Separating the effects of internal friction and transition state energy to explain the slow, frustrated folding of spectrin domains

Abstract

The elongated three-helix bundle domains spectrin R16 and R17 fold some two to three orders of magnitude more slowly than their homologue R15. We have shown that this slow folding is due, at least in part, to roughness in the free-energy landscape of R16 and R17. We have proposed that this roughness is due to a frustrated search for the correct docking of partly preformed helices. However, this accounts for only a small part of the slowing of folding and unfolding. Five residues on the A helix of R15, when inserted together into R16 or R17, increase the folding rate constants, reduce landscape roughness, and alter the folding mechanism to one resembling R15. The effect of each of these mutations individually is investigated here. No one mutation causes the behavior seen for the five in combination. However, two mutations, E18F and K25V, significantly increase the folding and unfolding rates of both R16 and R17 but without a concomitant loss in landscape roughness. E18F has the greatest effect on the kinetics, and a Φ-value analysis of the C helix reveals that the folding mechanism is unchanged. For both E18F and K25V the removal of the charge and resultant transition state stabilization is the main origin of the faster folding. Consequently, the major cause of the unusually slow folding of R16 and R17 is the non-native burial of the two charged residues in the transition state. The slowing due to landscape roughness is only about fivefold.free energy landscape | frustration | phi value | protein folding T he 15th, 16th, and 17th domains of chicken brain α-spectrin (R15, R16, and R17) have very similar structures, stabilities, and β-Tanford (β T ) values (which reflect the compactness of the transition state for folding and unfolding) (1-6). However, the folding of R15 differs from that of R16 and R17 in a number of respects. R15 folds and unfolds two orders of magnitude faster than R16 and three orders of magnitude faster than R17. R16 and R17 have two sequential transition states, and for both domains the first of these (TS1) shows significant landscape roughness (or "internal friction") (7). This has not been seen for any other domain of comparable size and folding kinetics, although theory has long predicted the possibility of such landscape roughness (8-15). R15 has a broad transition state (characterized by "rollover" in the unfolding limb for some mutants and for wild type under some conditions), but due to the speed of folding and unfolding it is not known whether it has two sequential transition states (16). However, the early transition state of R15 (which corresponds to TS1 of R16 and R17 and will be referred to as such) has a smoother, less frustrated landscape (7).Φ-value analysis shows that for all three domains the A and C helices are partially structured at TS1, whereas the B helix is relatively unstructured (16-18). R16 and R17 fold via a framework-like mechanism, with some tertiary contacts formed but more extensive secondary structure that extends throughout the A and C helices. In contrast, R1...

Folding pathway of a multidomain protein depends on its topology of domain connectivity

“…Using these values for α and Q E ðMÞ with τ 0 = 100 ps, the simulated timescales are τ A ≈ 67 μs, τ B ≈ 380 μs, and δ C ≈ 57 ms, which agree semiquantitatively with the experimentally observed values of τ 7 ≤ 35 μs, τ 6 ≈ 550 μs, and τ 5 ≈ 200 ms (19). It should be noted that in multidomain proteins, the heterogeneous distribution of frustration (44,45), particularly at the domain interface, should make the internal friction more evident than in small proteins. Thus, t 0 should nonlinearly depend on Q E ðMÞ, which further enhances the timescale lengthening effect during the late phases than that in the present estimations.…”

Protein Folding: Adding a Nucleus to Guide Helix Docking Reduces Landscape Roughness

“…The return of V65 and L97 either alone or in concert did not significantly slow the folding of these minimal core-swapped R16 variants. Second, we changed each of the residues in the A-helix, individually, in the background of the wild-type protein 14 . Mutation of three of the residues has little effect on the folding kinetics (E19D, I22L and V29L), whereas mutation of two (E18F and K25V) speeds folding; in the case of E18F, this speeding of folding and unfolding is significant.…”

“… 3–5,13 These slow-folding domains have been shown to have a rough energy landscape at TS1, which is responsible in part for the reduction in both folding and unfolding rate constants 6 . In fact, landscape roughness acts to reduce the folding and unfolding rate constants around 5-fold 14 . Recent characterization of a single point mutant (E18F) with a landscape that remains as rough as that of its R16 parent protein and folds via the same mechanism but with vastly increased rate constants has identified the remainder (majority) of the “slowing” to traditional effects of burial of charge on the transition state 14 .…”

Mapping Protein Conformational Landscapes under Strongly Native Conditions with Hydrogen Exchange Mass Spectrometry

“…For example, the recent report from Clarke et al detailing the ruggedness of the spectrin folding landscape by doing chevron analysis on a series of mutants required hundreds of separate measurements. 42 Furthermore, extrapolations in chevron analysis are often made from highly nonphysiological denaturant conditions, although physically based models for denaturant action are providing more confidence and ability to capture different behaviors. 43 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 …”