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

Abstract

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.

Disordered Proteins and Network Disorder in Network Descriptions of Protein Structure, Dynamics and Function: Hypotheses and a Comprehensive Review

“…Loops are important in folding and conformational transitions. In agreement with this, loops were shown to provide a major contribution to the slowest, most cooperative modes of motion in elastic network models [Arold et al, 1998;Kurkcuoglu et al, 2006;Orevi et al, 2009]. Finally, the effect of intrinsically disordered segments may also appear locally.…”

Segmental Conformational Disorder and Dynamics in the Intrinsically Disordered Protein α-Synuclein and Its Chain Length Dependence

“…The values of the diffusion coefficients observed here for a fully disordered natural polypeptide under folding conditions, which mimic the situation of a protein backbone at the moment of initiation of refolding, support the assumption that loop closure can be a significant very early pre-transition state event at the initiation of protein folding. 77 The chain length dependence of the dimensions of the ensemble of disordered segments of the domain studied here is steeper than that predicted by the theoretical model of Flory and the simulations of Goldenberg. This is probably due to the contribution of the excluded volume effect, which is stronger for the short segments, yet follows the power law as suggested by Flory.…”

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

“…A systematic labeling plan was designed in which pairs of probes were introduced at the ends of long loop structures and at the ends of several known secondary structure elements (Fig. 3) (Orevi et al 2009). Stopped flow mixing was used for the initiation of refolding (Ben Ishay et al 2012b).…”