PubMed · 13630350
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1959-01-10. [Infiltration].. https://pubmed.ncbi.nlm.nih.gov/13630350/
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The perturbation induced by mono- and divalent cations on the thermophilicity and thermostability of Solfolobus solfataricus beta-glycosidase, a hyperthermophilic tetrameric enzyme, has been investigated by spectroscopic and computational simulation methods to ascertain the Hofmeister effects on two strategic protein regions identified previously. Specifically, (1). an extra segment (83-124), present only in the sequence of hyperthermophilic glycosidases and recognized as an important thermostability determinant for the enzyme structure; and (2). a restricted area of the subunit interface responsible for the quaternary structure maintenance. Mono- and divalent cations inhibit to a different extent the beta-glycosidase activity, whose kinetic constants show an apparent competitive inhibition of the catalytic process that reflects the Hofmeister order. The thermostability is also affected by the nature and charge of the cations, reaching maximal effects for the case of Mg(2+). Fourier transform infrared spectroscopy has revealed very small changes in the protein secondary structure in the presence of the investigated cations at 20 degrees C, while large effects on the protein melting temperatures are observed. Computational analysis of the enzyme structure has identified negative patches on the accessible surface of the two identified regions. Following the Hofmeister series, cations weaken the existing electrostatic network that links the extra segment to the remaining protein matrix. In particular, the perturbing action of cations could involve the ionic pair interactions E107-R245 and E109-R185, thus leading to a local destructuring of the extra segment as a possible starting event for thermal destabilization. A detailed investigation of the electrostatic network at the A-C intermolecular interface of Sbetagly after energy minimization suggests that cations could cause a strong attenuation of the ion pair interactions E474-K72 and D473-R402, with consequent partial dissociation of the tetrameric structure.
One of the most recurring questions in protein folding refers to the interplay between formation of secondary structure and hydrophobic collapse. In contrast with secondary structure, it is hard to isolate hydrophobic collapse from other folding events. We have directly measured the dynamics of protein hydrophobic collapse in the absence of competing processes. Collapse was triggered with laser-induced temperature jumps in the acid-denatured form of a simple protein and monitored by fluorescence resonance energy transfer between probes placed at the protein ends. The relaxation time for hydrophobic collapse is only approximately equal to 60 ns at 305 K, even faster than secondary structure formation. At higher temperatures, as the protein becomes increasingly compact by a stronger hydrophobic force, we observe a slowdown of the dynamics of collapse. This dynamic hydrophobic effect is a high-temperature analogue of the dynamic glass transition predicted by theory. Our results indicate that in physiological conditions many proteins will initiate folding by collapsing to an unstructured globule. Local motions will presumably drive the following search for native structure in the collapsed globule.
Green fluorescent protein has revolutionized cell labeling and molecular tagging, yet the driving force and mechanism for its spontaneous fluorophore synthesis are not established. Here we discover mutations that substantially slow the rate but not the yield of this posttranslational modification, determine structures of the trapped precyclization intermediate and oxidized postcyclization states, and identify unanticipated features critical to chromophore maturation. The protein architecture contains a dramatic approximately 80 degrees bend in the central helix, which focuses distortions at G67 to promote ring formation from amino acids S65, Y66, and G67. Significantly, these distortions eliminate potential helical hydrogen bonds that would otherwise have to be broken at an energetic cost during peptide cyclization and force the G67 nitrogen and S65 carbonyl oxygen atoms within van der Waals contact in preparation for covalent bond formation. Further, we determine that under aerobic, but not anaerobic, conditions the Gly-Gly-Gly chromophore sequence cyclizes and incorporates an oxygen atom. These results lead directly to a conjugation-trapping mechanism, in which a thermodynamically unfavorable cyclization reaction is coupled to an electronic conjugation trapping step, to drive chromophore maturation. Moreover, we propose primarily electrostatic roles for the R96 and E222 side chains in chromophore formation and suggest that the T62 carbonyl oxygen is the base that initiates the dehydration reaction. Our molecular mechanism provides the basis for understanding and eventually controlling chromophore creation.