The Route from the Folded to the Amyloid State: Exploring the Potential Energy Surface of a Drug-Like Miniprotein

The amyloid formation of the folded segment of a variant of Exenatide (a marketed drug for type-2 diabetes mellitus) was studied by electronic circular dichroism (ECD) and NMR spectroscopy. We found that the optimum temperature for E5 protein amyloidosis coincides with body temperature and requires well below physiological salt concentration. Decomposition of the ECD spectra and its barycentric representation on the folded-unfolded-amyloid potential energy surface allowed us to monitor the full range of molecular transformation of amyloidogenesis. We identified points of no return (e.g.; T=37 °C, pH 4.1, c_E5=250 μm , c_NaCl=50 mm , t>4–6 h) that will inevitably gravitate into the amyloid state. The strong B-type far ultraviolet (FUV)-ECD spectra and an unexpectedly strong near ultraviolet (NUV)-ECD signal (Θ_{≈275–285 nm}) indicate that the amyloid phase of E5 is built from monomers of quasi-elongated backbone structure (φ≈−145°, ψ≈+145°) with strong interstrand Tyr↔Trp interaction. Misfolded intermediates and the buildup of “toxic” early-stage oligomers leading to self-association were identified and monitored as a function of time. Results indicate that the amyloid transition is triggered by subtle misfolding of the α-helix, exposing aromatic and hydrophobic side chains that may provide the first centers for an intermolecular reorganization. These initial clusters provide the spatial closeness and sufficient time for a transition to the β-structured amyloid nucleus, thus the process follows a nucleated growth mechanism.     

Chemical Perturbation of Oncogenic Protein Folding: from the Prediction of Locally Unstable Structures to the Design of Disruptors of Hsp90–Client Interactions

Protein folding quality control in cells requires the activity of a class of proteins known as molecular chaperones. Heat shock protein-90 (Hsp90), a multidomain ATP driven molecular machine, is a prime representative of this family of proteins. Interactions between Hsp90, its co-chaperones, and client proteins have been shown to be important in facilitating the correct folding and activation of clients. Hsp90 levels and functions are elevated in tumor cells. Here, we computationally predict the regions on the native structures of clients c-Abl, c-Src, Cdk4, B-Raf and Glucocorticoid Receptor, that have the highest probability of undergoing local unfolding, despite being ordered in their native structures. Such regions represent potential ideal interaction points with the Hsp90-system. We synthesize mimics spanning these regions and confirm their interaction with partners of the Hsp90 complex (Hsp90, Cdc37 and Aha1) by Nuclear Magnetic Resonance (NMR). Designed mimics selectively disrupt the association of their respective clients with the Hsp90 machinery, leaving unrelated clients unperturbed and causing apoptosis in cancer cells. Overall, selective targeting of Hsp90 protein–protein interactions is achieved without causing indiscriminate degradation of all clients, setting the stage for the development of therapeutics based on specific chaperone:client perturbation.     

Reduction of the Search Space for the Folding of Proteins at the Level of Formation and Assembly of Secondary Structures: A New View on the Solution of Levinthal′s Paradox

The complete volume of the protein conformation space is, by many orders of magnitude, smaller at the level of secondary structure elements than that at the level of amino acid residues; the latter, according to Levinthal′s estimate, scales approximately as 10^{2 L}, with L being the number of residues in the chain, whereas the former, as demonstrated in this paper, scales no faster than ～L^N, with N being the number of the secondary structure elements, which is approximately equal to L/15. This drastic decrease in the exponent (L/15 instead of 2 L) explains why sampling of the conformation space does not contradict the ability of the protein chain to find its most stable fold.     

Screening the molecular surface of human anticoagulant protein C: A search for interaction sites

Protein C (PC), a 62 kDa multi-modular zymogen, is activated to an anticoagulant serine protease (activated PC or APC) by thrombin bound to thrombomodulin on the surface of endothelial cells. PC/APC interacts with many proteins and the characterisation of these interactions is not trivial. However, molecular modelling methods help to study these complex biological processes and provide basis for rational experimental design and interpretation of the results. PC/APC consists of a Gla domain followed by two EGF modules and a serine protease domain. In this report, we present two structural models for full-length APC and two equivalent models for full-length PC, based on the X-ray structures of Gla-domainless APC and of known serine protease zymogens. The overall elongated shape of the models is further cross-validated using size exclusion chromatography which allows evaluation of the Stokes radius (r_s for PC = 33.15 Å r_s for APC = 34.19 Å), frictional ratio and axial ratio. We then propose potential binding sites at the surface of PC/APC using surface hydrophobicity as a determinant of the preferred sites of intermolecular recognition. Most of the predicted binding sites are consistent with previously reported experimental data, while some clusters highlight new regions that should be involved in protein-protein interactions.     

