Role of cofactors in metalloprotein folding

Metals are commonly found as natural constituents of proteins. Since many such metals can interact specifically with their corresponding unfolded proteins in vitro , cofactor-binding prior to polypeptide folding may be a biological path to active metalloproteins. By interacting with the unfolded polypeptide, the metal may create local structure that initiates and directs the polypeptide-folding process. Here, we review recent literature that addresses the involvement of metals in protein-folding reactions in vitro . To date, the best characterized systems are simple one such as blue-copper proteins, heme-binding proteins, iron-sulfur-cluster proteins and synthetic metallopeptides. Taken together, the available data demonstrates that metals can play diverse roles: it is clear that many cofactors bind before polypeptide folding and influence the reaction; yet, some do not bind until a well-structured active site is formed. The significance of characterizing the effects of metals on protein conformational changes is underscored by the many human diseases that are directly linked to anomalous protein-metal interactions.     

An approach to quality management in structural biology: biophysical selection of proteins for successful crystallization

Aggregation, incorrect folding and low stability are common obstacles for protein structure determination, and are often discovered at a very late state of protein production. In many cases, however, the reasons for failure to obtain diffracting crystals remain entirely unknown. We report on the contribution of systematic biophysical characterization to the success in structural determination of human proteins of unknown fold. Routine analysis using dynamic light scattering (DLS), differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) was employed to evaluate fold and stability of 263 purified protein samples (98 different human proteins). We found that FTIR-monitored temperature scanning may be used to detect incorrect folding and discovered a positive correlation between unfolding enthalpy measured with DSC and the size of small, globular proteins that may be used to estimate the quality of protein preparations. Furthermore, our work establishes that the risk of aggregation during concentration of proteins may be reduced through DLS monitoring. In summary, our study demonstrates that biophysical characterization provides an ideal tool to facilitate quality management for structural biology and many other areas of biological research.     

Over‐expression of secreted proteins from mammalian cell lines

Secreted mammalian proteins require the development of robust protein over‐expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large‐scale growth of cells in a variety of formats.     

Role of copper in folding and stability of cupredoxin-like copper-carrier protein CopC

CopC is a periplasmic copper carrier that, in contrast to cytoplasmic copper chaperones, has a beta-barrel fold and two metal-binding sites distinct for Cu(II) and Cu(I). The copper sites are located in each end of the molecule: the Cu(I) site involves His and Met coordination whereas the Cu(II) site consists of charged residues. To reveal biophysical properties of this protein, we have explored the effects of the cofactors on CopC unfolding in vitro. We demonstrate that Cu(II) coordination affects both protein stability and unfolding pathway, whereas Cu(I) has only a small effect on stability. Apo-CopC unfolds in a two-state reaction between pH 4 and 7.5 with maximal stability at pH 6. In contrast, Cu(II)-CopC unfolds in a three-state reaction at pH6 that involves a partly folded intermediate that retains Cu(II). This intermediate exhibits high thermal and chemical stability. Unique energetic and structural properties of different metalated CopC forms may help facilitate metal transport to many partners in vivo.     

