Protein engineering using circular permutation - structure, function, stability, and applications.

Protein engineering is important for creating novel variants from natural proteins, enabling a wide range of applications. Approaches such as rational design and directed evolution are routinely used to make new protein variants. Computational tools like de novo design can introduce new protein folds. Expanding the amino acid repertoire to include unnatural amino acids with non-canonical side chains in vitro by native chemical ligation and in vivo via codon expansion methods broadens sequence and structural possibilities. Circular permutation (CP) is an invaluable approach to redesigning a protein by rearranging the amino acid sequence, where the connectivity of the secondary structural elements is altered without changing the overall structure of the protein. Artificial CP proteins (CPs) are employed in various applications such as biocatalysis, sensing of small molecules by fluorescence, genome editing, ligand-binding protein switches, and optogenetic engineering. Many studies have shown that CP can lead to either reduced or enhanced stability or catalytic efficiency. The effects of CP on a protein's energy landscape cannot be predicted a priori. Thus, it is important to understand how CP can affect the thermodynamic and kinetic stability of a protein. In this review, we discuss the discovery and advancement of techniques to create protein CP, and existing reviews on CP. We delve into the plethora of biological applications for designed CP proteins. We subsequently discuss the experimental and computational reports on the effects of CP on the thermodynamic and kinetic stabilities of proteins of various topologies. An understanding of the various aspects of CP will allow the reader to design robust CP proteins for their specific purposes.

Circular permutation at azurin's active site slows down its folding.

Circular permutation (CP) is a technique by which the primary sequence of a protein is rearranged to create new termini. The connectivity of the protein is altered but the overall protein structure generally remains unperturbed. Understanding the effect of CP can help design robust proteins for numerous applications such as in genetic engineering, optoelectronics, and improving catalytic activity. Studies on different protein topologies showed that CP usually affects protein stability as well as unfolding rates. Though a significant number of proteins contain metals or other cofactors, reports of metalloprotein CPs are rare. Thus, we chose a bacterial metalloprotein, azurin, and its CP within the metal-binding site (cpF114). We studied the stabilities, folding, and unfolding rates of apo- and Zn2+-bound CP azurin using fluorescence and circular dichroism. The introduced CP had destabilizing effects on the protein. Also, the folding of the Zn2+-CP protein was much slower than that of the Zn2+-WT or apo-protein. We compared this study to our previously reported azurin-cpN42, where we had observed an equilibrium and kinetic intermediate. cpF114 exhibits an apparent two-state equilibrium unfolding but has an off-pathway kinetic intermediate. Our study hinted at CP as a method to modify the energy landscape of proteins to alter their folding pathways. WT azurin, being a faster folder, may have evolved to optimize the folding rate of metal-bound protein compared to its CPs, albeit all of them have the same structure and function. Our study underscores that protein sequence and protein termini positions are crucial for metalloproteins. TOC Figure. (Top) Zn2+-azurin WT structure (PDB code: 1E67) and 2-D topology diagram of Zn2+-cpF114 azurin. (Bottom) Cartoon diagram representing folding (red arrows) and unfolding (blue arrows) of apo- and Zn2+- WT and cpF114 azurins. The width of the arrows represents the rate of the corresponding processes.

Phase Separation and Aggregation of a Globular Folded Protein Small Ubiquitin-like Modifier 1 (SUMO1).

Liquid-liquid phase separation (LLPS) plays a crucial role in cellular organization, primarily driven by intrinsically disordered proteins (IDPs) leading to the formation of biomolecular condensates. A folded protein SUMO that post-translationally modifies cellular proteins has recently emerged as a regulator of LLPS. Given its compact structure and limited flexibility, the precise role of SUMO in condensate formation remains to be investigated. Here, we show the rapid phase separation of SUMO1 into micrometer-sized liquid-like condensates in inert crowders under physiological conditions. Subsequent time-dependent conformational changes and aggregation are probed by label-free methods (tryptophan fluorescence and Raman spectroscopy). Remarkably, experiments on a SUMO1 variant lacking the N-terminal disordered region further corroborate the role of its structured part in phase transitions. Our findings highlight the potential of folded proteins to engage in LLPS and emphasize further investigation into the influence of the SUMO tag on IDPs associated with membrane-less assemblies in cells.

