Anna L. Mallam

Experimental detection of knotted conformations in denatured proteins

Structures that contain a knot formed by the path of the polypeptide backbone represent some of the most complex topologies observed in proteins. How or why these topological knots arise remains unclear. By developing a method to experimentally trap and detect knots in nonnative polypeptide chains, we find that two knotted methyltransferases, YibK and YbeA, can exist in a trefoil-knot conformation even in their chemically unfolded states. The unique denatured-state topology of these molecules explains their ability to efficiently fold to their native knotted structures in vitro and offers insights into the potential role of knots in proteins. Furthermore, the high prevalence of the denatured-state knots identified here suggests that they are either difficult to untie or that threading of any untied molecules is rapid and spontaneous. The occurrence of such knotted topologies in unfolded polypeptide chains raises the possibility that they could play an important, and as yet unexplored, role in folding and misfolding processes in vivo.

Exploring knotting mechanisms in protein folding

One of the most striking topological features to be found in a protein is that of a distinct knot formed by the path of the polypeptide backbone. Such knotted structures represent some of the smallest “self-tying” knots observed in Nature. Proteins containing a knot deep within their structure add an extra complication to the already challenging protein-folding problem; it is not obvious how, during the process of folding, a substantial length of polypeptide chain manages to spontaneously thread itself through a loop. Here, we probe the folding mechanism of YibK, a homodimeric α/β-knot protein containing a deep trefoil knot at its carboxy terminus. By analyzing the effect of mutations made in the knotted region of the protein we show that the native structure in this area remains undeveloped until very late in the folding reaction. Single-site destabilizing mutations made in the knot structure significantly affect only the folding kinetics of a late-forming intermediate and the slow dimerization step. Furthermore, we find evidence to suggest that the heterogeneity observed in the denatured state is not caused by isomerization of the single cis proline bond as previously thought, but instead could be a result of the knotting mechanism. These results allow us to propose a folding model for YibK where the threading of the polypeptide chain and the formation of native structure in the knotted region of the protein occur independently as successive events.

Knotted Fusion Proteins Reveal Unexpected Possibilities in Protein Folding

Proteins that contain a distinct knot in their native structure are impressive examples of biological self-organization. Although this topological complexity does not appear to cause a folding problem, the mechanisms by which such knotted proteins form are unknown. We found that the fusion of an additional protein domain to either the amino terminus, the carboxy terminus, or to both termini of two small knotted proteins did not affect their ability to knot. The multidomain constructs remained able to fold to structures previously thought unfeasible, some representing the deepest protein knots known. By examining the folding kinetics of these fusion proteins, we found evidence to suggest that knotting is not rate limiting during folding, but instead occurs in a denatured-like state. These studies offer experimental insights into when knot formation occurs in natural proteins and demonstrate that early folding events can lead to diverse and sometimes unexpected protein topologies.

Solution structures of DEAD-box RNA chaperones reveal conformational changes and nucleic acid tethering by a basic tail

The mitochondrial DEAD-box proteins Mss116p of Saccharomyces cerevisiae and CYT-19 of Neurospora crassa are ATP-dependent helicases that function as general RNA chaperones. The helicase core of each protein precedes a C-terminal extension and a basic tail, whose structural role is unclear. Here we used small-angle X-ray scattering to obtain solution structures of the full-length proteins and a series of deletion mutants. We find that the two core domains have a preferred relative orientation in the open state without substrates, and we visualize the transition to a compact closed state upon binding RNA and adenosine nucleotide. An analysis of complexes with large chimeric oligonucleotides shows that the basic tails of both proteins are attached flexibly, enabling them to bind rigid duplex DNA segments extending from the core in different directions. Our results indicate that the basic tails of DEAD-box proteins contribute to RNA-chaperone activity by binding nonspecifically to large RNA substrates and flexibly tethering the core for the unwinding of neighboring duplexes.

