Tertiary Structure Formation Research Articles

Structural Basis of Folding Cooperativity in Model Proteins: Insights from a Microcanonical Perspective

Two-state cooperativity is an important characteristic in protein folding. It is defined by a depletion of states that lie energetically between folded and unfolded conformations. There are different ways to test for two-state cooperativity; however, most of these approaches probe indirect proxies of this depletion. Generalized-ensemble computer simulations allow us to unambiguously identify this transition by a microcanonical analysis on the basis of the density of states. Here, we present a detailed characterization of several helical peptides obtained by coarse-grained simulations. The level of resolution of the coarse-grained model allowed to study realistic structures ranging from small α-helices to a de novo three-helix bundle without biasing the force field toward the native state of the protein. By linking thermodynamic and structural features, we are able to show that whereas short α-helices exhibit two-state cooperativity, the type of transition changes for longer chain lengths because the chain forms multiple helix nucleation sites, stabilizing a significant population of intermediate states. The helix bundle exhibits signs of two-state cooperativity owing to favorable helix-helix interactions, as predicted from theoretical models. A detailed analysis of secondary and tertiary structure formation fits well into the framework of several folding mechanisms and confirms features that up to now have been observed only in lattice models.

Relation between duration of incubation period of prion infections and prion protein conformation

In this paper, we propose the hypothesis that the long incubation period of prion infections is dependent upon a low rate of pathological prion formation and accumulation. Reduced pathological prion formation might be caused by the high content of β-sheets in the molecule. β-Sheet folding appears to proceed more slowly than folding of α-helices; the former are a major component of the prion secondary structure. This hypothesis strongly agrees with the data about folding of the artificial protein l-polylysine. This protein exists in two subforms: a rapidly folding α-helix-enriched form and a β-sheet-rich form having a very slow speed of secondary and tertiary structure formation. According to our hypothesis, the limiting factor for prion infection propagation is the speed of β-sheet folding in molecules of pathological prion but not the speed of migration of this protein through the host organism.

Observation of Two Families of Folding Pathways of BBL

BBL is an independent folding domain of a large multienzyme complex, 2-oxoglutarate dehydrogenase. The folding mechanism of BBL is under debate between the views of noncooperative downhill-type and classical two-state. Extensive replica exchange molecular dynamics simulations of BBL in explicit solvent have shown some non-two-state behaviors despite no definitive evidence of downhill folding. In this work, we postprocess the replica exchange data using our roadmap-based MaxFlux reaction path algorithm to reveal atomically detailed folding pathways. A connected graph is used to organize and visualize the folding pathways initiated from random coils. High structural and transition heterogeneity is seen in the early stage of folding. Two main parallel folding pathways emerge in the later stage; one path shows that tertiary contact and helix formation develop at different stages of folding, whereas the other path exhibits concurrence of secondary and tertiary structure formation to some extent. Because the native state of BBL is sensitive to experimental conditions, we speculate that the relative predominance of the two pathways may vary with the protein construct and solvent conditions, possibly leading to the seeming discrepancy of experimental results. Our roadmap-based reaction path algorithm is a general tool to extract path information from replica exchange.

Effects of Mg2+ on the Free Energy Landscape for Folding a Purine Riboswitch RNA

There are potentially several ways Mg2+ might promote formation of an RNA tertiary structure: by causing a general "collapse" of the unfolded ensemble to more compact conformations, by favoring a reorganization of structure within a domain to a form with specific tertiary contacts, and by enhancing cooperative linkages between different sets of tertiary contacts. To distinguish these different modes of action, we have studied Mg2+ interactions with the adenine riboswitch, in which a set of tertiary interactions that forms around a purine-binding pocket is thermodynamically linked to the tertiary "docking" of two hairpin loops in another part of the molecule. Each of four RNA forms with different extents of tertiary structure were characterized by small-angle X-ray scattering. The free energy of interconversion between different conformations in the absence of Mg2+ and the free energy of Mg2+ interaction with each form have been estimated, yielding a complete picture of the folding energy landscape as a function of Mg2+ concentration. At 1 mM Mg2+ (50 mM K+), the overall free energy of stabilization by Mg2+ is large, -9.8 kcal/mol, and about equally divided between its effect on RNA collapse to a partially folded structure and on organization of the binding pocket. A strong cooperative linkage between the two sets of tertiary contacts is intrinsic to the RNA. This quantitation of the effects of Mg2+ on an RNA with two distinct sets of tertiary interactions suggests ways that Mg2+ may work to stabilize larger and more complex RNA structures.