Metal-binding and circular permutation-dependent thermodynamic and kinetic stability of azurin.

Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key ß-barrel, ß-sandwich, ß-α-ß, and all α-Greek key. CP mainly affects the protein stability and unfolding kinetics, while folding kinetics remains mostly unaltered. However, the effect of CP on metalloproteins is yet to be elaborately studied. The active site of metalloproteins poses an additional complexity in studying protein folding. Here, we investigate a CP variant (cpN42) of azurin-in both metal-free and metal-bound (holo) forms. As observed earlier in other proteins, apo-forms of wild-type (WT) and cpN42 fold with similar rates. In contrast, zinc-binding accelerates the folding of WT but decelerates the folding of cpN42. On zinc-binding, the spontaneous folding rate of WT increases by >250 times that of cpN42, which is unprecedented and the highest for any CP to date. On the other hand, zinc-binding reduces the spontaneous unfolding rate of cpN42 by ~100 times, making the WT and CP azurins unfold at similar rates. Our study demonstrates metal binding as a novel way to modulate the unfolding and folding rates of CPs compared to their WT counterparts. We hope our study increases the understanding of the effect of CP on the folding mechanism and energy landscape of metalloproteins.

Long-Range Charge Delocalization Mediates the Ultrafast Ligand-to-Metal Charge Transfer Dynamics at the Cu2+-Active Site in Azurin.

The blue color in metalloprotein azurin has traditionally been attributed to the intense cysteine-to-Cu2+ ligand-to-metal charge transfer transition centered at 628 nm. Although resonance Raman measurements of the Cu2+ active site have implied that the LMCT transition electronically couples to the protein scaffold well beyond its primary metal-ligand coordination shell, the structural extent of this electronic coupling and visualization of the protein-mediated charge transfer dynamics have remained elusive. Here, using femtosecond broadband transient absorption and impulsive Raman spectroscopy, we provide direct evidence for a rapid relaxation between two distinct charge transfer states, having different spatial delocalization, within ∼300 fs followed by recombination of charges in subpicosecond time scales. We invoke the formation of a protein-centered radical cation, possibly Trp48 or a Phe residue, within 100 fs substantiating the long-range electronic coupling for the first time beyond the traditional copper active site. The Raman spectra of the excited CT state show the presence of protein-centric vibrations along with the vibrational modes assigned to the copper active site. Our results demonstrate a large delocalization length scale of the initially populated CT state, thereby highlighting the possibility of exploiting azurin photochemistry for energy conversion techniques.

Native Salt Bridges Are a Key Regulator of Ubiquitin's Mechanical Stability.

Although it is known that various intramolecular interactions determine protein mechanical stability, a detailed molecular-level understanding of the key regulators of protein mechanical stability is still lacking. Here, we present evidence for salt bridges in ubiquitin as important intramolecular interactions that can affect protein mechanical stability. Ubiquitin has two salt bridges: one relatively surface-exposed (SB1:K11-E34) and the other relatively buried (SB2:K27-D52). Ubiquitin is a reversible post-translational modifier and is stable mechanically (Favgu = 185 pN). On breaking SB1, the mechanical stability of ubiquitin is slightly enhanced (Favgu = 193 pN). In contrast, the mechanical stability significantly decreased upon breaking SB2 (Favgu = 158 pN). These results suggest that SB1 are SB2 are regulators of the mechanical stability of ubiquitin. Interestingly, the mechanical stability decreased further (Favgu = 145 pN) for the double salt bridge (DB) null variant. Monte Carlo simulations elucidate that the main regulating factor is the spontaneous unfolding rate constant (ku0), being the highest for the DB null variant followed by the SB2 null variant, and it remains unaltered for the SB1 null variant, while the native-to-transition-state distance (xu) remains unchanged. Our study provides mechanistic understanding on how two native salt bridges can independently regulate the mechanical stability in a protein, which has implications in designing protein-based robust biomaterials in the future.