Mechanism of the kinetically-controlled folding reaction of subtilisin

Like many secreted proteases, subtilisin is kinetically stable in the mature form but unable to fold without assistance from its prodomain. The existence of high kinetic barriers to folding challenges many widely accepted ideas, namely, the thermodynamic determination of native structure and the sufficiency of thermodynamic stability to determine a pathway. The purpose of this article is to elucidate the physical nature of the kinetic barriers to subtilisin folding and to show how the prodomain overcomes these barriers. To address these questions, we have studied the bimolecular folding reaction of the subtilisin prodomain and a series of subtilisin mutants, which were designed to explore the steps in the folding reaction. Our analysis shows that inordinately slow folding of the mature form of subtilisin results from the accrued effects of two slow and sequential processes: (1) the formation of an unstable and topologically challenged intermediate and (2) the proline-limited isomerization of the intermediate to the native state. The low stability of nascent folding intermediates results in part from subtilisin's high dependence on metal binding for stability. Native subtilisin is thermodynamically unstable in the absence of bound metals. Because the two metal binding sites are formed late in folding, however, they contribute little to the stability of folding intermediates. The formation of productive folding intermediates is further hindered by the topological challenge of forming a left-handed crossover connection between beta-strands S2 and S3. This connection is critical to propagate the folding reaction. In the presence of the prodomain, folding proceeds through one major intermediate, which is stabilized by prodomain binding, independent of metal concentration and proline isomerization state. The prodomain also catalyzes the late proline isomerizations needed to form metal site B. Rate-limiting proline isomerization is common in protein folding, but its effect in slowing subtilisin folding is amplified because of the instability of the intermediate and an apparent need for simultaneous isomerization of multiple prolines in order to create metal site B. Thus, the kinetically controlled folding reaction of subtilisin, although unusual, is explained by the accrued effects of events found in other proteins.     

Trace metals in the brain: allosteric modulators of ligand-gated receptor channels,the case of ATP-gated P2X receptors

Zinc and copper are indispensable trace metals for life with a recognized role as catalysts in enzyme actions. We now review evidence supporting the role of trace metals as novel allosteric modulators of ionotropic receptors: a new and fundamental physiological role for zinc and copper in neuronal and brain excitability. The review is focussed on ionotropic receptor channels including nucleotide receptors, in particular the P2X receptor family. Since zinc and copper are stored within synaptic vesicles in selected brain regions, and released to the synaptic cleft upon electrical nerve ending depolarization, it is plausible that zinc and copper reach concentrations in the synapse that profoundly affect ligand-gated ionic channels, including the ATP-gated currents of P2X receptors. The identification of key P2X receptor amino acids that act as ligands for trace metal coordination, carves the structural determinants underlying the allosteric nature of the trace metal modulation. The recognition that the identified key residues such as histidines, aspartic and glutamic acids or cysteines in the extracellular domain are different for each P2X receptor subtype and may be different for each metal, highlights the notion that each P2X receptor subtype evolved independent strategies for metal coordination, which form upon the proper three-dimensional folding of the receptor channels. The understanding of the molecular mechanism of allosteric modulation of ligand-operated ionic channels by trace metals is a new contribution to metallo-neurobiology.     

Bacterial ATP-driven transporters of transition metals: physiological roles, mechanisms of action, and roles in bacterial virulence

Maintaining adequate intracellular levels of transition metals is fundamental to the survival of all organisms. While all transition metals are toxic at elevated intracellular concentrations, metals such as iron, zinc, copper, and manganese are essential to many cellular functions. In prokaryotes, the concerted action of a battery of membrane-embedded transport proteins controls a delicate balance between sufficient acquisition and overload. Representatives from all major families of transporters participate in this task, including ion-gradient driven systems and ATP-utilizing pumps. P-type ATPases and ABC transporters both utilize the free energy of ATP hydrolysis to drive transport. Each of these very different families of transport proteins has a distinct role in maintaining transition metal homeostasis: P-type ATPases prevent intracellular overloading of both essential and toxic metals through efflux while ABC transporters import solely the essential ones. In the present review we discuss how each system is adapted to perform its specific task from mechanistic and structural perspectives. Despite the mechanistic and structural differences between P-type ATPases and ABC transporters, there is one important commonality: in many clinically relevant bacterial pathogens, transporters of transition metals are essential for virulence. Here we present several such examples and discuss how these may be exploited for future antibacterial drug development.     

