Early Closure of a Long Loop in the Refolding of Adenylate Kinase: A Possible Key Role of Non-Local Interactions in the Initial Folding Steps

Tomer Orevi, Eldad Ben Ishay, Menachem Pirchi, Maik H. Jacob, Dan Amir, Elisha Haas

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Most globular protein chains, when transferred from high to low denaturant concentrations, collapse instantly before they refold to their native state. The initial compaction of the protein molecule is assumed to have a key effect on the folding pathway, but it is not known whether the earliest structures formed during or instantly after collapse are defined by local or by non-local interactions-that is, by secondary structural elements or by loop closure of long segments of the protein chain. Stable closure of one or several long loops can reduce the chain entropy at a very early stage and can prevent the protein from following non-productive pathways whose number grows exponentially with the length of the protein chain. In Escherichia coli adenylate kinase (AK), about seven long loops define the topology of the native structure. We selected four loop-forming sections of the chain and probed the time course of loop formation during refolding of AK. We labeled the termini of the loop segments with tryptophan and cysteine-5-amidosalicylic acid. This donor-acceptor pair of probes used with fluorescence resonance excitation energy transfer spectroscopy (FRET) is suitable for detecting very short distances and thus is able to distinguish between random and specific compactions. Refolding of AK was initiated by stopped-flow mixing, followed simultaneously by donor and acceptor fluorescence, and analyzed in terms of energy transfer efficiency and distance. In the collapsed state of AK, observed after the 5-ms dead time of the instrument, one of the selected segments shows a native-like separation of its termini; it forms a loop already in the collapsed state. A second segment that includes the first but is longer by 15 residues shows an almost native-like separation of its termini. In contrast, a segment that is shorter but part of the second segment shows a distance separation of its termini as high as a segment that spans almost the whole protein chain. We conclude that a specific network of non-local interactions, the closure of one or several loops, can play an important role in determining the protein folding pathway at its early phases.

Original languageEnglish
Pages (from-to)1230-1242
Number of pages13
JournalJournal of Molecular Biology
Issue number4
StatePublished - 30 Jan 2009

Bibliographical note

Funding Information:
This work was supported by research and equipment grants from the Israel Science Foundation, the United States–Israel Binational Foundation, the Marie Curie Transfer of Knowledge Grant from the European Union, and the Damadian Center for Magnetic Resonance Research of the Bar-Ilan University. The continuous technical support of Mr. Eli Zimermann is gratefully acknowledged. We thank Dr. Gershon Hazan for his continuous help and suggestions and Eitan Lerner for invaluable assistance.


  • FRET
  • adenylate kinase
  • fast collapse
  • protein folding
  • stopped flow


Dive into the research topics of 'Early Closure of a Long Loop in the Refolding of Adenylate Kinase: A Possible Key Role of Non-Local Interactions in the Initial Folding Steps'. Together they form a unique fingerprint.

Cite this