How does a knotted protein fold

The issue of how a newly synthesized polypeptide chain folds to form a protein with a unique three‐dimensional structure, otherwise known as the ‘protein‐folding problem’, remains a fundamental question in the life sciences. Over the last few decades, much information has been gathered about the mechanisms by which proteins fold. However, despite the vast topological diversity observed in biological structures, it was thought improbable, if not impossible, that a polypeptide chain could ‘knot’ itself to form a functional protein. Nevertheless, such knotted structures have since been identified, raising questions about how such complex topologies can arise during folding. Their formation does not fit any current folding models or mechanisms, and therefore represents an important piece of the protein‐folding puzzle. This article reviews the progress made towards discovering how nature codes for, and contends with, knots during protein folding, and examines the insights gained from both experimental and computational studies. Mechanisms to account for the formation of knotted structures that were previously thought unfeasible, and their implications for protein folding, are also discussed.

Untangling the folding mechanism of the 52-knotted protein UCH-L3

Proteins possessing deeply embedded topological knots in their structure add a stimulating new challenge to the already complex protein‐folding problem. The most complicated knotted topology observed to date belongs to the human enzyme ubiquitin C‐terminal hydrolase UCH‐L3, which is an integral part of the ubiquitin–proteasome system. The structure of UCH‐L3 contains five distinct crossings of its polypeptide chain, and it adopts a 52‐knotted topology, making it a fascinating target for folding studies. Here, we provide the first in depth characterization of the stability and folding of UCH‐L3. We show that the protein can unfold and refold reversibly in vitro without the assistance of molecular chaperones, demonstrating that all the information necessary for the protein to find its knotted native structure is encoded in the amino acid sequence, just as with any other globular protein, and that the protein does not enter into any deep kinetic traps. Under equilibrium conditions, the unfolding of UCH‐L3 appears to be two‐state, however, multiphasic folding and unfolding kinetics are observed and the data are consistent with a folding pathway in which two hyperfluorescent intermediates are formed. In addition, a very slow phase in the folding kinetics is shown to be limited by proline‐isomerization events. Overall, the data suggest that a knotted topology, even in its most complex form, does not necessarily limit folding in vitro, however, it does seem to require a complex folding mechanism which includes the formation of several distinct intermediate species.

Use of protein engineering techniques to elucidate protein folding pathways.

Publisher Summary This chapter describes different approaches to studying protein-folding pathways that have employed protein-engineering techniques. The chapter discusses the early works on tryptophan synthase and dihydrofolate reductase through the development and widespread application of Φ-value analysis to the folding of small model systems to the more recent works on larger proteins with complex topologies. The applications of protein-engineering methods to study specific processes linked with protein folding—such as proline isomerization and disulfide-bond formation—are discussed in the chapter. The most powerful tool that has revolutionized the study of protein-folding pathways is protein engineering. Protein-engineering techniques have advanced over the past two decades, and new developments—such as the use of larger and more diverse protein libraries and selection methods; the incorporation of novel amino acids into proteins using engineered- and expanded-genetic codes; and the combination of semisynthetic methods and protein-engineering techniques—have increased the experimental possibilities for studying folding pathways.

Backbone NMR assignments of a topologically knotted protein in urea-denatured state

YbeA is a 3-methylpseudoridine methyltransferase from Escherichia coli that forms a stable homodimer in solution. It is one of the deeply trefoil 31 knotted proteins, of which the knot encompasses the C-terminal helix that threads through a long loop. Recent studies on the knotted protein folding pathways using YbeA have suggested that the protein knot remains present under chemically denaturing conditions. Here, we report 1H, 13C and 15N chemical shift assignments for urea-denatured YbeA, which will serve as the basis for further structural characterisations using solution state NMR spectroscopy with paramagnetic spin labeled and partial alignment media.

Backbone 1H, 13C and 15N assignments of YibK and avariant containing a unique cysteine residue at C-terminus in 8 M urea-denatured states [corrected].

YibK is a tRNA methyltransferase from Haemophilus influenzae, which forms a stable homodimer in solution and contains a deep trefoil 31 knot encompassing the C-terminal helix that threads through a long loop. It has been a model system for investigating knotted protein folding pathways. Recent data have shown that the polypeptide chain of YibK remains loosely knotted under highly denaturing conditions. Here, we report (1)H, (13)C and (15)N chemical shift assignments for YibK and its variant in the presence of 8 M urea. This work forms the basis for further analysis using NMR techniques such as paramagnetic relaxation enhancement, residual dipolar couplings and spin-relaxation dynamics analysis.