Proteins of Diverse Function and Subcellular Location Are Lysine Acetylated in Arabidopsis

Acetylation of the ε-amino group of lysine (Lys) is a reversible posttranslational modification recently discovered to be widespread, occurring on proteins outside the nucleus, in most subcellular locations in mammalian cells. Almost nothing is known about this modification in plants beyond the well-studied acetylation of histone proteins in the nucleus. Here, we report that Lys acetylation in plants also occurs on organellar and cytosolic proteins. We identified 91 Lys-acetylated sites on 74 proteins of diverse functional classes. Furthermore, our study suggests that Lys acetylation may be an important posttranslational modification in the chloroplast, since four Calvin cycle enzymes were acetylated. The plastid-encoded large subunit of Rubisco stands out because of the large number of acetylated sites occurring at important Lys residues that are involved in Rubisco tertiary structure formation and catalytic function. Using the human recombinant deacetylase sirtuin 3, it was demonstrated that Lys deacetylation significantly affects Rubisco activity as well as the activities of other central metabolic enzymes, such as the Calvin cycle enzyme phosphoglycerate kinase, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase, and the tricarboxylic acid cycle enzyme malate dehydrogenase. Our results demonstrate that Lys acetylation also occurs on proteins outside the nucleus in Arabidopsis (Arabidopsis thaliana) and that Lys acetylation could be important in the regulation of key metabolic enzymes.

Interplay Between Secondary and Tertiary Structure Formation in Protein Folding Cooperativity

Protein folding cooperativity is defined by the nature of the finite-size thermodynamic transition exhibited upon folding: two-state transitions show a free energy barrier between the folded and unfolded ensembles, while downhill folding is barrierless. A microcanonical analysis, where the energy is the natural variable, has shown better suited to unambiguously characterize the nature of the transition compared to its canonical counterpart. Replica exchange molecular dynamics simulations of a high resolution coarse-grained model allow for the accurate evaluation of the density of states, in order to extract precise thermodynamic information, and measure its impact on structural features. The method is applied to three helical peptides: a short helix shows sharp features of a two-state folder, while a longer helix and a three-helix bundle exhibit downhill and two-state transitions, respectively. Extending the results of lattice simulations and theoretical models, we find that it is the interplay between secondary structure and the loss of non-native tertiary contacts which determines the nature of the transition.

Probing the Kinetic Network of Folding-Unfolding Transitions in Proteins

The formation of secondary and tertiary structure elements in protein folding are intrinsically complex processes, notoriously difficult to study in a systematic manner. We construct coarse master equations for helix formation processes based on data from atomistic molecular dynamics simulations of helix-rich proteins. By carefully controlling the effects of fast, non-Markovian transitions, on one hand, and the typically limited sampling of slow relaxation processes on the other hand, we probe the underlying network of folding-unfolding transitions between the various configuration states of a protein. This systematic analysis reveals the transition states and the associated folding pathways at multiple levels, from atomistic to coarse-grained representations. We validate our approach in folding studies of short helix-forming polyalanine peptides, as well as of a larger, helix-turn-helix subdomain of a viral scaffolding protein. Our analysis of local, site-specific formation of intra- and inter-chain interactions is a first step towards understanding the elementary stages of secondary and tertiary structure formation in the folding of large proteins, and it allows a direct comparison to data from recent infrared vibrational spectroscopy studies.

