Together, these results show that designed repeat-protein folding is governed by seed formation, interface propagation, and terminal boundary conditions, and establish intramolecular crosslinking as a strategy for rationally reshaping folding landscapes in designed proteins.
The structural feasibility of chain folding within an accepted cellulose-II structure is investigated in this study. We incorporate 3-residue and 5-residue folded glucosyl turns into a cellulose-II fibril crystal model to assess if the dominant structure can be retained. Ten candidate turn models were generated by metadynamics simulations and ranked by semiempirical quantum mechanical energies. Across >30 independent 300 ns molecular dynamics trajectories, the crystalline core retains its unit cell parameters, X-ray diffraction pattern, hydrogen bonding network, and canonical 4C1 ring conformation. This suggests that, should such folds be present, they need not compromise bulk crystalline integrity. No significant fibril twisting was observed in either the unfolded or chain-fold models. At the chain-folding ends of the fibril, high-energy boat and skew-boat conformations drive hydroxymethyl rotamer redistribution and noncanonical hydrogen bonding contacts. The greater structural flexibility of the 5-residue folded turns allows the chain-fold ends to access a broader conformational space, yet the structural perturbation remains localized within the same 3-residue region as the 3-residue folded turns. The 3-residue turn is therefore considered the more plausible chain-fold structure for cellulose-II, should such a structure actually exist in reality.
Linghan Kong, Stephen J. Eichhorn, Richard A. Bryce· Biomacromolecules· 0 citations
Protein folding is the process by which a polypeptide chain organizes into its three-dimensional structure through a balance of stabilizing and destabilizing interactions encoded by the sequence. A central question in protein biophysics is how thermodynamic factors guide a polypeptide toward its native folded state despite the rugged energy landscape and the competing influence of nonnative interactions. In many biomolecular processes, cooperativity provides a mechanism by which multiple weak interactions act collectively to generate a robust response. In the context of protein folding, such cooperative effects may arise when the formation of one native contact enhances the stability or likelihood of nearby native contacts, thereby promoting collective organization toward the folded state. At the same time, folding is opposed by the much larger number of non-native interactions, whose heterogeneity can introduce frustration and destabilize folding even when the average native bias favors the folded phase. The interplay of these competing effects in determining foldability remains unclear in statistical-mechanical models. Here, we address this problem using a one-dimensional spin-glass model of protein folding with explicit shared-residue cooperative interactions encoded through wedge-based motifs. We show that modest cooperative bias can stabilize folding even where the noncooperative system remains unfolded, whereas non-native energetic fluctuation suppresses folding and shifts the transition to higher cooperative strengths. We further find that partial cooperative coverage is sufficient to lower the folding threshold. Therefore, the model provides a mean-field framework for incorporating cooperative interaction strength into the native one-dimensional model of protein folding and for describing how local cooperativity reshapes the folding transition.
A fundamental question in structural biology centres around understanding protein evolution. Key to this process is mutational robustness, defined as the protein fold’s ability to absorb sequence changes without collapsing its structure. Here, we show that robustness is systematically shaped by simple features such as protein size, geometry, and oligomeric state. We used Foldseek-identified (structural) homologs to quantify family size across monomers and higher homo-oligomers. We found that proteins in larger families are consistently larger in size, more compact in atomic density, and less exposed to solvent. Strikingly, homo-oligomers occupy systematically larger families than monomers, revealing quaternary structure itself as a driver of mutational tolerance, not merely a functional supplement. This signature of robustness can be further linked to increasing functional complexity in proteins; those with adaptive, multifaceted biological roles belong to larger structural families than those with specific roles, thereby linking structural flexibility directly to evolutionary versatility. In short, simple yet overlooked features of protein geometry can explain mutational robustness and evolvability, offering a structural rationale for why certain protein families have diversified extensively while others remain in evolutionary stasis. eTOC blurb (short summary) Why do some protein folds diversify into thousands of variants while others remain rare and rigid? This work shows that the answer lies in geometry: proteins with denser cores, larger size, and higher-order oligomeric assembly tolerate mutations more readily, occupy larger structural families, and support more versatile biological roles. This reveals that protein size, shape, and self-assembly, not just sequence, are fundamental drivers of evolvability.
De novo protein design is advancing rapidly1,2. This is being driven by AI to generate protein backbones, sequences, and structural models3–7. As a result, de novo designed proteins are becoming larger and more complex8–10, and increasingly explore new protein structures11,12. By contrast, natural proteins have evolved structural and functional complexity by modular combination of recurring protein domains13. Approximately 25% of these natural domains are mostly α-helical structures14. Here we show how these can be expanded using rational computational design. Following the domain classification scheme CATH15, we build complex all-α de novo proteins hierarchically using sequence-to-structure relationships for helix-helix interactions, systematic rules to connect helices, computational tools to design loops, and in silico evaluation. The pipeline starts with a target architecture of free-standing helices. These are connected into a topology by considering local arrangements of helical bundles using understood sequence-to-structure relationships for helix packing. Single-chain sequences are completed using template- and AI-based methods. Finally, AlphaFold models are assessed to give small numbers of designs for experimental validation. We test 31 designs for 14 different architectures and 25 topologies. 75% of these express as stable, monomeric, water-soluble proteins; and >30% yield X-ray crystal structures matching the designs to atomic accuracy and with new-to-nature structures. Finally, several of the scaffolds are functionalised through one-shot designs to deliver ion, small-molecule and protein binders.
K. I. Albanese, Joel J. Chubb, L. Gutierrez-Rus et al.· bioRxiv· 0 citations
How the earliest structure within the unfolded state is formed during biosynthesis on the ribosome and whether it has any consequences for downstream folding remain open questions. Here, we combine 15N paramagnetic relaxation enhancement NMR with all-atom molecular dynamics simulations to characterise the unfolded state of a folding-competent immunoglobulin-like domain on the ribosome at the cusp of folding initiation. We identify three structurally distinct sub-ensembles that differ in compaction and ribosome interactions. Non-native contacts, together with ribosome interactions, likely delay folding, yet their persistence alongside early native-like contacts within a sparsely populated compact sub-ensemble suggests they may also facilitate the formation of a co-translational folding nucleus, whose contacts overlap with those of the downstream intermediates. From these findings we infer a mechanistic model of de novo folding initiation during biosynthesis and, by linking the folding nucleus to downstream partially structured intermediates and the native state, provide a complete atomistic description of a co-translational folding pathway.
Ivana V. Bukvin, Julian O. Streit, Tomasz Włodarski et al.· bioRxiv· 0 citations
Understanding how proteins evolve requires moving beyond the classical sequence-structure paradigm toward a framework that incorporates conformational dynamics as a substrate of innovation. Within this framework, function emerges not only from single structures but from an ensemble encoded in the sequence. In this context, single-molecule Förster resonance energy transfer, which resolves heterogeneous and transient conformations inaccessible to ensemble methods, has shown that dynamics can tune activity, specificity, and regulation. Importantly, disordered proteins - which maintain biochemical properties rather than precise sequences - represent the limiting case of this principle, where dynamics become the primary substrate for evolution. Recent advances have extended these principles to protein design, demonstrating that dynamics are not only evolvable but also programmable. Together, these insights position conformational dynamics at the center of understanding and engineering the evolutionary logic of protein function, opening the door to study how proteins are tuned to operate under the nonequilibrium conditions of living cells.
Sixto M. Herrera, Elías Manríquez-Benítez, Exequiel Medina· Current Opinion in Structura...· 0 citations