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Thermal Unfolding of Bacteriophage T4 Short Tail Fibers
Author(s) -
Jayaraman Arul,
De Bernardez Clark Eliana,
Goldberg Edward
Publication year - 1997
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp9701067
Subject(s) - thermolysin , bacteriophage , denaturation (fissile materials) , chemistry , protease , proteases , thermostability , hydrolysis , biophysics , crystallography , enzyme , biochemistry , trypsin , biology , gene , escherichia coli , nuclear chemistry
Abstract The short tail fibers of bacteriophage T4 are composed of a homotrimer of the product of gene 12 (P12) with a molecular weight of 165 000. P12 is capable of reconstituting defective phage particles lacking gene 12. After heating to 75 °C, P12 was able to retain 90% of its ability to reconstitute T412 − particles. When heated above 75 °C, P12 was no longer capable of fully reconstituting defective phage particles. By 95 °C, the reconstitution efficiency of the P12 preparation was reduced by 4 orders of magnitude. Thermal unfolding was also monitored by heating the protein in the presence of SDS to freeze partially unfolded states; by protease hydrolysis; and by intrinsic fluorescence changes. Exposure to SDS had little effect for temperatures up to 55 °C, but by 65 °C, the reconstitution efficiency of P12 treated with 0.01% SDS dropped to less than 1% of the original titer. Thermolysin digestion of P12 heated to various temperatures showed that treated P12 started to inactivate before 45 °C and inactivation was essentially complete by 55 °C. Intrinsic fluorescence data of heated P12 indicated that the protein begins to unfold by 45 °C and exhibits distinct peak shifts at 60 °C and above 80 °C. We conclude that, in the absence of SDS or proteases, P12 heated to 75 °C can refold back to an active conformation. Trimeric P12 undergoes some irreversible denaturation between 75 and 85 °C, and heating between 85 and 95 °C results in dissociation of P12 into monomers.