Stabilization of <i>Bacillus stearothermophilus</i> neutral protease by introduction of prolines

Hardy, Florence; Vriend, Gerrit; Veltman, Oene R.; Vinne, Bernard van der; Venema, Gerard; Eijsink, Vincent G. H.

doi:10.1016/0014-5793(93)81497-n

Cited by 98 publications

(64 citation statements)

References 34 publications

(13 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…There are various methods of stabilizing proteins 24) : introduction of a disulfide bond 25,26) or a proline residue, 27) removal of a glycine residue, 27,28) glycosylation of a protein, 6,17) etc. However, we do not always obtain the results we expect.…”

Section: Discussionmentioning

confidence: 99%

Thermostability of Doubly Glycosylated Recombinant Lysozyme.

Hashimoto

Munemura

Masumoto

et al. 2001

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

Proteins containing covalently bonded oligosaccharidechains are widely distributed in cells. The oligosaccharidechains are called the third biological chain after nucleic acids and proteins and have more diverse function than nucleic acids or proteins. This diversity decides the function, characterization and distribution of glycoproteins. The diversity is attributable to the absence of template in the biosynthesis, though DNA replication, mRNA transcription, or protein translation is carried out in the presence of the template. Oligosaccharide-chains of glycoproteins are related to the retention of active structure, extension of retention time within the body, selective transport of proteins in cells, signal transduction, cell adhesion, differentiation of cell, control of cell proliferation, etc. 1,2)There is, however, little information on the addition site of the oligosaccharide-chain, compared to information on the chain itself. In spite of the presence of the Asn-type glycosylation signal sequence, Asn-Xaa-Ser/Thr (Xaa: any amino acids except proline), non-glycosylated protein is present. 3,4) On the other hand, when the glycosylation signal sequence was introduced into non-glycosylated protein by gene engineering, glycosylation was confirmed and the glycosylated protein was analyzed. 5,6) Proper selection and combination of the glycosylated positions enables us to prepare highly stable proteins. Thermodynamic stabilization by artificial double glycosylation on a protein has not yet been reported.In this report, we selected hen lysozyme as a model protein and prepared two single mutants, Q41S and R61S, which are mutant lysozymes in which Gln-41 and Arg-61 were substituted with Ser, respectively (Fig. 1). We previously screened the proper sites to introduce glycosylation on the basis of each asparagine residue in lysozyme (unpublished data). As a result, Q41S and R61S were glycosylated and thermodynamically more stable than the wild type. Thus, the double mutant Q41S/R61S, into which the double glycosylation can be introduced, was prepared and characterized. MATERIALS AND METHODS MaterialsPolymerase chain reaction (PCR) was carried out with an "Expand High Fidelity PCR System" (EHF) kit from Roche (Germany). T4 DNA ligase and restriction enzymes were from Fermentas MBI (Lithuania). Oligonucleotides were synthesized by Life Technologies, Inc. (MD, U.S.A.). Micrococcus luteus (M. luteus), a substrate of lysozyme, was purchased from Seikagaku Kogyo (Tokyo, Japan), and CM-Toyopearl 650M, a cation-exchange resin, was from Tosoh (Tokyo). N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)-trypsin was from Sigma Chemicals (St. Louis, U.S.A.). Site-Directed Mutagenesis and Construction of Yeast Expression Vectors for Q41S and R61S MutantsLysozyme mutant genes, Q41S and R61S, were prepared as reported 7) with a slight modification in the PCR method. A thermal cycler used for PCR was the "Mini Cycler" (MJ Research). The first PCR (10 cycles of 45 s at 95°C, 1 min at 50°C and 1 min at 72°C, followed by 15 cycles of 45 s ...

show abstract

Section: Discussionmentioning

confidence: 99%

Thermostability of Doubly Glycosylated Recombinant Lysozyme.

Hashimoto

Munemura

Masumoto

et al. 2001

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

show abstract

“…In an attempt to optimize these interactions we focused on mutations of the solvent-exposed Ser65. The stabilization obtained after the rigidifying Ser65--+Pro mutation (&50 = +4.7"C) had already shown the feasibility of stabilizing TLP-ste by mutating this residue [17]. Here we examined the possibility of stabilizing TLP-ste by engineering a salt bridge between residue 65 and Argll in the p-sheet.…”

Section: Mutationsmentioning

confidence: 99%

Mutational Analysis of a Surface Area that is Critical for the Thermal Stability of Thermolysin‐Like Proteases

