In a recent article in PNAS, Zhuravlev et al. (1) determine unfolding trajectories of the Src tyrosine kinase SH3 (Src homology 3) domain [Protein Data Base (PDB) ID code 1SRL]. Using laser optical tweezing, constant force was applied to the SH3 N and C termini (residues 9 and 59), and f-dependent unfolding rates were computed. Notably, a switch between distinct unfolding pathways was detected. The question arises as to what extent such molecular mechanisms apply to living cells/physiological settings.
The approach by Zhuravlev et al. (1) relies on proteins unfolding upon application of force to N and C protein termini. A notable parallel appears to be the folding/unfolding cycles, as guided by chaperones, such as Hsp70 or GroES/GroEL (2). Chaperone interactions indeed greatly lower the activation energy required for protein unfolding, through a sequential sliding of progressively unfolded/folding polypeptides in folding apparatuses.
The issue of whether N and C termini anchoring for folding/unfolding can occur in living cells was thus challenged. Such an occurrence rests on a basic tenet, that is, that N and C termini for anchored folding/unfolding should be structurally available for binding (i.e., they should be exposed to the solvent on the protein surface). This model was tested on a sample of randomly chosen protein crystals (Table 1). Rather remarkably, all examined proteins were found to possess surface-accessible, exposed N and C termini. Distinct protein domains can behave as independent folding units (3). Consistent, surface-exposed N and C termini were identified in SH2, SH3, PH, PTB, EGF, and DNA-binding domains [including the PDB ID code 1SRL SH3 domain in (1)].
Table 1.
Protein surface-exposure of N and C termini
| Protein species | N terminus | C terminus |
| SH2, SH3 domains | ||
| Src SH3 1SRL | Surface | Surface |
| SEM-5 C-terminal SH3 1SEM | Surface | Surface |
| SH2 1cwd | Surface | Surface |
| PH, PTB domains | ||
| PLCδ PH 1mai | Surface | Surface |
| Pleckstrin PH 1pls | Surface | Surface |
| Dynamin PH 1dyn | Surface | Surface |
| Spectrin PH 1btn | Surface | Surface |
| Spectrin PH 1dro | Surface | Surface |
| PTB 1shc | Surface | Surface |
| EGF domains | ||
| IGF-BP5 1boe | Surface | Surface |
| E-selectin 1esl | Surface | Surface |
| Gromos 1apo | Surface | Surface |
| Signaling complexes | ||
| Gαβγ 1got (all subunits) | Surface | Surface |
| Gβ 2trc | Surface | Surface |
| Cytoskeleton | ||
| β-actin 2oan | Surface | Surface |
| Profilin 1hlu | Surface | Surface |
| Gelsolin 1d0n | Surface | Surface |
| Severin 1svq | Surface | Surface |
| Spectrin repeat 2spc | Surface | Surface |
| Villin 1vil | Surface | Surface |
| Enzymes | ||
| Acetylcholinesterase 2ace | Surface | Surface |
| Cathepsin D 1lya | Surface | Surface |
| Ferredoxin 1awd | Surface | Surface |
| Fructose-1,6-bisphosphatase 1fpi | Surface | Surface |
| Glucose oxidase 1gog | Surface | Surface |
| Glutaredoxin (phage T4 thioredoxin) 1aba | Surface | Surface |
| Inositol polyphosphate 1-phosphatase 1inp | Surface | Surface |
| PI-specific PLC 1gym | Surface | Surface |
| PLCδ1 1djx | Surface | Surface |
| Insulin receptor catalytic domain 1irk | Surface | Surface |
| PKA 1cmk | Surface | Surface |
| PKCβ1 1rlw | Surface | Surface |
| Metallothionein-2 2mhu (EGF domain) | Surface | Surface |
| Transcription factors | ||
| 1a1 | Surface | Surface |
| 1a5t | Surface | Surface |
| 1a6b | Surface | Surface |
| 1aaf | Surface | Surface |
| 1aay | Surface | Surface |
| 1ard | Surface | Surface |
| 1are | Surface | Surface |
| 1arf | Surface | Surface |
| 1bbo | Surface | Surface |
| 1bhi | Surface | Surface |
| 1bj6 | Surface | Surface |
| 1dsq | Surface | Surface |
| 1dsv | Surface | Surface |
| 1dvp | Surface | Surface |
| 1fre | Surface | Surface |
| 1gnf | Surface | Surface |
| 1hcp | Surface | Surface |
| 1hra | Surface | Surface |
| 1hvn | Surface | Surface |
| 1hvo | Surface | Surface |
| 1ile | Surface | Surface |
| 1mey | Surface | Surface |
| 1mfs | Surface | Surface |
| 1nc8 | Surface | Surface |
| 1ncs | Surface | Surface |
| 1paa | Surface | Surface |
| 1pyi | Surface | Surface |
| 1qf8 | Surface | Surface |
| 1rgd | Surface | Surface |
| 1rmd | Surface | Surface |
| 1sp1 | Surface | Surface |
| 1sp2 | Surface | Surface |
| 1tf3 | Surface | Surface |
| 1tf6 | Surface | Surface |
| 1ubd | Surface | Surface |
| 1zaa | Surface | Surface |
| 1zfd | Surface | Surface |
| 1zin | Surface | Surface |
| 1znf | Surface | Surface |
| 1znm | Surface | Surface |
| 2adr | Surface | Surface |
| 2gli | Surface | Surface |
| 2znf | Surface | Surface |
| 3znf | Surface | Surface |
| 4znf | Surface | Surface |
| 5znf | Surface | Surface |
| 7znf | Surface | Surface |
| DNA binding proteins | ||
| GCN4 1A02 | Surface | Surface |
| TAF12 1QB3 | Surface | Surface |
| RFC2 1IQP (all subunits) | Surface | Surface |
| Cytoplasmic proteins | ||
| Haemoglobin 1nih | Surface | Surface |
| MCP-1 1don | Surface | Surface |
| Myoglobin 1do1 | Surface | Surface |
| NEF 1efn | Surface | Surface |
| Streptavidin 1rst | Surface | Surface |
| GFP 1ema | Surface | Surface |
However, cotranslational protein folding (4, 5) occurs in a strictly sequential manner, from the N- to C-terminal ends of newly synthesized polypeptides. Investigations on the speed limit of protein folding have identified examples where folding occurs in microseconds to nanoseconds (6). Main conformational changes of a protein backbone can be complete after only 20 ps (7). The protein synthesis apparatus can add one amino acid every 50 ms. Thus, protein synthesis is several orders of magnitude slower than the folding process. Translation-coupled folding is thus rate-limiting, leading to a quasiequilibrium, restricted sampling of the conformational space during translation, which plays a key role in folding (5). Correspondingly, protein folding was shown to proceed through a compact conformation in the peptide tunnel, to then reach a native-like structure after emergence from the ribosome (5). Such processivity is largely preserved also in unfolding/folding chaperone-assisted processes (2, 8), suggesting this feature to be fundamental for folding in living cells.
Such N to C terminus folding processivity is entirely missing in typical ensemble folding or unfolding experiments (1, 7, 9). Nucleation mechanisms can be shared by the ensemble vs. sequential folding processes (3, 10). However, all subsequent folding steps remain missing. Hence, additional technology quantum leaps are called for, to extend the validity of the mechanisms investigated by Zhuravlev et al. (1) to physiological settings. The potential is there for fundamental insights into protein folding/unfolding processes.
Footnotes
The author declares no conflict of interest.
References
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