AI Article Synopsis

  • The SiaA protein in Streptococcus pyogenes is involved in heme uptake and was studied through mutations to understand how these changes affect heme binding.
  • pH titrations revealed that mutations led to similar pKa values compared to the wild-type, but resonant Raman spectra indicated a different heme environment in the mutants.
  • The study suggests that the unfolding of SiaA is influenced by the interaction strength of its amino acid residues with heme, proposing a mechanism where minor structural changes allow for heme release while maintaining overall protein stability for ongoing heme trafficking.

Article Abstract

The protein SiaA (HtsA) is part of a heme uptake pathway in Streptococcus pyogenes. In this report, we present the heme binding of the alanine mutants of the axial histidine (H229A) and methionine (M79A) ligands, as well as a lysine (K61A) and cysteine (C58A) located near the heme propionates (based on homology modeling) and a control mutant (C47A). pH titrations gave pKa values ranging from 9.0 to 9.5, close to the value of 9.7 for WT SiaA. Resonance Raman spectra of the mutants suggested that the ferric heme environment may be distinct from the wild-type; spectra of the ferrous states were similar. The midpoint reduction potential of the K61A mutant was determined by spectroelectrochemical titration to be 61±3mV vs. SHE, similar to the wild-type protein (68±3mV). The addition of guanidine hydrochloride showed two processes for protein denaturation, consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket (the half-life for the slower process ranged from less than half a day to two days). The ease of protein unfolding was related to the strength of interaction of the residues with the heme. We hypothesize that kinetically facile but only partial unfolding, followed by a very slow approach to the completely unfolded state, may be a fundamental attribute of heme trafficking proteins. Small motions to release/transfer the heme accompanied by resistance to extensive unfolding may preserve the three dimensional form of the protein for further uptake and release.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943329PMC
http://dx.doi.org/10.1016/j.jinorgbio.2015.10.016DOI Listing

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