Structural and dynamic effects of α‐Helix deletion in Sso7d: Implications for protein thermal stability

Merlino, Antonello; Graziano, Giuseppe; Mazzarella, L.

doi:10.1002/prot.20270

Cited by 24 publications

(19 citation statements)

References 62 publications

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“…Indeed the main difference between the two proteins, as revealed by the simulations, is the finding that ONC samples similar essential subspaces at 300 and 350 K (RMSIP, 0.69), whereas the conformational subspaces sampled by (C87S,des103-104)-ONC at the studied temperatures are much less overlapped (RMSIP, 0.55). Interestingly similar results were obtained when comparing the dynamics of the hyperthermophilic protein Sso7d and its truncated and less stable variant at two different temperatures (59). To show the dynamic differences between ONC and (C87S,des103-104)-ONC at 350 K in terms of conformational space sampling, the projection of the C␣ atom trajectory along the first two eigenvectors derived from the essential dynamic analysis for ONC and (C87S,des103-104)-ONC is reported in Fig.…”

Section: X-ray Structure Of M23l-onc and (C87sdes103-104)-onc-supporting

confidence: 69%

“…High temperature simulations have been increasingly performed in the last few years to detect the first steps of the unfolding pathway of proteins (65,66) and to address features of protein thermal stability (59,60,67). At this temperature the root mean square deviations of the C␣ atoms from the starting (C87S,des103-104)-ONC (B) at 350 K. 2D, two-dimensional.…”

Section: X-ray Structure Of M23l-onc and (C87sdes103-104)-onc-mentioning

confidence: 99%

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The Importance of Dynamic Effects on the Enzyme Activity

Merlino

Mazzarella

Carannante

et al. 2005

Journal of Biological Chemistry

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Onconase (ONC), a member of the RNase A superfamily extracted from oocytes of Rana pipiens, is an effective cancer killer. It is currently used in treatment of various forms of cancer. ONC antitumor properties depend on its ribonucleolytic activity that is low in comparison with other members of the superfamily. The most damaging side effect from Onconase treatment is renal toxicity, which seems to be caused by the unusual stability of the enzyme. Therefore, mutants with reduced thermal stability and/or increased catalytic activity may have significant implications for human cancer chemotherapy. In this context, we have determined the crystal structures of two Onconase mutants (M23L-ONC and C87S,des103-104-ONC) and performed molecular dynamic simulations of ONC and C87S,des103-104-ONC with the aim of explaining on structural grounds the modifications of the activity and thermal stability of the mutants. The results also provide the molecular bases to explain the lower catalytic activity of Onconase compared with RNase A and the unusually high thermal stability of the amphibian enzyme.Ribonucleases are widely found in living organisms and have been proposed to function in RNA metabolism and gene expression (1). Several ribonucleases have been isolated from organs of various animals and have been well characterized. Bovine pancreatic ribonuclease (RNase A) 1 is the most studied member of the family (2). It has been used as a model for the study of the fundamental aspects of protein structure and function (2, 3). Several members of the RNase A superfamily exhibit additional biological functions in connection with their intrinsic ribonucleolytic activities (4, 5). In humans, eosinophil-derived neurotoxin and eosinophil cationic protein (6) exert neurotoxicity as well as antiparasitic activity (7). Bovine seminal ribonuclease (BS-RNase) has been found to induce apoptosis of human thyroid carcinoma cell lines (8, 9). Onconase (ONC) from Rana pipiens is cytotoxic (10) and cytostatic to several tumor lines and is currently in phase III clinical trials for the treatment of malignant mesothelioma (8,11,12). ONC shares 30% of identity with the sequence of RNase A, and its three-dimensional structure exhibits a highly conserved ribonuclease-like topology (13), which comprises a characteristic V-shaped ␤-sheet motif surrounded by three ␣-helices. Differences are mainly localized in the loop regions, which are significantly shorter in ONC, and at the C terminus where a disulfide bond, unique to amphibian ribonucleases, tethers the C-terminal residue Cys 104 to Cys 87 located in one strand of the ␤-sheet (13). Another distinct property of ONC is the presence of an N-terminal pyroglutamate (Pyr 1 ), which folds back against the N-terminal helix (13). The overall active site architecture closely resembles that of RNase A and includes His 10 , Lys 31 , and His 97 , which form the catalytic triad corresponding to His 12 , Lys 41 , and His 119 , respectively, of the pancreatic enzyme. Despite this similarity and the activating effe...