Rational Design of Protein-Specific Folding Modifiers.

Protein-folding can go wrong in vivo and in vitro, with significant consequences for the living organism and the pharmaceutical industry, respectively. Here we propose a design principle for small-peptide-based protein-specific folding modifiers. The principle is based on constructing a "xenonucleus", which is a prefolded peptide that mimics the folding nucleus of a protein. Using stopped-flow kinetics, NMR spectroscopy, Förster resonance energy transfer, single-molecule force measurements, and molecular dynamics simulations, we demonstrate that a xenonucleus can make the refolding of ubiquitin faster by 33 ± 5%, while variants of the same peptide have little or no effect. Our approach provides a novel method for constructing specific, genetically encodable folding catalysts for suitable proteins that have a well-defined contiguous folding nucleus.

Disruption of desmosome function leads to increased centrosome clustering in 14-3-3γ-knockout cells with supernumerary centrosomes.

14-3-3 proteins are conserved, dimeric, acidic proteins that regulate multiple cellular pathways. Loss of either 14-3-3ε or 14-3-3γ leads to centrosome amplification. However, we find that while the knockout of 14-3-3ε leads to multipolar mitoses, the knockout of 14-3-3γ results in centrosome clustering and pseudo-bipolar mitoses. 14-3-3γ knockouts demonstrate compromised desmosome function and a decrease in keratin levels, leading to decreased cell stiffness and an increase in centrosome clustering. Restoration of desmosome function increased multipolar mitoses, whereas knockdown of either plakoglobin or keratin 5 led to decreased cell stiffness and increased pseudo-bipolar mitoses. These results suggest that the ability of the desmosome to anchor keratin filaments maintains cell stiffness, thus inhibiting centrosome clustering, and that phenotypes observed upon 14-3-3 loss reflect the dysregulation of multiple pathways.

Azurin-Derived Peptides: Comparison of Nickel- and Copper-Binding Properties.

Metalloproteins are an important class of proteins involved in metal uptake, transport, and electron-transfer reactions. Mimicking the active sites of these proteins through miniaturization is an active area of research with applications in biotechnology and medicine. Azurin is a 128-residue copper-binding cupredoxin protein involved in electron-transfer reactions. Previous studies have reported on the copper-binding-induced spectroscopic and structural properties of peptide loops (11 and 13 residues) from azurin. These azurin peptides exhibited novel stoichiometries. However, the underlying mechanism of fluorescence quenching upon copper binding remains to be understood, whether it is due to electron transfer, energy transfer, or both. Here, we report nickel-binding-associated spectroscopic and structural properties of the azurin peptides. They develop a ß-turn upon nickel binding as seen in circular dichroism and exhibit electronic transitions centered at 270 and 450 nm. Unlike copper, which exhibited 1:1 and 1:2 peptide:metal stoichiometries, nickel exhibited only a 1:1 stoichiometry. Tryptophan-containing peptides showed fluorescence quenching upon nickel binding, which is due to electron transfer. These results further suggest that the quenching in copper-bound peptides is also due to electron transfer, which could not be ascertained in previous studies. Overall, azurin peptides provide a platform for studying metal-induced structural and spectroscopic properties using transition-metal ions.

Applications of atomic force microscopy in modern biology.