An Off‐Pathway Folding Intermediate of an Acyl Carrier Protein Domain Coexists with the Folded and Unfolded States under Native Conditions

A protein can exist in multiple states under native conditions and those states with low populations are often critical to biological function and self‐assembly. To investigate the role of the minor states of an acyl carrier protein, NMR techniques were applied to determine the number of minor states and characterize their structures and kinetics. The acyl carrier protein from Micromonospora echinospora was found to exist in one major folded state (95.2 %), one unfolded state (4.1 %), and one intermediate state (0.7 %) under native conditions. The three states are in dynamic equilibrium and the intermediate state very likely adopts a native‐like structure and is an off‐pathway folding product. The intermediate state may mediate the formation of oligomers in vitro and play an important role in the recognition of partner enzymes in vivo.     

Decoding the components of dynamics in three‐domain proteins

In this study, we examine the feasibility and limitations of describing the motional behavior of three‐domain proteins in which the domains are linearly connected. In addition to attempting the determination of the internal and overall reorientational correlation times, we investigate the existence of correlations in the motions between the three domains. Since in linearly arranged three‐domain proteins, there are typically no experimental data that can directly report on motional correlation between the first and the third domain, we address this question by dynamics simulations. Two limiting cases occur: (1) for weak repulsive potentials and (2) when strong repulsive potentials are applied between sequential domains. The motions of the terminal domains become correlated in the case of strong interdomain repulsive potentials when these potentials do not allow the angle between the sequential domains to be smaller than about 60°. Using the model‐free (MF) and extended MF formalisms of Lipari and Szabo, we find that the motional behavior can be separated into two components; the first component represents the concerted overall motion of the three domains, and the second describes the independent component of the motion of each individual domain. We find that this division of the motional behavior of the protein is maintained only when their timescales are distinct and can be made when the angles between sequential domains remain between 60° and 160°. In this work, we identify and quantify interdomain motional correlations. © 2013 Wiley Periodicals, Inc.     

Radiation Damage and Racemic Protein Crystallography Reveal the Unique Structure of the GASA/Snakin Protein Superfamily

Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63‐residue member of this family, snakin‐1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin‐1, determined by a novel combination of racemic protein crystallization and radiation‐damage‐induced phasing (RIP), is reported. Racemic crystals of snakin‐1 and quasi‐racemic crystals incorporating an unnatural 4‐iodophenylalanine residue were prepared from chemically synthesized d ‐ and l ‐proteins. Breakage of the C?I bonds in the quasi‐racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces.     

Possibilities and Limitations of Different Separation Techniques for the Analysis of the Protein Corona

One of the biggest challenges in the field of nanomedicine is the adsorption of biomolecules on the nanomaterial upon contact with a biological medium. The interactions of the resulting protein corona are essential for their behavior in a biological system. Thus, it is now commonly accepted that understanding the formation and consequently understanding the influence of the protein corona on the biological response is crucial. However, the outcome of the protein corona characterization cannot easily be compared between different studies and techniques, since many different sample preparation procedures exist that are suitable for different materials or methods. Depending on the applied procedure, the nanomaterial–protein system will be altered in a certain way, so that it is necessary to consider the individual influence on the protein corona. Accordingly, the aim of this Minireview is to give an overview of the applied sample preparation methods for the analysis of the protein corona and to evaluate their influence on the outcome of the results especially with regard to the introduced terms “soft” and “hard protein corona”. Special focus will be placed on the comparison of the most commonly used techniques such as centrifugation, magnetic, and chromatographic separation.     

