Thermal unfolding of bacteriophage T4 short tail fibers.
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 degrees C, P12 was able to retain 90% of its ability to reconstitute T412- particles. When heated above 75 degrees C, P12 was no longer capable of fully reconstituting defective phage particles. By 95 degrees 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 degrees C, but by 65 degrees 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 degrees C and inactivation was essentially complete by 55 degrees C. Intrinsic fluorescence data of heated P12 indicated that the protein begins to unfold by 45 degrees C and exhibits distinct peak shifts at 60 degrees C and above 80 degrees C. We conclude that, in the absence of SDS or proteases, P12 heated to 75 degrees C can refold back to an active conformation. Trimeric P12 undergoes some irreversible denaturation between 75 and 85 degrees C, and heating between 85 and 95 degrees C results in dissociation of P12 into monomers.
Jayaraman
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A
Department of Chemical Engineering, Tufts University, Medford, Massachusetts 02155, USA.
De Bernardez Clark
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Goldberg
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E
D017122:Bacteriophage T4 / Q000737:chemistry*; D006358:Hot Temperature; D006868:Hydrolysis; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D014764:Viral Proteins; C071684:gene 12 protein, Enterobacteria phage T4; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin; D013820:Thermolysin