Single-molecule force spectroscopy (SMFS) is an emerging tool to investigate mechanical properties of biomolecules and their responses to mechanical forces, and one of the most-used techniques for mechanical manipulation is the atomic force microscope (AFM). AFM was invented as an imaging tool which can be used to image biomolecules in sub-molecular resolution in physiological conditions. It can also be used as a molecular force probe for applying mechanical forces on biomolecules. In this brief review, we will provide exciting examples from recent literature which show how the advances in AFM have enabled us to gain deep insights into mechanical properties and mechanobiology of biomolecules. AFM has been applied to study mechanical properties of cells, tissues, microorganisms, viruses as well as biological macromolecules such as proteins. It has found applications in biomedical fields like cancer biology, where it has been used both in the diagnostic phases as well as drug discovery. AFM has been able to answer questions pertaining to mechanosensing by neurons, and mechanical changes in viruses during infection by the viral particles as well as the fundamental processes such as cell division. Fundamental questions related to protein folding have also been answered by SMFS like determination of energy landscape properties of variety of proteins and their correlation with their biological functions. A multipronged approach is needed to diversify the research, as a combination with optical spectroscopy and computer-based steered molecular dynamic simulations along with SMFS can help us gain further insights into the field of biophysics and modern biology.

Role of Ligand Binding Site in Modulating the Mechanical Stability of Proteins with ß-Grasp Fold.

Despite many studies on ligand-modulated protein mechanics, a comparative analysis of the role of ligand binding site on any specific protein fold is yet to be made. In this study, we explore the role of ligand binding site on the mechanical properties of ß-grasp fold proteins, namely, ubiquitin and small ubiquitin related modifier 1 (SUMO1). The terminal segments directly connected through hydrogen bonds constitute the ß-clamp geometry (or mechanical clamp), which confers high mechanical resilience to the ß-grasp fold. Here, we study ubiquitin complexed with CUE2-1, a ubiquitin-binding domain (UBD) from yeast endonuclease protein Cue2, using a combination of single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations. Our study reveals that CUE2-1 does not alter the mechanical properties of ubiquitin, despite directly interacting with its ß-clamp. To explore the role of ligand binding site, we compare the mechanical properties of the ubiquitin/CUE2-1 complex with that of previously studied SUMO1/S12, another ß-grasp protein complex, using SMD simulations. Simulations on the SUMO1/S12 complex corroborate previous experimentally observed enhancement in the mechanical stability of SUMO1, even though S12 binds away from the ß-clamp. Differences in ligand binding-induced structural impact at the transition state of the two complexes explain the differences in ligand modulated protein mechanics. Contrary to previous reports, our study demonstrates that direct binding of ligands to the mechanical clamp does not necessarily alter the mechanical stability of ß-grasp fold proteins. Rather, binding interactions away from the clamp can reinforce protein stability provided by the ß-grasp fold. Our study highlights the importance of binding site and binding modes of ligands in modulating the mechanical stability of ß-grasp fold proteins.

An evolutionary non-conserved motif in Helicobacter pylori arginase mediates positioning of the loop containing the catalytic residue for catalysis.

The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (-153ESEEKAWQKLCSL165-) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122-Trp159-Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more significant, indicating they are crucial to function. Our data suggest that the Lys57-Glu155-Ser152 interaction influences the positioning of the loop containing the catalytic His133 so that this His can participate in catalysis, thereby providing a mechanistic understanding into the role of this motif in catalytic function. Lys57 was also found only in the arginases of other Helicobacter gastric pathogens. Based on the non-conserved motif, we found a new molecule, which specifically inhibits this enzyme. Thus, the present study not only provides a molecular basis into the role of this motif in function, but also offers an opportunity for the design of inhibitors with greater efficacy.

The unfolding transition state of ubiquitin with charged residues has higher energy than that with hydrophobic residues.

