Sequential Closure of Loop Structures Forms the Folding Nucleus during the Refolding Transition of the Escherichia coli Adenylate Kinase Molecule

Tomer Orevi, Gil Rahamim, Dan Amir, Sagar Kathuria, Osman Bilsel, C. Robert Matthews, Elisha Haas

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


The ensemble of conformers of globular protein molecules immediately following transfer from unfolding to folding conditions is assumed to be collapsed though still disordered, as the first steps of the folding pathway are initiated. In order to test the hypothesis that long loop closure transitions are part of the initiation of the folding pathway, our groups are studying the initiation of the folding transition of a model protein by time-resolved excitation energy transfer (trFRET) detected fast kinetics experiments. Site-specific double labeling is used to study the timing of conformational transitions of individual loop forming chain segments at the microsecond time regime. Previously, it was shown that at least three long loops in the Escherichia coli adenylate kinase (AK) molecule close within the first 5 ms of folding of AK, while the main global folding transition occurs in a time regime of seconds. In order to enhance the time resolution of the kinetics experiments to the microsecond time regime and determine the rate of closure of the two N terminal loops (loop I residues 1-26 and loop II residues 29-72), we applied a continuous flow based double kinetics experiment. These measurements enabled us to obtain a microsecond series of transient time dependent distributions of distances between the ends of the labeled loops. Analysis of the trFRET experiments show that the N terminal loop (loop I) is closed within less than 60 μs after the initiation of refolding. Loop II is also mostly closed within that time step but shows an additional small reduction of the mean end-to-end distance in a second phase at a rate of 0.005 μs-1. This second phase can either reflect tightening of a loosely closed loop in the ensemble of conformers or may reflect two subpopulations in the ensemble, which differ in the rate of closure of loop II, but not in the rate of closure of loop I. This study shows the very fast closure of long loops in the otherwise disordered backbone and fine details of the very early hidden pretransition state steps that are essential for the fast and efficient folding of the protein molecule.

Original languageEnglish
Pages (from-to)79-91
Number of pages13
Issue number1
Early online date14 Dec 2015
StatePublished - 12 Jan 2016

Bibliographical note

Publisher Copyright:
© 2015 American Chemical Society.


This study was supported by grants from the Israel Science Foundation (ISF1464/10 and the I-CORE 1902/12), the United States-Israel Binational Science Foundation (Grant No. 2011143), and grants from the National Science Foundation (MCB1121942 to C.R.M. and O.B. and NSF IDBR1353942 to O.B.). This collaborative work was enhanced by a Research Coordination Network Grant MCB1051344, from the National Science Foundation. We are grateful to Mr. E. Zimerman, M. Schneeberg, and D. Freedman for excellent technical assistance. We thank Gershon Hazan, Eitan Lerner, and Asaf Grupi for their contributions and discussions.

FundersFunder number
National Science FoundationIDBR1353942, MCB1051344, MCB1121942, 1353942
United States-Israel Binational Science Foundation2011143
Israel Science FoundationISF1464/10, I-CORE 1902/12


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