Research

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10 pages, 634 KiB

Open AccessArticle

Conformational Entropy as a Potential Liability of Computationally Designed Antibodies

by Thomas Löhr, Pietro Sormanni and Michele Vendruscolo

Biomolecules 2022, 12(5), 718; https://0-doi-org.brum.beds.ac.uk/10.3390/biom12050718 - 18 May 2022

Cited by 8 | Viewed by 2463

In silico antibody discovery is emerging as a viable alternative to traditional in vivo and in vitro approaches. Many challenges, however, remain open to enabling the properties of designed antibodies to match those produced by the immune system. A major question concerns the [...] Read more.

In silico antibody discovery is emerging as a viable alternative to traditional in vivo and in vitro approaches. Many challenges, however, remain open to enabling the properties of designed antibodies to match those produced by the immune system. A major question concerns the structural features of computer-designed complementarity determining regions (CDRs), including the role of conformational entropy in determining the stability and binding affinity of the designed antibodies. To address this problem, we used enhanced-sampling molecular dynamics simulations to compare the free energy landscapes of single-domain antibodies (sdAbs) designed using structure-based (DesAb-HSA-D3) and sequence-based approaches (DesAbO), with that of a nanobody derived from llama immunization (Nb10). Our results indicate that the CDR3 of DesAbO is more conformationally heterogeneous than those of both DesAb-HSA-D3 and Nb10, and the CDR3 of DesAb-HSA-D3 is slightly more dynamic than that of Nb10, which is the original scaffold used for the design of DesAb-HSA-D3. These differences underline the challenges in the rational design of antibodies by revealing the presence of conformational substates likely to have different binding properties and to generate a high entropic cost upon binding. Full article

(This article belongs to the Special Issue Protein Folding Stability and Dynamics: Commemorative Issue in Honor of Professor Sir Christopher Dobson (1949–2019))

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12 pages, 2567 KiB

Open AccessArticle

Under Conditions of Amyloid Formation Bovine Carbonic Anhydrase B Undergoes Fragmentation by Acid Hydrolysis

by Victor Marchenkov, Natalya Ryabova, Vitaly Balobanov, Anatoly Glukhov, Nelly Ilyina and Natalya Katina

Biomolecules 2021, 11(11), 1608; https://0-doi-org.brum.beds.ac.uk/10.3390/biom11111608 - 30 Oct 2021

Cited by 2 | Viewed by 1364

Abstract

The development of many severe human diseases is associated with the formation of amyloid fibrils. Most of the available information on the process of amyloid formation has been obtained from studies of small proteins and peptides, wherein the features of complex proteins’ aggregation [...] Read more.

The development of many severe human diseases is associated with the formation of amyloid fibrils. Most of the available information on the process of amyloid formation has been obtained from studies of small proteins and peptides, wherein the features of complex proteins’ aggregation remain insufficiently investigated. Our work aimed to research the amyloid aggregation of a large model protein, bovine carbonic anhydrase B (BCAB). It has previously been demonstrated that, when exposed to an acidic pH and elevated temperature, this protein forms amyloid fibrils. Here, we show that, under these conditions and before amyloid formation, BCAB undergoes fragmentation by acid hydrolysis to give free individual peptides and associated peptides. Fragments in associates contain a pronounced secondary structure and act as the main precursor of amyloid fibrils, wherein free peptides adopt mostly unstructured conformation and form predominantly irregular globular aggregates. Reduced acidity decreases the extent of acid hydrolysis, causing BCAB to form amorphous aggregates lacking the thioflavin T binding β-structure. The presented results provide new information on BCAB amyloid formation and show the importance of protein integrity control when working even in mildly acidic conditions. Full article

(This article belongs to the Special Issue Protein Folding Stability and Dynamics: Commemorative Issue in Honor of Professor Sir Christopher Dobson (1949–2019))

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26 pages, 3640 KiB

Open AccessFeature PaperArticle

The Right-Handed Parallel β-Helix Topology of Erwinia chrysanthemi Pectin Methylesterase Is Intimately Associated with Both Sequential Folding and Resistance to High Pressure

by Jessica Guillerm, Jean-Marie Frère, Filip Meersman and André Matagne

Biomolecules 2021, 11(8), 1083; https://0-doi-org.brum.beds.ac.uk/10.3390/biom11081083 - 22 Jul 2021

Cited by 4 | Viewed by 1733

Abstract

The complex topologies of large multi-domain globular proteins make the study of their folding and assembly particularly demanding. It is often characterized by complex kinetics and undesired side reactions, such as aggregation. The structural simplicity of tandem-repeat proteins, which are characterized by the [...] Read more.