The native-state structure and folding pathways of a protein are encoded in its amino acid sequence. Ubiquitin, a post-translational modifier, primarily noted for its role in intracellular protein degradation, has two salt bridges: one relatively exposed (SB1:K11-E34) and the other relatively buried (SB2:K27-D52). Here, we study the role of hydrophobic interactions and sequence specificity in protein folding, by mutating the salt-bridge residues in ubiquitin with hydrophobic residues. Equilibrium chemical denaturation using GdnHCl shows that the SB1 null variant is thermodynamically stabilised whereas the SB2 null variant is destabilised only slightly. The thermodynamic stability of the double salt-bridge (DB) null variant is an additive effect of the individual salt bridges. Kinetic experiments show that all the salt-bridge null variants fold through a more stable intermediate with relatively faster folding rates than the wild-type. The SB2 null variant has a highly stabilised unfolding transition state (TS) and a slightly destabilised native state, leading to its kinetic instability, whereas the kinetic stability of the SB1 null variant is not compromised as its TS and native state are stabilized to a similar extent. The TS stabilisation is also additive for the DB null variant, which has the most stabilised TS and high kinetic instability. Our results underscore the importance of kinetic stability in optimising the protein energy landscape. Our study establishes the fact that the TSs can be stabilized by hydrophobic residues in the place of buried charged residues. It further highlights the role of charged residues in the protein interior in dictating the folding pathway.

A new tension induction paradigm unravels tissue response and the importance of E-cadherin in the developing epidermis.

The epidermis, being the outermost epithelial layer in metazoans, experiences multiple external and self-generated mechanical stimuli. The tissue-scale response to these mechanical stresses has been actively studied in the adult stratified epidermis. However, the response of the developing bi-layered epidermis to differential tension and its molecular regulation has remained poorly characterised. Here we report an oil injection based method, which in combination with atomic force microscopy (AFM), allows manipulation as well as estimation of tension in the developing epidermis. Our results show that the injection of mineral oil into the brain ventricle of developing zebrafish embryos stretches the overlying epidermis. The epidermal tension increases linearly with the injected volume of oil and the injection of 14-17 nL oil results in a two-fold increase in epidermal tension. This increase in epidermal tension is sufficient to elicit a physiological response characterised by temporal changes in the cell cross-sectional area and an increase in cell proliferation. Our data further indicate that the depletion of E-cadherin in the epidermis is detrimental for tissue integrity under increased mechanical stress. The application of this experimental paradigm in a genetically tractable organism such as zebrafish can be useful in uncovering mechanisms of tension sustenance in the developing epidermis.

Variance of Atomic Coordinates as a Dynamical Metric to Distinguish Proteins and Protein-Protein Interactions in Molecular Dynamics Simulations.

Protein dynamics is a manifestation of the complex trajectories of these biomolecules on a multidimensional rugged potential energy surface (PES) driven by thermal energy. At present, computational methods such as atomistic molecular dynamics (MD) simulations can describe thermal protein conformational changes in fully solvated environments over millisecond timescales. Despite these advances, a quantitative assessment of protein dynamics remains a complicated topic, intricately linked to issues such as sampling convergence and the identification of appropriate reaction coordinates/structural features to describe protein conformational states and motions. Here, we present the cumulative variance of atomic coordinate fluctuations (CVCF) along trajectories as an intuitive PES sensitive metric to assess both the extent of sampling and protein dynamics captured in MD simulations. We first examine the sampling problem in model one- (1D) and two-dimensional (2D) PES to demonstrate that the CVCF when traced as a function of the sampling variable (time in MD simulations) can identify local and global equilibria. Further, even far from global equilibrium, a situation representative of standard MD trajectories of proteins, the CVCF can distinguish different PES and therefore resolve the resultant protein dynamics. We demonstrate the utility of our CVCF analysis by applying it to distinguish the dynamics of structurally homologous proteins from the ubiquitin family (ubiquitin, SUMO1, SUMO2) and ubiquitin protein-protein interactions. Our CVCF analysis reveals that differential side-chain dynamics from the structured part of the protein (the conserved ß-grasp fold) present distinct protein PES to distinguish ubiquitin from SUMO isoforms. Upon binding to two functionally distinct protein partners (UBCH5A and UEV), intrinsic ubiquitin dynamics changes to reflect the binding context even though the two proteins have similar binding modes, which lead to negligible (sub-angstrom scale) structural changes.

