Influence of Ca2+ on conformation and stability of three bacterial hybrid glucanases.


Abstract

The three hybrid glucanases (1-12)AMY x MAC(13-214), (1-12)AMY x des-Tyr13MAC(14-214); (1-16)AMY x MAC(17-214) are composed of short N-terminal segments of 12 or 16 amino acid residues derived from the Bacillus amyloliquefaciens glucanase (AMY) and of residues 13-214, 14-214 and 17-214, respectively, derived from the Bacillus macerans enzyme (MAC). The three proteins have similar conformational features as shown by the similar characteristics of their CD spectra in the far- and near-ultraviolet region. A metal-ion-binding site was identified in the hybrid glucanase (1-16)AMY x MAC(17-214) by a crystal structure analysis [Keitel, T., Simon, O., Borriss, R. & Heinemann, U. (1993) Proc. Natl Acad. Sci. USA 90, 5287-5291]. Only minor conformational changes of the three hybrid glucanases were observed depending on the presence or absence of Ca2+ ions but for (1-16)AMY x MAC(17-214) and (1-12)AMY x des-Tyr13MAC(14-214) the occupation of this metal-binding site by a Ca2+ ion is connected with a large increase of the stability against thermal and chemical unfolding. Surprisingly, for (1-12)AMY x MAC(13-214), which differs from (1-12)AMY x des-Tyr13MAC(14-214) by only one additional amino acid in an N-terminal loop region, the effect of Ca2+ ions on the stability is small. The exchange of a few amino acid residues near the N-terminus of the B. macerans glucanase against amino acids found at comparable positions in the B. amyloliquefaciens glucanase seems to influence very strongly the strength of the Ca2+ binding site and concomitantly the stability of the hybrid glucanases. Study holds ProTherm entries: 10085, 10086, 10087, 10088, 10089, 10090, 10091, 10092, 10093, 10094, 10095, 10096, 10097, 10098, 10099, 10100, 10101, 10102, 10103, 10104, 10105, 10106 Extra Details: additive : Gdn/HCl(1.5 M),Hybrid I (1-16)AMY MAC(17-214) containing 16 N-terminal amino acids derived from Bacillus amyloliquefaciens (1,3-1,4)-beta-gluconase; with the C-terminal sequence segments 17-214 from Bacillus macerans (1,3-1,4)-beta-gluconase. hybrid Glucanases; conformation; thermostability;,unfolding

Submission Details

ID: qZ8pYvwk

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:39 p.m.

Version: 1

Publication Details
Welfle K;Misselwitz R;Welfle H;Politz O;Borriss R,Eur. J. Biochem. (1995) Influence of Ca2+ on conformation and stability of three bacterial hybrid glucanases. PMID:7758469
Additional Information

Number of data points 47
Proteins Beta-glucanase ; Beta-glucanase
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dG ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: dG ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: dHcal temp:25.0 C, units:kcal/mol ; Experimental Assay: dG details:Additives , ionic:CaCl2: 1 mM ; Experimental Assay: dCp ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: dHcal ionic:: , units:kJ/mol·K, details:Additives EDTA (1 mM), ; Experimental Assay: dCp ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: dHvH units:kJ/mol·K, ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: dCp units:kJ/mol·K, ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: dHcal ionic:: , units:kJ/mol, details:Additives EDTA (1 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: dHvH units:kJ/mol·K, details:Additives , ionic:CaCl2: 1 mM ; Experimental Assay: dCp details:Additives , ionic:CaCl2: 1 mM ; Experimental Assay: Tm details:Additives , ionic:CaCl2: 1 mM ; Experimental Assay: dHvH units:kcal/mol, details:Additives , ionic:CaCl2: 1 mM ; Experimental Assay: dCp details:Additives , ionic:CaCl2: 1 mM ; Experimental Assay: dHcal units:kJ/mol·K ; Experimental Assay: Tm details:Additives , ionic:CaCl2: 1 mM
Libraries Mutations for sequence FDCAEYRSTNIYGYGLYEVSMKPAKNTGIVSSFFTYTGPAHGTQWDEIDIEFLGKDTTKVQFNYYTNGVGGHEKVISLGFDASKGFHTYAFDWQPGYIKWYVDGVLKHTATANIPSTPGKIMMNLWNGTGVDDWLGSYNGANPLYAEYDWVKYTSNQTGGSFFEPFNSYNSGTWEKADGYSNGGVFNCTWRANNVNFTNDGKLKLGLTSSAYNA
Sequence Assay Result Units