The complex topologies of large multi-domain globular proteins make the study of their folding and assembly particularly demanding. It is often characterized by complex kinetics and undesired side reactions, such as aggregation. The structural simplicity of tandem-repeat proteins, which are characterized by the repetition of a basic structural motif and are stabilized exclusively by sequentially localized contacts, has provided opportunities for dissecting their folding landscapes. In this study, we focus on the Erwinia chrysanthemi pectin methylesterase (342 residues), an all-β pectinolytic enzyme with a right-handed parallel β-helix structure. Chemicals and pressure were chosen as denaturants and a variety of optical techniques were used in conjunction with stopped-flow equipment to investigate the folding mechanism of the enzyme at 25 °C. Under equilibrium conditions, both chemical- and pressure-induced unfolding show two-state transitions, with average conformational stability (ΔG° = 35 ± 5 kJ·mol⁻¹) but exceptionally high resistance to pressure (P_m = 800 ± 7 MPa). Stopped-flow kinetic experiments revealed a very rapid (τ < 1 ms) hydrophobic collapse accompanied by the formation of an extended secondary structure but did not reveal stable tertiary contacts. This is followed by three distinct cooperative phases and the significant population of two intermediate species. The kinetics followed by intrinsic fluorescence shows a lag phase, strongly indicating that these intermediates are productive species on a sequential folding pathway, for which we propose a plausible model. These combined data demonstrate that even a large repeat protein can fold in a highly cooperative manner. Full article

(This article belongs to the Special Issue Protein Folding Stability and Dynamics: Commemorative Issue in Honor of Professor Sir Christopher Dobson (1949–2019))

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18 pages, 5209 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Parallel and Sequential Pathways of Molecular Recognition of a Tandem-Repeat Protein and Its Intrinsically Disordered Binding Partner

by Ben M. Smith, Pamela J. E. Rowling, Christopher M. Dobson and Laura S. Itzhaki

Biomolecules 2021, 11(6), 827; https://0-doi-org.brum.beds.ac.uk/10.3390/biom11060827 - 01 Jun 2021

Cited by 3 | Viewed by 3328

Abstract

The Wnt signalling pathway plays an important role in cell proliferation, differentiation, and fate decisions in embryonic development and the maintenance of adult tissues. The twelve armadillo (ARM) repeat-containing protein β-catenin acts as the signal transducer in this pathway. Here, we investigated the [...] Read more.

The Wnt signalling pathway plays an important role in cell proliferation, differentiation, and fate decisions in embryonic development and the maintenance of adult tissues. The twelve armadillo (ARM) repeat-containing protein β-catenin acts as the signal transducer in this pathway. Here, we investigated the interaction between β-catenin and the intrinsically disordered transcription factor TCF7L2, comprising a very long nanomolar-affinity interface of approximately 4800 Å² that spans ten of the twelve ARM repeats of β-catenin. First, a fluorescence reporter system for the interaction was engineered and used to determine the kinetic rate constants for the association and dissociation. The association kinetics of TCF7L2 and β-catenin were monophasic and rapid (7.3 ± 0.1 × 10⁷ M⁻¹·s⁻¹), whereas dissociation was biphasic and slow (5.7 ± 0.4 × 10⁻⁴ s⁻¹, 15.2 ± 2.8 × 10⁻⁴ s⁻¹). This reporter system was then combined with site-directed mutagenesis to investigate the striking variability in the conformation adopted by TCF7L2 in the three different crystal structures of the TCF7L2–β-catenin complex. We found that the mutation had very little effect on the association kinetics, indicating that most interactions form after the rate-limiting barrier for association. Mutations of the N- and C-terminal subdomains of TCF7L2 that adopt relatively fixed conformations in the crystal structures had large effects on the dissociation kinetics, whereas the mutation of the labile sub-domain connecting them had negligible effect. These results point to a two-site avidity mechanism of binding with the linker region forming a “fuzzy” complex involving transient contacts that are not site-specific. Strikingly, the two mutations in the N-terminal subdomain that had the largest effects on the dissociation kinetics showed two additional phases, indicating partial flux through an alternative dissociation pathway that is inaccessible to the wild type. The results presented here provide insights into the kinetics of the molecular recognition of a long intrinsically disordered region with an elongated repeat-protein surface, a process found to involve parallel routes with sequential steps in each. Full article