Experimental comparison of energy landscape features of ubiquitin family proteins.

Small ubiquitin-related modifiers (SUMO1 and SUMO2) are ubiquitin family proteins, structurally similar to ubiquitin, differing in terms of their amino acid sequence and functions. Therefore, they provide a great platform for investigating sequence-structure-stability-function relationship. Here, we used chemical denaturation in comparing the folding-unfolding pathways of the SUMO proteins with their structural homologue ubiquitin (UF45W-pseudo wild-type [WT] tryptophan variant) with structurally analogous tryptophan mutations (SUMO1 [S1F66W], SUMO2 [S2F62W]). Equilibrium denaturation studies report that ubiquitin is the most stable protein among the three. The observed denaturant-dependent folding rates of SUMOs are much lower than ubiquitin and primarily exhibit a two-state folding pathway unlike ubiquitin, which has a kinetic folding intermediate. We hypothesize that, as SUMO proteins start off as slow folders, they avoid stabilizing their folding intermediates and the presence of which might further slow-down their folding rates. The denaturant-dependent unfolding of ubiquitin is the fastest, followed by SUMO2, and slowest for SUMO1. However, the spontaneous unfolding rate constant is the lowest for ubiquitin (~40 times), and similar for SUMOs. This correlation between thermodynamic stability and kinetic stability is achieved by having different unfolding transition state positions with respect to the solvent-accessible surface area, as quantified by the Tanford ß u values: ubiquitin (0.42) > SUMO2 (0.20) > SUMO1 (0.16). The results presented here highlight the unique energy landscape features which help in optimizing the folding-unfolding rates within a structurally homologous protein family.

Insight into the Excitation-Dependent Fluorescence of Carbon Dots.

High quantum yield, photoluminescence tunability, and sensitivity to the environment are a few distinct trademarks that make carbon nanodots (CDs) interesting for fundamental research, with potential to replace the prevalent inorganic semiconductor quantum dots. Currently, application and fundamental understanding of CDs are constrained because it is difficult to make a quantitative comparison among different types of CDs simply because their photoluminescence properties are directly linked to their size distribution, the surface functionalization, the carbon core structures (graphitic or amorphous) and the number of defects. Herein, we report a facile one-step synthesis of mono-dispersed and highly fluorescent nanometre size CDs from a 'family' of glucose-based sugars. These CDs are stable in aqueous solutions with photoluminescence in the visible range. Our results show several common features in the family of CDs synthesized in that the fluorescence, in the visible region, is due to a weak absorption in the 300-400 nm from a heterogeneous population of fluorophores. Fluorescence quenching experiments suggest the existence of not only surface-exposed fluorophores but more importantly solvent inaccessible fluorophores present within the core of CDs. Interestingly, time-resolved fluorescence anisotropy experiments directly suggest that a fast exchange of excitation energy occurs that results in a homo-FRET based depolarization within 150 ps of excitation.

Mechanical Softening of a Small Ubiquitin-Related Modifier Protein Due to Temperature Induced Flexibility at the Core.