Veltman

Vriend²,

Hardy

et al. 1997

European Journal of Biochemistry

View full text Add to dashboard Cite

Site-directed mutagenesis was used to assess the contribution of individual residues and a bound calcium in the 55 -69 region of the thermolysin-like protease of Bacillus stearothermophilus (TLP-ste) to thermal stability. The importance of the 55-69 region was reflected by finding that almost all mutations had drastic effects on stability. These effects (both stabilizing and destabilizing) were obtained by mutations affecting main chain flexibility, as well as by mutations affecting the interaction between the 55-69 region and the rest of the protease molecule. The calcium-dependency of stability could be largely abolished by mutating one of its ligands (Asp57 or Asp.59). In the case of the AspS7+Ser mutation, the accompanying loss in stability was modest compared with the effects of other destabilizing mutations or the effects of (combinations of) stabilizing mutations. The detailed knowledge of the stability-determining region of TLP-ste permits effective rational design of stabilizing mutations, which, presumably, are also useful for related TLP such as thermolysin. This is demonstrated by the successful design of a stabilizing salt bridge involving residues 65 and 11.Keywords: thermal stability; thermolysin; autolysis ; calcium binding; unfolding pathway.Bacillus thermolysin-li ke proteases (TLP) are metallo-endopeptidases consisting of 300-3 19 residues. These enzymes contain one zinc ion that is essential for catalysis and they bind a varying number of calcium ions for which it is known that they contribute to stability [I -21. The amino acid sequences of many TLP are known and the crystal structures of thermolysin (the TLP of Bacillus thermoproteolyticus; [3,41) and TLP-cer (the TLP of Bacillus cereus; [S]) have been determined. TLP consist of an N-terminal domain (residues 1 -154; thermolysin numbering) characterized by a predominance of P-pleated sheet and a C-terminal domain (residues 155-31 6) that is mainly a-helical [4, 51. TLP differ in thermal stability [6] and the structural determinants of these differences have been analyzed by several sitedirected mutagenesis studies [7-111. At elevated temperatures TLP are irreversibly inactivated as a result of autolysis. Considering the broad specificity of TLP [ 12, 131, conformational Enzyme. Thermolysin (EC 3.4.24.27).proteolytic attack [14]. It has been shown that local unfolding processes that render the protein susceptible towards autolysis are the rate-limiting steps in thermal inactivation [6,[15][16]. It has been proposed that these thermally induced local unfolding processes mainly involve surface located regions of the protein 16, 9, 141. Accordingly, it has been shown that the changes in the stability of TLP-ste is most easily effected by mutation of surface-located residues that are clustered in one particular region (residues 55-69) of the protein [9, 111 (and see Figs 1 and 2). The difference in stability between thermolysin (tso = 86.5 "C ; see below for definition of tS0) and the TLP of B. stemothermophilus CU21 (TLP-ste; t50 = 73.4"C) ...

show abstract

“…Serendipity plays a major role (Imanaka et al, 1986;Pantoliano et al, 1989;Mrabet et al, 1992;Hardy et al, 1993), even though the probability of finding a stabilizing mutation by chance is small and methods have to be devised to reduce the search space (Serrano et al, 1993;Heringa et al, 1995). a-Helices may respond favorably to the stabilization of the helix dipole (Nicholson et al, 1988;Goodenough et al, 1991;Eijsink et al, 1992;Walter et al, 1995) or to the provision of N-cap structural elements Reprint requests to: Boris Steipe, Genzentrum, Feodor-Lynen-Str.…”

mentioning

confidence: 99%

β‐Turn propensities as paradigms for the analysis of structural motifs to engineer protein stability

et al. 1997

View full text Add to dashboard Cite

The thermodynamic stability of a protein provides an experimental metric for the relationship of protein sequence and native structure. We have investigated an approach based on an analysis of the structural database for stability engineering of an immunoglobulin variable domain. The most frequently occurring residues in specific positions of p-turn motifs were predicted to increase the folding stability of mutants that were constructed by site-directed mutagenesis. Even in positions in which different residues are conserved in immunoglobulin sequences, the predictions were confirmed. Frequently, mutants with increased p-turn propensities display increased folding cooperativities, suggesting pronounced effects on the unfolded state independent of the expected effect on conformational entropy. We conclude that structural motifs with predominantly local interactions can serve as templates with which patterns of sequence preferences can be extracted from the database of protein structures. Such preferences can predict the stability effects of mutations for protein engineering and design.

show abstract

Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines

Cited by 98 publications

References 34 publications

Thermostability of Doubly Glycosylated Recombinant Lysozyme.

Thermostability of Doubly Glycosylated Recombinant Lysozyme.

Mutational Analysis of a Surface Area that is Critical for the Thermal Stability of Thermolysin‐Like Proteases

β‐Turn propensities as paradigms for the analysis of structural motifs to engineer protein stability

Contact Info

Product

Resources

About