(This article belongs to the Special Issue Protein Folding Stability and Dynamics: Commemorative Issue in Honor of Professor Sir Christopher Dobson (1949–2019))

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13 pages, 28174 KiB

Open AccessArticle

The ‘Shape-Shifter’ Peptide from the Disulphide Isomerase PmScsC Shows Context-Dependent Conformational Preferences

by Lorna J. Smith, Chloe W. Green and Christina Redfield

Biomolecules 2021, 11(5), 642; https://0-doi-org.brum.beds.ac.uk/10.3390/biom11050642 - 26 Apr 2021

Cited by 1 | Viewed by 1959

Abstract

Multiple crystal structures of the homo-trimeric protein disulphide isomerase PmScsC reveal that the peptide which links the trimerization stalk and catalytic domain can adopt helical, β-strand and loop conformations. This region has been called a ‘shape-shifter’ peptide. Characterisation of this peptide using NMR [...] Read more.

Multiple crystal structures of the homo-trimeric protein disulphide isomerase PmScsC reveal that the peptide which links the trimerization stalk and catalytic domain can adopt helical, β-strand and loop conformations. This region has been called a ‘shape-shifter’ peptide. Characterisation of this peptide using NMR experiments and MD simulations has shown that it is essentially disordered in solution. Analysis of the PmScsC crystal structures identifies the role of intermolecular contacts, within an assembly of protein molecules, in stabilising the different linker peptide conformations. These context-dependent conformational properties may be important functionally, allowing for the binding and disulphide shuffling of a variety of protein substrates to PmScsC. They also have a relevance for our understanding of protein aggregation and misfolding showing how intermolecular quaternary interactions can lead to β-sheet formation by a sequence that in other contexts adopts a helical structure. This ‘shape-shifting’ peptide region within PmScsC is reminiscent of one-to-many molecular recognition features (MoRFs) found in intrinsically disordered proteins which are able to adopt different conformations when they fold upon binding to their protein partners. Full article

(This article belongs to the Special Issue Protein Folding Stability and Dynamics: Commemorative Issue in Honor of Professor Sir Christopher Dobson (1949–2019))

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16 pages, 1612 KiB

Open AccessArticle

Extremely Thermostabilizing Core Mutations in Coiled-Coil Mimetic Proteins of HIV-1 gp41 Produce Diverse Effects on Target Binding but Do Not Affect Their Inhibitory Activity

by Mario Cano-Muñoz, Samuele Cesaro, Bertrand Morel, Julie Lucas, Christiane Moog and Francisco Conejero-Lara

Biomolecules 2021, 11(4), 566; https://0-doi-org.brum.beds.ac.uk/10.3390/biom11040566 - 12 Apr 2021

Cited by 7 | Viewed by 1809

Abstract

A promising strategy to neutralize HIV-1 is to target the gp41 spike subunit to block membrane fusion with the cell. We previously designed a series of single-chain proteins (named covNHR) that mimic the trimeric coiled-coil structure of the gp41 N-terminal heptad repeat (NHR) [...] Read more.