Effects of macromolecular crowding agents on protein folding in vitro and in silico

Proteins fold and function inside cells which are environments very different from that of dilute buffer solutions most often used in traditional experiments. The crowded milieu results in excluded-volume effects, increased bulk viscosity and amplified chances for inter-molecular interactions. These environmental factors have not been accounted for in most mechanistic studies of protein folding executed during the last decades. The question thus arises as to how these effects—present when polypeptides normally fold in vivo—modulate protein biophysics. To address excluded volume effects, we use synthetic macromolecular crowding agents, which take up significant volume but do not interact with proteins, in combination with strategically selected proteins and a range of equilibrium and time-resolved biophysical (spectroscopic and computational) methods. In this review, we describe key observations on macromolecular crowding effects on protein stability, folding and structure drawn from combined in vitro and in silico studies. As expected based on Minton’s early predictions, many proteins (apoflavodoxin, VlsE, cytochrome c, and S16) became more thermodynamically stable (magnitude depends inversely on protein stability in buffer) and, unexpectedly, for apoflavodoxin and VlsE, the folded states changed both secondary structure content and, for VlsE, overall shape in the presence of macromolecular crowding. For apoflavodoxin and cytochrome c, which have complex kinetic folding mechanisms, excluded volume effects made the folding energy landscapes smoother (i.e., less misfolding and/or kinetic heterogeneity) than in buffer.     

Understanding the folding of GFP using biophysical techniques

Green fluorescent protein (GFP) and its many variants are probably the most widely used proteins in medical and biological research, having been extensively engineered to act as markers of gene expression and protein localization, indicators of protein–protein interactions and biosensors. GFP first folds, before it can undergo an autocatalytic cyclization and oxidation reaction to form the chromophore, and in many applications the folding efficiency of GFP is known to limit its use. Here, we review the recent literature on protein engineering studies that have improved the folding properties of GFP. In addition, we discuss in detail the biophysical work on the folding of GFP that is beginning to reveal how this large and complex structure forms.     

Transition metal homeostasis: from yeast to human disease

Transition metal ions are essential nutrients to all forms of life. Iron, copper, zinc, manganese, cobalt and nickel all have unique chemical and physical properties that make them attractive molecules for use in biological systems. Many of these same properties that allow these metals to provide essential biochemical activities and structural motifs to a multitude of proteins including enzymes and other cellular constituents also lead to a potential for cytotoxicity. Organisms have been required to evolve a number of systems for the efficient uptake, intracellular transport, protein loading and storage of metal ions to ensure that the needs of the cells can be met while minimizing the associated toxic effects. Disruptions in the cellular systems for handling transition metals are observed as a number of diseases ranging from hemochromatosis and anemias to neurodegenerative disorders including Alzheimer??s and Parkinson??s disease. The yeast Saccharomyces cerevisiae has proved useful as a model organism for the investigation of these processes and many of the genes and biological systems that function in yeast metal homeostasis are conserved throughout eukaryotes to humans. This review focuses on the biological roles of iron, copper, zinc, manganese, nickel and cobalt, the homeostatic mechanisms that function in S. cerevisiae and the human diseases in which these metals have been implicated.     

Understanding the folding of GFP using biophysical techniques

Green fluorescent protein (GFP) and its many variants are probably the most widely used proteins in medical and biological research, having been extensively engineered to act as markers of gene expression and protein localization, indicators of protein-protein interactions and biosensors. GFP first folds, before it can undergo an autocatalytic cyclization and oxidation reaction to form the chromophore, and in many applications the folding efficiency of GFP is known to limit its use. Here, we review the recent literature on protein engineering studies that have improved the folding properties of GFP. In addition, we discuss in detail the biophysical work on the folding of GFP that is beginning to reveal how this large and complex structure forms.     

A high-performance liquid chromatography method for determining transition metal content in proteins

Transition metals are common components of cellular proteins and the detailed study of metalloproteins necessitates the identification and quantification of bound metal ions. Screening for metals is also an informative step in the initial characterization of the numerous unknown and unclassified proteins now coming through the proteomic pipeline. We have developed a high-performance liquid chromatography method for the quantitative determination of the most prevalent biological transition metals: manganese, iron, cobalt, nickel, copper, and zinc. The method is accurate and simple and can be adapted for automated high-throughput studies. The metal analysis involves acid hydrolysis to release the metal ions into solution, followed by ion separation on a mixed-bead ion-exchange column and absorbance detection after postcolumn derivatization with the metallochromic indicator 4-(2-pyridylazo)resorcinol. The potential interferences by common components of protein solutions were investigated. The metal content of a variety of metalloproteins was analyzed and the data were compared to data obtained from inductively coupled plasma-atomic emission spectroscopy. The sensitivity of the assay allows for the detection of 0.1-0.8 nmol, depending on the metal. The amount of protein required is governed by the size of the protein and the fraction of protein with metal bound. For routine analysis 50 microg was used but for many proteins 10 microg would be sufficient. The advantages, disadvantages, and possible applications of this method are discussed.     