Despite the growing interest in the thermal softening of proteins, the mechanistic details of it are far from understood. ß-Grasp proteins have globular shape with compact structure and they are mechanically resilient. The ß-clamp or mechanical clamp in them formed by the interactions between the terminal ß-strands is generally associated with the protein mechanical resistance. Although previous studies showed that temperature can perturb the protein mechanical stability, the structural changes leading to the lowered mechanical resistance are not known. Here, we investigated the temperature dependent mechanical stability of small ubiquitin-related modifier 2 (SUMO2) using single-molecule force spectroscopy (SMFS) and the corresponding conformational changes using ensemble experiments. SMFS studies on the polyprotein of SUMO2 estimate a decrease in the spring constant of the protein from 4.50 to 1.35 N/m upon increasing the temperature from 5 to 45 °C. Interestingly, near-UV circular dichroism spectroscopy reveals a decrease in tertiary structure content while the overall secondary structure of the protein remains unchanged. Steady-state fluorescence and quenching studies on SUMO2 with a tryptophan mutation at the core (F60W) show that the nonpolar environment of the tryptophan is unchanged and the protein core is inaccessible to the bulk solvent, in the same temperature range. We attribute the thermal softening observed in atomic force microscopy (AFM) experiments to the reduction in tertiary structure of SUMO2. Our results provide evidence for the importance of the intramolecular interactions at the protein core along with the ß-clamp or mechanical clamp in providing the mechanical resistance as well as in modulating the protein stiffness.

Differences in the mechanical unfolding pathways of apo- and copper-bound azurins.

Metalloproteins carry out diverse biological functions including metal transport, electron transfer, and catalysis. At present, the influence of metal cofactors on metalloprotein stability is not well understood. Here, we report the mechanical stability and unfolding pathway of azurin, a cupredoxin family protein with ß-barrel topology and type I copper-binding centre. Single-molecule force spectroscopy (SMFS) experiments reveal 2-state and 3-state unfolding pathways for apo-azurin. The intermediate in the 3-state pathway occurs at an unfolding contour length of 7.5 nm from the native state. Steered molecular dynamics (SMD) simulations show that apo-azurin unfolds via a first transition state (TS) where ß2Β-ß8 and ß7-ß8 strand pairs rupture to form the intermediate, which subsequently unfolds by the collective rupture of remaining strands. SMFS experiments on holo-azurin exhibit an additional 4-state pathway besides the 2-state and 3-state pathways. The unfolding contour length leading to the first intermediate is 6.7 nm suggesting a sequestration of ~1 nm polypeptide chain length by the copper. SMD simulations reveal atomistic details of the copper sequestration and predict a combined ß4-ß7 pair and copper coordination sphere rupture to create the third TS in the 4-state pathway. Our systematic studies provide detailed mechanistic insights on modulation of protein mechanical properties by metal-cofactors.

Force Spectroscopy of the Plasmodium falciparum Vaccine Candidate Circumsporozoite Protein Suggests a Mechanically Pliable Repeat Region.

The most effective vaccine candidate of malaria is based on the Plasmodium falciparum circumsporozoite protein (CSP), a major surface protein implicated in the structural strength, motility, and immune evasion properties of the infective sporozoites. It is suspected that reversible conformational changes of CSP are required for infection of the mammalian host, but the detailed structure and dynamic properties of CSP remain incompletely understood, limiting our understanding of its function in the infection. Here, we report the structural and mechanical properties of the CSP studied using single-molecule force spectroscopy on several constructs, one including the central region of CSP, which is rich in NANP amino acid repeats (CSPrep), and a second consisting of a near full-length sequence without the signal and anchor hydrophobic domains (CSPΔHP). Our results show that the CSPrep is heterogeneous, with 40% of molecules requiring virtually no mechanical force to unfold (<10 piconewtons (pN)), suggesting that these molecules are mechanically compliant and perhaps act as entropic springs, whereas the remaining 60% are partially structured with low mechanical resistance (∼70 pN). CSPΔHP having multiple force peaks suggests specifically folded domains, with two major populations possibly indicating the open and collapsed forms. Our findings suggest that the overall low mechanical resistance of the repeat region, exposed on the outer surface of the sporozoites, combined with the flexible full-length conformations of CSP, may provide the sporozoites not only with immune evasion properties, but also with lubricating capacity required during its navigation through the mosquito and vertebrate host tissues. We anticipate that these findings would further assist in the design and development of future malarial vaccines.