Structural Insights into the Trp‐Cage Folding Intermediate Formation

Abstract

The 20 residue long Trp-cage is the smallest protein known, and thus has been the subject of several in vitro and in silico folding studies. Here, we report the multistate folding scenario of the miniprotein in atomic detail. We detected and characterized different intermediate states by temperature dependent NMR measurements of the (15)N and (13)C/(15)N labeled protein, both at neutral and acidic pH values. We developed a deconvolution technique to characterize the invisible--fully folded, unfolded and intermediate--fast exchanging states. Using nonlinear fitting methods we can obtain both the thermodynamic parameters (ΔH(F-I), T(m)(F-I), ΔC(p)(F-I) and ΔH(I-U), T(m)(I-U), ΔC(p)(I-U)) and the NMR chemical shifts of the conformers of the multistate unfolding process. During the unfolding of Trp-cage distinct intermediates evolve: a fast-exchanging intermediate is present under neutral conditions, whereas a slow-exchanging intermediate-pair emerges at acidic pH. The fast-exchanging intermediate has a native-like structure with a short α-helix in the G(11)-G(15) segment, whereas the slow-exchanging intermediate-pair presents elevated dynamics, with no detectable native-like residue contacts in which the G(11)-P(12) peptide bond has either cis or trans conformation. Heteronuclear relaxation studies combined with MD simulations revealed the source of backbone mobility and the nature of structural rearrangements during these transitions. The ability to detect structural and dynamic information about folding intermediates in vitro provides an excellent opportunity to gain new insights into the energetic aspects of the energy landscape of protein folding. Our new experimental data offer exceptional testing ground for further computational simulations.

Folding Dynamics and Pathways of the Trp-Cage Miniproteins

“…This structural element has been a subject of discussion in MD simulations, 2,28,32 and experimental evidence for residual flexibility at this site has been reported based on both NMR structure ensembles 8,14,19,33 and crystal structures. 23 Gai and co-workers 7 have observed a more rapid phase (ca.…”

“…While there are unanswered questions concerning the Trp-cage folding pathway, with a number of groups having reported data suggesting that Trp-cage folding is not a two-state system, 19,28,33−35 these data are largely associated with structure melting analysis rather dynamics measurements. Both T-jump and NMR relaxation experiments are consistent with a net two-state process, with fewer exceptions.…”

Dynamic Folding Pathway Models of the Trp-Cage Protein

“…Also, other computational studies on Trp-cage reported the partial formations of the helical elements in the unfolded state [15–17, 20, 22, 23]. Similarly, experimental studies showed the existence of the helical elements in the unfolded state of Trp-cage [3, 11], and another experimental study emphasized the importance of preformed structure in the unfolded state for its fast folding [4]. The conformation at j = 300 shows the RMSD value of 5.6 Å, quite different from the native structure.…”

“…Due to the difficulties in the understanding of folding dynamics for large proteins, fragments of proteins (e.g., α -helix and β -hairpin) and small proteins have been mainly used to investigate protein folding dynamics. Recently, the 20-residue Trp-cage protein [1] with a fast folding rate [2] has attracted many researchers, both experimentalists [1–11] and theoreticians [12–24], in the protein-folding research community.…”

Preorganization of the catalytic Zn2+-binding site in the HNH nuclease motif—A solution study

“…The role of the tryptophan residues in the structure stabilization has already been demonstrated for a number of peptides [25][26][27] and proteins [28,29]. Examples include e.g.…”