Transmembrane Helical Protein Folding: Lipid Modulation and Folding Transition States

General folding principles have emerged from studies on water-soluble proteins, but it is unclear how these ideas will translate to transmembrane proteins, which expose rather than hide their hydrophobic surfaces. We combine kinetic and thermodynamic studies of the reversible unfolding of helical membrane proteins to provide a definitive value for the reaction free energy and a means to probe the transition state. Efficient systems also need to be developed to stabilise, unfold and re-fold a wider range of alpha helical membrane proteins. We have been developing in vitro lipid bicelle and bilayer folding systems for membrane proteins. Bicelle properties, as well as the stored curvature elastic stress of model bilayers can be used to optimise the rate, yield and stability of folded protein. We have shown that events such as transmembrane helix insertion, as well as tertiary and quaternary structure formation are altered by the stored curvature stress of the bilayer. Either changing the lipid chains or introducing a different headgroup such as phosphoethanolamine alters the curvature stress of a phosphatoidylcholine lipid bilayer. Our results on a variety of monomeric, multidomain and multi-subunit proteins will be described.

Enhanced Specificity against Misfolding in a Thermostable Mutant of the Tetrahymena Ribozyme

Structured RNAs encode native conformations that are more stable than the vast ensembles of alternative conformations, but how this specificity is evolved is incompletely understood. Here we show that a variant of the Tetrahymena group I intron ribozyme that was generated previously by in vitro selection for enhanced thermostability also displays modestly enhanced specificity against a stable misfolded structure that is globally similar to the native state, despite the absence of selective pressure to increase the energy gap between these structures. The enhanced specificity for native folding arises from mutations in two nucleotides that are close together in space in the native structure, and additional experiments show that these two mutations do not affect the stability of the misfolded conformation relative to the largely unstructured transition state ensemble for interconversion between the native and misfolded conformers. Thus, they selectively stabilize the native state, presumably by strengthening a local tertiary contact network that cannot form in the misfolded conformation. The stabilization is larger in the presence of the peripheral element P5abc, suggesting that cooperative tertiary structure formation plays a key role in the enhanced stability. The increased specificity in the absence of explicit selection suggests that the large energy gap in the wild-type RNA may have arisen analogously, a consequence of selective pressure for stability of the functional structure. More generally, the structural rigidity and intricate networks of contacts in structured RNAs may allow them to evolve substantial structural specificity without explicit negative selection, even against closely related alternative structures.

Cytomegalovirus Latency Promotes Cardiac Lymphoid Neogenesis and Accelerated Allograft Rejection in CMV Naïve Recipients

Human cytomegalovirus (HCMV) infection is associated with the acceleration of transplant vascular sclerosis (TVS) and chronic allograft rejection (CR). HCMV-negative recipients of latently HCMV infected donor grafts are at highest risk for developing CMV disease. Using a rat heart transplant CR model, we have previously shown that acute rat CMV (RCMV) infection following transplantation significantly accelerates both TVS and CR. Here, we report that RCMV-naïve recipients of heart allografts from latently RCMV-infected donors undergo acceleration of CR with similar kinetics as acutely infected recipients. In contrast to acutely infected recipients, treatment of recipients of latently infected donor hearts with ganciclovir did not prevent CR or TVS. We observed the formation of tertiary lymphoid structures (TLOs) containing macrophages and T cells in latently infected hearts prior to transplantation but not in uninfected rats. Moreover, pathway analysis of gene expression data from allografts from latently infected donors indicated an early and sustained production of TLO-associated genes compared to allografts from uninfected donors. We conclude that RCMV-induced TLO formation and alteration of donor tissue T cell profiles prior to transplantation in part mediate the ganciclovir-insensitive rejection of latently infected donor allografts transplanted into naïve recipients by providing a scaffold for immune activation.

Insight into the structural organization of the omega leader of TMV RNA: The role of various regions of the sequence in the formation of a compact structure of the omega RNA

The 5′-untranslated sequence of tobacco mosaic virus RNA – the so called omega leader – is a well-known translational enhancer. The structure of the omega RNA has unusual features. Despite the absence of extensive secondary structure of the Watson–Crick type, the omega RNA possesses a stable compact conformation. The central part of the omega sequence contains many CAA repeats and is flanked by U-rich regions. In this work we synthesized the polyribonucleotides containing modified omega sequences, and studied them using analytical ultracentrifugation and thermal melting techniques. It was demonstrated that changes made in both the central and the 3′-proximal part of the sequence led to a strong destabilization of the omega RNA structure. We conclude that the regular (CAA)n core region and the 3′-proximal AU-rich region of the omega RNA interact with each other and contribute together to the formation of a stable tertiary structure.