Flexible Folding: Disulfide-Containing Peptides and Proteins Choose the Pathway Depending on the Environments

In the last few decades, development of novel experimental techniques, such as new types of disulfide (SS)-forming reagents and genetic and chemical technologies for synthesizing designed artificial proteins, is opening a new realm of the oxidative folding study where peptides and proteins can be folded under physiologically more relevant conditions. In this review, after a brief overview of the historical and physicochemical background of oxidative protein folding study, recently revealed folding pathways of several representative peptides and proteins are summarized, including those having two, three, or four SS bonds in the native state, as well as those with odd Cys residues or consisting of two peptide chains. Comparison of the updated pathways with those reported in the early years has revealed the flexible nature of the protein folding pathways. The significantly different pathways characterized for hen-egg white lysozyme and bovine milk α-lactalbumin, which belong to the same protein superfamily, suggest that the information of protein folding pathways, not only the native folded structure, is encoded in the amino acid sequence. The application of the flexible pathways of peptides and proteins to the engineering of folded three-dimensional structures is an interesting and important issue in the new realm of the current oxidative protein folding study.     

Inversion of the Side‐Chain Stereochemistry of Indvidual Thr or Ile Residues in a Protein Molecule: Impact on the Folding,Stability, and Structure of the ShK Toxin

ShK toxin is a cysteine‐rich 35‐residue protein ion‐channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side‐chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo‐Thr or an allo‐Ile residue. The three allo‐Thr or allo‐Ile‐containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi‐racemic X‐ray crystallography and were similar to wild‐type ShK. All three ShK analogues retained ion‐channel blocking activity.     

Maltose-binding Protein Improving the Crystallizability of C2 Domain of Human Coagulation Factor V

Human coagulation Factor V（FV）, together with Factor Xa, assembles to prothrombinase complex on activated cell surface, which converts prothrombin into thrombin, leading to fibrin deposition. The C2 domain of FV is believed to be a primary anchor for the assembly of pro- thrombinase on the cell surface, and was proposed as a target to intervene with pathological thrombotic events. We report here the crystal structure of the C2 domain of FV fused to maltose-binding protein（MBP）. The fusion tag of MBP is critical to generate the crystal for this study. There is no strong interaction between MBP and FVC2. The overall structure of FVC2 is similar to the previous FVC2 structures, suggesting the MBP fusion does not perturb the molecular structure of FVC2. This crystal form of FVC2 can be used for future study of molecular interaction between FVC2 and its inhibitors.     

Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.     

Amyloid Polymorphism in the Protein Folding and Aggregation Energy Landscape

Protein folding involves a large number of steps and conformations in which the folding protein samples different thermodynamic states characterized by local minima. Kinetically trapped on‐ or off‐pathway intermediates are metastable folding intermediates towards the lowest absolute energy minima, which have been postulated to be the natively folded state where intramolecular interactions dominate, and the amyloid state where intermolecular interactions dominate. However, this view largely neglects the rich polymorphism found within amyloid species. We review the protein folding energy landscape in view of recent findings identifying specific transition routes among different amyloid polymorphs. Observed transitions such as twisted ribbon→crystal or helical ribbon→nanotube, and forbidden transitions such helical ribbon?crystal, are discussed and positioned within the protein folding and aggregation energy landscape. Finally, amyloid crystals are identified as the ground state of the protein folding and aggregation energy landscape.     

Palladium in the Chemical Synthesis and Modification of Proteins

The field of site‐specific modification of proteins has drawn significant attention in recent years owing to its importance in various research areas such as the development of novel therapeutics and understanding the biochemical and cellular behaviors of proteins. The presence of a large number of reactive functional groups in the protein of interest and in the cellular environment renders modification at a specific site a highly challenging task. With the development of sophisticated chemical methodologies it is now possible to target a specific site of a protein with a desired modification, however, many challenges remain to be solved. In this context, transition metals in particular palladium‐mediated C−C bond‐forming and C−O bond‐cleavage reactions gained great interest owing to the unique catalytic properties of palladium. Palladium chemistry is being explored for protein modifications in vitro, on the cell surface, and within the cell. Very recently, palladium complexes have been applied for the rapid deprotection of several widely utilized cysteine protecting groups as well as in the removal of solubilizing tags to facilitate chemical protein synthesis. This Minireview highlights these advances and how the accumulated knowledge of palladium chemistry for small molecules is being impressively transferred to synthesis and modification of chemical proteins.