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Section: X-ray Structure Of M23l-onc and (C87sdes103-104)-onc-supporting

confidence: 69%

Section: X-ray Structure Of M23l-onc and (C87sdes103-104)-onc-mentioning

confidence: 99%

The Importance of Dynamic Effects on the Enzyme Activity

Merlino

Mazzarella

Carannante

et al. 2005

Journal of Biological Chemistry

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“…The models produced by the server were then manually adjusted to built missing residues on the N-terminal α-helical region and energy-minimized in vacuo by means of the GROMOS96 force-field, following a procedure previously reported [44,45].…”

Section: Homology Modellingmentioning

confidence: 99%

A new RNase sheds light on the RNase/angiogenin subfamily from zebrafish

Pizzo

Merlino

Turano

et al. 2010

Biochemical Journal

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Recently, extracellular RNases of the RNase A superfamily, with the characteristic CKxxNTF sequence signature, have been identified in fish. This has led to the recognition that these RNases are present in the whole vertebrate subphylum. In fact, they comprise the only enzyme family unique to vertebrates. Four RNases from zebrafish (Danio rerio) have been previously reported and have a very low RNase activity; some of these are endowed, like human angiogenin, with powerful angiogenic and bactericidal activities. In the present paper, we report the three-dimensional structure, the thermodynamic behaviour and the biological properties of a novel zebrafish RNase, ZF-RNase-5. The investigation of its structural and functional properties, extended to all other subfamily members, provides an inclusive description of the whole zebrafish RNase subfamily

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“…[34][35][36] Many mutation studies have reported that thermostabilizing mutations are located in terminal regions and that proteins can be stabilized by restricting the flexibility of terminal regions. 35,36 Thus, flexible residues in terminal regions can be good target sites to increase protein thermostability.…”

Section: Structural Interpretation Of Mutationsmentioning

confidence: 99%

The development of a thermostable CiP (Coprinus cinereus peroxidase) through in silico design

Kim

Lee

Joo

et al. 2010

Biotechnology Progress

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Protein thermostability is a crucial issue in the practical application of enzymes, such as inorganic synthesis and enzymatic polymerization of phenol derivatives. Much attention has been focused on the enhancement and numerous successes have been achieved through protein engineering methods. Despite fruitful results based on random mutagenesis, it was still necessary to develop a novel strategy that can reduce the time and effort involved in this process. In this study, a rapid and effective strategy is described for increasing the thermal stability of a protein. Instead of random mutagenesis, a rational strategy was adopted to theoretically stabilize the thermo labile residues of a protein using computational methods. Protein residues with high flexibility can be thermo labile due to their large range of movement. Here, residue B factor values were used to identify putatively thermo labile residues and the RosettaDesign program was applied to search for stable sequences. Coprinus cinereus (CiP) heme peroxidase was selected as a model protein for its importance in commercial applications, such as the polymerization of phenolic compounds. Eleven CiP residues with the highest B factor values were chosen as target mutation sites for thermostabilization, and then redesigned using RosettaDesign to identify sequences. Eight mutants based on the redesigns, were produced as functional enzymes and two of these (S323Y and E328D) showed increased thermal stability over the wild-type in addition to conserved catalytic activity. Thus, this strategy can be used as a rapid and effective in silico design tool for obtaining thermostable proteins.

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Structural and dynamic effects of α‐Helix deletion in Sso7d: Implications for protein thermal stability

Cited by 24 publications

References 62 publications

The Importance of Dynamic Effects on the Enzyme Activity

The Importance of Dynamic Effects on the Enzyme Activity

A new RNase sheds light on the RNase/angiogenin subfamily from zebrafish

The development of a thermostable CiP (Coprinus cinereus peroxidase) through in silico design

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