A promising strategy to neutralize HIV-1 is to target the gp41 spike subunit to block membrane fusion with the cell. We previously designed a series of single-chain proteins (named covNHR) that mimic the trimeric coiled-coil structure of the gp41 N-terminal heptad repeat (NHR) region and potently inhibit HIV-1 cell infection by avidly binding the complementary C-terminal heptad repeat (CHR) region. These proteins constitute excellent tools to understand the structural and thermodynamic features of this therapeutically important interaction. Gp41, as with many coiled-coil proteins, contains in core positions of the NHR trimer several highly conserved, buried polar residues, the role of which in gp41 structure and function is unclear. Here we produced three covNHR mutants by substituting each triad of polar residues for the canonical isoleucine. The mutants preserve their helical structure and show an extremely increased thermal stability. However, increased hydrophobicity enhances their self-association. Calorimetric analyses show a marked influence of mutations on the binding thermodynamics of CHR-derived peptides. The mutations do not affect however the in vitro HIV-1 inhibitory activity of the proteins. The results support a role of buried core polar residues in maintaining structural uniqueness and promoting an energetic coupling between conformational stability and NHR–CHR binding. Full article

(This article belongs to the Special Issue Protein Folding Stability and Dynamics: Commemorative Issue in Honor of Professor Sir Christopher Dobson (1949–2019))

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Review

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21 pages, 1066 KiB

Open AccessReview

Protein Fibrillation under Crowded Conditions

by Annelise H. Gorensek-Benitez, Bryan Kirk and Jeffrey K. Myers

Biomolecules 2022, 12(7), 950; https://0-doi-org.brum.beds.ac.uk/10.3390/biom12070950 - 06 Jul 2022

Cited by 4 | Viewed by 2181

Abstract

Protein amyloid fibrils have widespread implications for human health. Over the last twenty years, fibrillation has been studied using a variety of crowding agents to mimic the packed interior of cells or to probe the mechanisms and pathways of the process. We tabulate [...] Read more.

Protein amyloid fibrils have widespread implications for human health. Over the last twenty years, fibrillation has been studied using a variety of crowding agents to mimic the packed interior of cells or to probe the mechanisms and pathways of the process. We tabulate and review these results by considering three classes of crowding agent: synthetic polymers, osmolytes and other small molecules, and globular proteins. While some patterns are observable for certain crowding agents, the results are highly variable and often depend on the specific pairing of crowder and fibrillating protein. Full article

(This article belongs to the Special Issue Protein Folding Stability and Dynamics: Commemorative Issue in Honor of Professor Sir Christopher Dobson (1949–2019))

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23 pages, 3522 KiB

Open AccessEditor’s ChoiceReview

Folding and Stability of Ankyrin Repeats Control Biological Protein Function

by Amit Kumar and Jochen Balbach

Biomolecules 2021, 11(6), 840; https://0-doi-org.brum.beds.ac.uk/10.3390/biom11060840 - 05 Jun 2021

Cited by 16 | Viewed by 5311

Abstract

Ankyrin repeat proteins are found in all three kingdoms of life. Fundamentally, these proteins are involved in protein-protein interaction in order to activate or suppress biological processes. The basic architecture of these proteins comprises repeating modules forming elongated structures. Due to the lack [...] Read more.

Ankyrin repeat proteins are found in all three kingdoms of life. Fundamentally, these proteins are involved in protein-protein interaction in order to activate or suppress biological processes. The basic architecture of these proteins comprises repeating modules forming elongated structures. Due to the lack of long-range interactions, a graded stability among the repeats is the generic properties of this protein family determining both protein folding and biological function. Protein folding intermediates were frequently found to be key for the biological functions of repeat proteins. In this review, we discuss most recent findings addressing this close relation for ankyrin repeat proteins including DARPins, Notch receptor ankyrin repeat domain, IκBα inhibitor of NFκB, and CDK inhibitor p19^INK4d. The role of local folding and unfolding and gradual stability of individual repeats will be discussed during protein folding, protein-protein interactions, and post-translational modifications. The conformational changes of these repeats function as molecular switches for biological regulation, a versatile property for modern drug discovery. Full article

(This article belongs to the Special Issue Protein Folding Stability and Dynamics: Commemorative Issue in Honor of Professor Sir Christopher Dobson (1949–2019))

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