Transient Tertiary Structure Formation within the Ribosome Exit Port

The exit tunnel of the ribosome is commonly considered to be sufficiently narrow that co-translational folding can begin only when specific segments of nascent chains are fully extruded from the tunnel. Here we show, on the basis of molecular simulations and comparison with experiment, that the long-range contacts essential for initiating protein folding can form within a nascent chain when it reaches the last 20 Å of the exit tunnel. We further show that, in this "exit port", a significant proportion of native and non-native tertiary structure can form without steric overlap with the ribosome itself, and provide a library of structural elements that our simulations predict can form in the exit tunnel and is amenable to experimental testing. Our results show that these elements of folded tertiary structure form only transiently and are at their midpoints of stability at the boundary region between the inside and the outside of the tunnel. These findings provide a framework for interpreting a range of recent experimental studies of ribosome nascent chain complexes and for understanding key aspects of the nature of co-translational folding.

CD20+ B Cells: The Other Tumor-Infiltrating Lymphocytes

Tumor-infiltrating CD8(+) T cells are strongly associated with patient survival in a wide variety of human cancers. Less is known about tumor-infiltrating CD20(+) B cells, which often colocalize with T cells, sometimes forming organized lymphoid structures. In autoimmunity and organ transplantation, T cells and B cells collaborate to generate potent, unrelenting immune responses that can result in extensive tissue damage and organ rejection. In these settings, B cells enhance T cell responses by producing Abs, stimulatory cytokines, and chemokines, serving as local APCs, and organizing the formation of tertiary lymphoid structures that sustain long-term immunity. Thus, B cells are an important component of immunological circuits associated with persistent, rampant tissue destruction. Engagement of tumor-reactive B cells may be an important condition for generating potent, long-term T cell responses against cancer.

The Osmolyte TMAO Stabilizes Native RNA Tertiary Structures in the Absence of Mg2+: Evidence for a Large Barrier to Folding from Phosphate Dehydration

The stabilization of RNA tertiary structures by ions is well known, but the neutral osmolyte trimethylamine oxide (TMAO) can also effectively stabilize RNA tertiary structure. To begin to understand the physical basis for the effects of TMAO on RNA, we have quantitated the TMAO-induced stabilization of five RNAs with known structures. So-called m values, the increment in unfolding free energy per molal of osmolyte at constant KCl activity, are ∼0 for a hairpin secondary structure and between 0.70 and 1.85 kcal mol−1m−1 for four RNA tertiary structures (30–86 nt). Further analysis of two RNAs by small-angle X-ray scattering and hydroxyl radical probing shows that TMAO reduces the radius of gyration of the unfolded ensemble to the same endpoint as seen in titration with Mg2+ and that the structures stabilized by TMAO and Mg2+ are indistinguishable. Remarkably, TMAO induces the native conformation of a Mg2+ ion chelation site formed in part by a buried phosphate, even though Mg2+ is absent. TMAO interacts weakly, if at all, with KCl, ruling out the possibility that TMAO stabilizes RNA indirectly by increasing salt activity. TMAO is, however, strongly excluded from the vicinity of dimethylphosphate (unfavorable interaction free energy, +211 cal mol−1m−1 for the potassium salt), an ion that mimics the RNA backbone phosphate. We suggest that formation of RNA tertiary structure is accompanied by substantial phosphate dehydration (loss of 66–173 water molecules in the RNA structures studied) and that TMAO works principally by reducing the energetic penalty associated with this dehydration. The strong parallels we find between the effects of TMAO and Mg2+ suggest that RNA sequence is more important than specific ion interactions in specifying the native structure.

