Advances in science have most often depended on the exploitation of some particularly favorable model system. The question then arises whether the particular model or the methods used make the results atypical. The work just described depends heavily on the Cyt c model. Are the Cyt c results misleading, orchestrated somehow by its covalently bound heme group? Do the results reflect artifacts that depend on the chemical denaturants used or the HX methods themselves ?
Although the bound heme group is unusual, Cyt c is a typical protein with the same primary, secondary, and tertiary structural elements and interactions common to all proteins. It may be that the presence of the bound heme group somehow aids the experimental discrimination of the different Cyt c foldons but it is hard to see how it could create foldons and orchestrate their sequential incorporation de novo.
In any case, analogous results have now been obtained in many laboratories for many proteins with and without prosthetic groups, using a variety of destabilants (urea, GdmCl, pH, temperature, pressure), or none at all, and even in experiments that did not use HX. Some examples can be noted.
8.1 Apomyoglobin (apoMb, heme removed)
The structure of an intermediate in the folding pathway of apoMb (eight helices, A–H) has been extensively studied by HX pulse labeling.
Initial study of a populated kinetic folding intermediate found that the A, G, and H helices are formed (Jennings & Wright, 1993
). Helix B may be formed in part. The intermediate is native-like, obligatory, and on the folding pathway, although somewhat malleable. Individual helices can be added or removed by mutational manipulation of their relative stability (Cavagnero et al. 1999
; Garcia et al. 2000
). As for the blocked Cyt c
intermediate, some misfolding is present (Jamin et al. 1999
; Nishimura et al. 2006
These results are much like the Cyt c
results described previously. A discrete on-pathway intermediate with native-like interacting N-terminal and C-terminal elements is formed as an initial step. Subsequent folding is slow, suggesting a barrier limited by the need to first repair a non-native misfolding that is seen to be present (Nishimura et al. 2006
) (see Section 9).
8.2 Ribonuclease H1 (RNase H)
Marqusee and co-workers extensively analyzed the folding behavior of RNase H, which consists of five helices (A–E) and five β-strands (I–V).
NHX results (Chamberlain et al. 1996
; Chamberlain & Marqusee, 2000
) for the native protein at equilibrium found two subglobal PUFs. In the lowest free-energy PUF strands I, II, III, V, and helix E are unfolded. The next higher PUF additionally unfolds helix B and strand IV, leaving helices A and D as the final unfolding unit. A similar ladder with the same order but slightly different groupings was found in a thermophilic homolog (Hollien & Marqusee, 1999
In agreement, folding experiments (HX pulse labeling, CD, mutational effects) connect the earliest kinetic phase to formation of helices A and D and strand IV and a second phase largely to β
-sheet formation, placing the PUFs found by NHX on the folding pathway (Raschke & Marqusee, 1997
; Raschke et al. 1999
). An HX protection study of the equilibrium acid molten globule shows that only the highest free-energy PUF with helices A and D and also B are well formed (Dabora et al. 1996
), just as for apoMb. Related work found that a large synthetic fragment containing the contiguous helices A–D and strand IV displays independent stability (Chamberlain et al. 1999
; Cecconi et al. 2005
). In the different kinds of experiment, analogous PUFs were found to have similar stability.
These kinetic and thermodynamic results reveal cooperative foldon units in RNase H, describe their composition in terms of the secondary structural elements of the native protein, and show their role in determining a stepwise folding pathway. As for apoMb, additional structural units can be added to or subtracted from any given PUF by adjusting condition-dependent local stabilities, but it appears that the rank order of PUF formation is robust, as expected from the sequential stabilization principle.
8.3 Apocytochrome b562 (apoCyt b562)
ApoCyt b562 is a four-helix bundle protein (I–IV) with the non-covalently bound heme group removed.
Fuentes & Wand (1998a
) studied apoCyt b562
by native state HX using both GdmCl and pressure as destabilants. The results imply a sequence in which the core bi-helices II+III fold first followed in unspecified order by helices I and IV. Bai and co-workers stabilized apoCyt b562
by multiple mutations which provided the additional dynamic range in ΔGHX
needed to more definitively distinguish the different foldons by NHX (Chu et al. 2002
). They also performed kinetic folding and phi analysis studies. All of the results consistently show that the core helices II+III fold first followed by helix IV and then helix I.
Bai and co-workers then created different constructs with the native state mutationally destabilized so that other states normally at higher free energy became dominantly populated, and solved their solution structures by NMR. Well-folded structures were found, nearly identical to the first and second PUFs found by NHX (Takei et al. 2002
; Feng et al. 2003a
). The helices maintain their near-native main-chain conformation but the newly exposed apolar side-chains energy minimize by significant non-native repacking.
These results independently document the foldon units found by NHX and demonstrate a sequential foldon-dependent folding pathway. They also yield the important demonstration that incomplete native forms (intermediates, transition states) are likely to include non-native interactions and illustrate one case in atomic detail.
8.4 Outer surface protein A (OspA)
The 28 kDa outer surface protein from B. burgdorferi consists of 21 adjacent antiparallel β-strands and one α-helix arranged as a flat nine-stranded β-sheet capped on both ends by globular domains.
Koide and co-workers used NHX in EX1 and EX2 modes to dissect OspA into five cooperative foldon units (Yan et al. 2002
). The NHX results and mutational phi analysis showed that at least two PUFs constructed from these foldons account for sequential intermediates in the folding pathway (Yan et al. 2004
8.5 Triosephosphate isomerase (TIM)
Triosephosphate isomerase is the archetypal TIM barrel protein, with eight alternating α
pairs. Silverman and Harbury studied the TIM homodimer by a technology that does not depend on HX (Silverman & Harbury, 2002b
). They measured the accessibility to attack of 47 Cys residues inserted at various buried sites, so that reactivity depends on transient exposure by dynamic structural opening reactions (Silverman & Harbury, 2002a
). Results found were exactly analogous to NHX results. With increasing denaturant, local fluctuational reaction pathways with zero m
values merge into larger subglobal unfolding reactions that define three distinct PUFs. Further mutational studies analogous to the stability labeling experiments in Section 5 showed that the foldons unfold in a sequential pathway manner.
These results do not depend on any possible artifacts based on HX behavior. They demonstrate subglobal foldon units and their role in constructing a stepwise folding pathway.
Amino-acid-resolved information obtained by HX and non-HX methods under native conditions for many proteins of all structural types indicate that proteins may be viewed quite generally as accretions of cooperative foldon units. Where sufficiently detailed information is available, the results show that the steps in folding pathways are accounted for by the formation and association of native-like foldon units, and that this occurs in a sequential stabilization process that progressively grows the native protein. The two motivating concepts, cooperative foldon units and sequential stabilization, can be seen to represent straightforward expressions of highly documented protein behaviors.