title: Sub-millisecond chain collapse of the Escherichia coli globin ApoHmpH.
abstract: Myoglobins are ubiquitous proteins that play a seminal role in oxygen storage, transport, and NO metabolism. The folding mechanism of apomyoglobins from different species has been studied to a fair extent over the last two decades. However, integrated investigations of the entire process, including both the early (sub-ms) and late (ms-s) folding stages, have been missing. Here, we study the folding kinetics of the single-Trp Escherichia coli globin apoHmpH via a combination of continuous-flow microfluidic and stopped-flow approaches. A rich series of molecular events emerges, spanning a very wide temporal range covering more than 7 orders of magnitude, from sub-microseconds to tens of seconds. Variations in fluorescence intensity and spectral shifts reveal that the protein region around Trp120 undergoes a fast collapse within the 8 μs mixing time and gradually reaches a native-like conformation with a half-life of 144 μs from refolding initiation. There are no further fluorescence changes beyond ca. 800 μs, and folding proceeds much more slowly, up to 20 s, with acquisition of the missing helicity (ca. 30%), long after consolidation of core compaction. The picture that emerges is a gradual acquisition of native structure on a free-energy landscape with few large barriers. Interestingly, the single tryptophan, which lies within the main folding core of globins, senses some local structural consolidation events after establishment of native-like core polarity (i.e., likely after core dedydration). In all, this work highlights how the main core of the globin fold is capable of becoming fully native efficiently, on the sub-millisecond time scale.
authors.affiliation: Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.forename: Neşe
authors.initials: N
authors.lastname: Kurt
authors.forename: Jennifer
authors.initials: J
authors.lastname: Choi
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Lapidus
authors.forename: Silvia
authors.initials: S
authors.lastname: Cavagnero
mesh_terms: D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005914:Globins / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D017510:Protein Folding
chemical_list: D029968:Escherichia coli Proteins; C585967:apoHmpH protein, E coli; D005914:Globins