Ser) were determined; they are not significantly different from the full-length mutant. The strategy described here provides a general approach for protein dissection and domain swapping studies, with the capacity both for rapid in vivo screening as well as in vitro characterization. Further, the strategy suggests a simple in vivo enzyme-based detection system for protein-protein interactions, which we illustrate with two examples: ras-GTPase and raf-ras-binding domain and FK506-binding proteinrapamycin complexed with the target of rapamycin TOR2.

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“…The functioning residues on the peptide backbone are then arranged to give the proper configuration for effective catalytic activity. Because enzymes cleaved at certain sites have been demonstrated to show catalytic activity as high as that of uncleaved enzyme (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), chain connectivity must not be absolutely required for catalytic activity. This means that chain connectivity is crucial for catalytic activity in some regions, but in others, it is not.…”

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Circular Permutation Analysis as a Method for Distinction of Functional Elements in the M20 Loop of Escherichia coliDihydrofolate Reductase

Nakamura

Iwakura

1999

Journal of Biological Chemistry

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A functional element of an enzyme can be defined as the smallest unit of the local peptide backbone of which the connectivity is crucial for the catalytic activity. In order to elucidate the distribution of functional elements in an active site flexible loop (the M20 loop) of Escherichia coli dihydrofolate reductase, systematic cleavage of main chain connectivity was performed using circular permutation. Our analysis is based on the assumption that a permutation within a functional element would significantly reduce enzyme function, whereas ones outside or at the boundaries of the elements would affect the function only slightly. Thus, a functional element would be assigned as the minimum peptide chain between the identified boundaries. Comparison of the activities of the circularly permuted variants revealed that the peptide chain around the M20 loop could be divided into four regions (regions 1-4). Region 1 was found to play an important role in overall tertiary fold because most variants permuted at region 1 did not accumulate in E. coli cells stably. A distinction between region 2 and region 3 was in agreement with the extent of movements calculated from the coordinates of ␣ carbons, supporting the idea that the movement of peptide backbone is a key feature of enzyme function. The boundary between region 3 and region 4 coincided with that between the M20 loop and the following ␣ helix. From equilibrium binding studies, region 2 was found to be involved in the binding of nicotinamide substrates, whereas region 4 appeared to be very important for the binding of pterin substrates.The active site of an enzyme contains amino acid residues involved in enzyme function. Some residues in the active site bind to the substrate or cofactor, and others are involved in the catalysis itself. In addition, residues away from the active site sometimes promote the catalytic reaction through intramolecular interactions (1, 2). Enzyme-catalyzed reactions proceed, in general, through multiple steps, including binding of the substrate, catalysis, and release of product. At each step, certain amino acid residues play critical roles. However, an isolated collection of these directly functioning residues is not sufficient to obtain catalytic activity. Amino acid residues should be connected covalently to make up a polypeptide chain with a specific amino acid sequence that determines a proper tertiary structure. The functioning residues on the peptide backbone are then arranged to give the proper configuration for effective catalytic activity. Because enzymes cleaved at certain sites have been demonstrated to show catalytic activity as high as that of uncleaved enzyme (3-16), chain connectivity must not be absolutely required for catalytic activity. This means that chain connectivity is crucial for catalytic activity in some regions, but in others, it is not. The detailed configuration of the functioning residues seems to depend on local peptide backbone rather than the overall polypeptide chain. In this paper, we define a "functional...

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Circularly permuted dihydrofolate reductase of E.coli has functional activity and a destabilized tertiary structure

Cited by 44 publications

References 0 publications

Intersubunit circular permutation of human hemoglobin

Intersubunit circular permutation of human hemoglobin

Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments

Circular Permutation Analysis as a Method for Distinction of Functional Elements in the M20 Loop of Escherichia coliDihydrofolate Reductase

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