Journal of Molecular Biology
The Folding Transition State of Protein L Is Extensive with Nonnative Interactions (and Not Small and Polarized)
Graphical Abstract
Highlights
► Protein L's transition state (TS) has the entire β sheet according to ψ analysis. ► This TS is much larger than the one identified by ϕ analysis and Gō models. ► The second hairpin is nonnative due to unfavorable backbone angles formed early. ► This agrees with molecular dynamics and Protein Data Bank-based ϕ,ψ sampling ItFix simulations. ► Accurate modeling of the folding transition states remains an ongoing challenge.
Introduction
The IgG binding domain of Protein L (Protein L) contains two hairpins and a central helix and has been a test bed for many experimental and theoretical studies of folding.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 Mutational ϕ analysis experiments indicate that the folding transition state ensemble (TSE) contains only the amino-terminal hairpin1, 2, 3, 4 (Fig. 1). The TSE of a protein with the same α/β fold, Protein G, is also assigned by ϕ analysis to have a single hairpin, but this hairpin is located at the carboxy terminus,12 a behavior attributed to different properties of the turn sequences.13, 14 The difference between the TSEs of these two proteins is cited as an example where the specific sequence, rather than just the protein's topology, influences the folding pathway. A variety of computational studies support this view.5, 6, 7, 8, 9, 11
Despite this broad consensus, we decided to reexamine the folding behavior of Protein L because a TSE with only a single hairpin seems inordinately small. A hairpin scarcely defines Protein L's topology, yet this protein obeys the well-known correlation between folding rate and topology [relative contact order (RCO)].15, 16 Our studies of three other proteins with disparate RCOs indicate that their TSEs acquire a similar level of native topology, RCOTSE ≈ 0.7∙RCONative.17, 18, 19, 20, 21 If this relationship is generally applicable, it would provide a simple rationalization for the kf–RCO correlation, as well as a constraint for possible TSE structures of other proteins.
In the case of Protein L, the presence of only a single hairpin in the TSE equates to an RCO fraction of only 25%, and even the inclusion of the helix would increase the RCO only to 40%. The TSE must minimally include long-range contacts between the amino- and carboxy-terminal strands in order to achieve an RCO fraction close to 70%. Furthermore, whereas a 1:1 relationship between hydrogen bond content and surface burial is found in the TSEs of other proteins,22, 23 the hydrogen bond content of a single hairpin is grossly inadequate to match the surface burial of the highly collapsed TSE of Protein L as determined by the denaturant dependence of the folding rates.
Here, we employ ψ analysis24 to characterize the TSE structure of Protein L. ψ is well suited for determining the structure because the methodology directly identifies pairwise residue–residue contacts. The methodology employs bi-histidine (biHis) metal ion binding sites on the surface of protein, which are stabilized by the addition of metal ions. The ion-induced stabilization of the TSE relative to the native state is represented by the ψ value that is high if the biHis site is present in the TSE. Data for a multitude of biHis sites (individually introduced) can be used to generate structural models of the TSE analogous to the use of nuclear Overhauser enhancement distance constraints in NMR-based structure determination.
These experiments demonstrate that Protein L's TSE contains the entire four-stranded β sheet. Although the amino-terminal hairpin is native-like, the carboxy hairpin and the long-range interactions between the two hairpins have nonnative properties. We conduct simulations of the individual hairpins using our ItFix folding algorithm where the side chains are represented by single Cβ atoms,25, 26 as well as all-atom explicit solvent molecular dynamics (MD) simulations. Without invoking any knowledge on the native state, both methods indicate that the carboxy-terminal hairpin forms rapidly, but with a nonnative turn. We discuss the implications of our findings with regard to TSE topology, the accuracy of ϕ analysis and its ability to validate theoretical studies.
Section snippets
Results
ψ analysis17, 19, 20, 24, 27, 28 proceeds by introducing biHis metal ion binding sites at positions across the protein's surface. A total of eight sites are individually introduced into Protein L to probe the formation of the three native strand–strand pairings and the helix (Fig. 1). Upon addition of metal ions, the biHis sites stabilize strand–strand pairings or the helix because an increase in metal ion concentration stabilizes the interaction between the two histidine partners. The changes
Discussion
The present study is motivated by the belief that a TSE for Protein L containing only a single hairpin, as suggested by ϕ analysis,1, 2, 3, 4 is unreasonably small because it scarcely defines the protein's topology or has enough hydrogen-bonded structure to be commensurate with the observed degree of surface burial. We have applied ψ analysis and demonstrated that the TSE is extensive, containing the entire β sheet network along with some nonnative structure associated with the carboxy hairpin.
Folding measurements
The pseudo-wild-type sequence used has 64 amino acids, MEEVTIKANL IFANGSTQTA EFKGTFEKAT SEAYAYADTL KKDNGEWTVD VADKGYTLNI KFAG, which contains a Y47W mutation to enable fluorescence-monitored folding and unfolding. All variants are verified by DNA sequencing prior to expression. Purification uses either reverse-phase HPLC (C8 and C18 columns) or ion exchange (Amersham Biosciences Q Sepharose® Fast Flow) followed by gel-filtration HPLC (GE Healthcare HiPrep™ 16/60 Sephacryl™ S-100) in series.
Acknowledgements
We thank members of the Freed and Sosnick groups for helpful discussions and C. Antoniou for assistance in protein production. This work was supported, in part, by the National Institutes of Health Grant GM55694 (T.R.S.), National Science Foundation Grant CHE-1111918 (K.F.) and The University of Chicago-Argonne National Laboratory Seed Grant Program (T.R.S., Mike Wilde). R.H.Z. acknowledges the financial support from the IBM Blue Gene Program.
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