Interplay between Secondary and Tertiary Structure Formation in Protein Folding Cooperativity

Protein folding cooperativity is defined by the nature of the finite-size thermodynamic transition exhibited upon folding: two-state transitions show a free-energy barrier between the folded and unfolded ensembles, while downhill folding is barrierless. A microcanonical analysis, where the energy is the natural variable, has proved to be better suited than its canonical counterpart to unambiguously characterize the nature of the transition. Replica-exchange molecular dynamics simulations of a high-resolution coarse-grained model allow for the accurate evaluation of the density of states in order to extract precise thermodynamic information and measure its impact on structural features. The method has been applied to three helical peptides: a short helix shows sharp features of a two-state folder, while a longer helix and a three-helix bundle exhibit downhill and two-state transitions, respectively. Extending the results of lattice simulations and theoretical models, we have found that it is the interplay between secondary structure and the loss of non-native tertiary contacts that determines the nature of the transition.

PS3-05 The chemokine network in the formation of tertiary lymphoid structures in colorectal cancer

PS3-05 The chemokine network in the formation of tertiary lymphoid structures in colorectal cancer

A novel mutation in the mitochondrial tRNA Ala gene (m.5636T>C) in a patient with progressive external ophthalmoplegia

We report a heteroplasmic novel mutation m.5636T>C in the mt-tRNA Ala in a patient with bilateral ptosis and ophthalmoparesis in whom a muscle biopsy showed cytochrome c oxdidase (COX) negative and ragged red fibers. Using laser capture microdissection we have isolated COX negative fibers and COX positive fibers from the muscle of the patient and determined that the mutation load was clearly increased in COX negative muscle fibers. Additionally, the mutated m.5636T nucleotide is conserved in all the mammal and non-mammal species analyzed and might be structurally relevant as it is located in a position involved in the formation of tertiary structure of canonical mitochondrial tRNAs.

Appraisal of translocation pathways for displaying ankyrin repeat protein on phage particles

Depending on the molecular properties of the proteins of interest (POI), the rate of success in displaying proteins on phage particles is unpredictable. Formation of polypeptide tertiary structure in the cytoplasm occasionally results in low level display on viral particles. Here we assessed the influence of different leader peptides on the display of a premature cytoplasmic folding protein, ankyrin repeat protein (ARP), via the minor coat protein pIII. These peptides include the Sec, SRP and Tat pathways. The results demonstrated that the Sec and SRP pathways were capable of displaying the protein on the viral particle, whereas the Tat pathway failed to do so. Interestingly, the Tat pathway efficiently directed ARP through its translocon without fusing with pIII. Furthermore, the soluble form of ARP was detected in Escherichia coli periplasm.

Context-Dependent Remodeling of Structure in Two Large Protein Fragments

Protein folding involves the formation of secondary structural elements from the primary sequence and their association with tertiary assemblies. The relation of this primary sequence to a specific folded protein structure remains a central question in structural biology. An increasing body of evidence suggests that variations in homologous sequence ranging from point mutations to substantial insertions or deletions can yield stable proteins with markedly different folds. Here we report the structural characterization of domain IV (D4) and ΔD4 (polypeptides with 222 and 160 amino acids, respectively) that differ by virtue of an N-terminal deletion of 62 amino acids (28% of the overall D4 sequence). The high-resolution crystal structures of the monomeric D4 and the dimeric ΔD4 reveal substantially different folds despite an overall conservation of secondary structure. These structures show that the formation of tertiary structures, even in extended polypeptide sequences, can be highly context dependent, and they serve as a model for structural plasticity in protein isoforms.

New insights into the development of lymphoid tissues

Secondary lymphoid organs are important locations for the initiation of adaptive immune responses. They develop before birth, and their formation requires interaction between lymphotoxin-α₁ß₂-expressing lymphoid-tissue inducer cells and lymphotoxin-ß receptor-expressing stromal organizer cells. Here, we discuss new insights into the earliest phases of peripheral lymph node and Peyer's patch formation that occur before lymphotoxin-ß receptor signalling and suggest a role for the developing nervous system. In addition, we discuss the differing requirements for the postnatal formation of mucosa-associated lymphoid tissues and tertiary lymphoid structures that develop at sites of chronic inflammation.