Copper and nickel binding in multi-histidinic peptide fragments

Multi-histidinic peptides have been investigated for Cu(II) and Ni(II) binding. We present spectroscopic evidence that, at low pH and from sub-stoichiometric to stoichiometric amounts of metals, macrochelate and multi-histidinic Cu(II) and Ni(II) complexes form; but, from neutral pH and above, both copper and nickel bind to individual histidine residues. NMR, EPR, UV–Visible (UV–Vis) and UV–Visible CD spectroscopy were used to understand about the variety of complexes obtained at low pHs, where amide deprotonation and coordination is unfavoured. A structural transition between two coordination geometries, as the pH is raised, was observed. Metal binds to N_δ of histidine imidazole when main-chain coordination is involved and coordinates via N_ε under mildly acidic conditions and sub-stoichiometric amounts of metals. From EPR results a distortion from planarity has been evidenced for the Cu(II) multi-histidinic macrochelate systems, which may be relevant to biological activity. The behaviour of our peptides was comparable to the pH dependent effect on Cu(II) coordination observed in octapeptide repeat domain in prion proteins and in amyloid precursor peptides involved in Alzheimer’s disease. Changes in pH and levels of metal affect coordination mode and can have implications for the affinity, folding and redox properties of proteins and peptide fragments.     

What is the role of thermodynamics on protein stability?

The most challenging and emerging field of biotechnology is the tailoring of proteins to attain the desired characteristic properties. In order to increase the stability of proteins and to study the function of proteins, the mechanism by which proteins fold and unfold should be known. It has been debated for a long time how exactly the linear form of a protein is converted into a stable 3-dimensional structure. The literature showed that many theories support the fact that protein folding is a thermodynamically controlled process. It is also possible to predict the mechanism of protein deactivation and stability to an extent from thermodynamic studies. This article reviewed various theories that have been proposed to explain the process of protein folding after its biosynthesis in ribosomes. The theories of the determination of the thermodynamic properties and the interpretation of thermodynamic data of protein stability are also discussed in this article.     

The six metal binding domains in human copper transporter,ATP7B: molecular biophysics and disease-causing mutations

Wilson Disease (WD) is a hereditary genetic disorder, which coincides with a dysfunctional copper (Cu) metabolism caused by mutations in ATP7B, a membrane-bound P_1B-type ATPase responsible for Cu export from hepatic cells. The N-terminal part (~ 600 residues) of the multi-domain 1400-residue ATP7B constitutes six metal binding domains (MBDs), each of which can bind a copper ion, interact with other ATP7B domains as well as with different proteins. Although the ATP7B’s MBDs have been investigated in vitro and in vivo intensively, it remains unclear how these domains modulate overall structure, dynamics, stability and function of ATP7B. The presence of six MBDs is unique to mammalian ATP7B homologs, and many WD causing missense mutations are found in these domains. Here, we have summarized previously reported in vitro biophysical data on the MBDs of ATP7B and WD point mutations located in these domains. Besides the demonstration of where the research field stands today, this review showcasts the need for further biophysical investigation about the roles of MBDs in ATP7B function. Molecular mechanisms of ATP7B are important not only in the development of new WD treatment but also for other aspects of human physiology where Cu transport plays a role.     

Co-expression for intracellular processing in microbial protein production

The biological activity of a recombinant protein is highly dependent on its biophysical properties including post-translational modifications, solubility, and stability. Production of active recombinant proteins requires careful design of the expression strategy and purification schemes. This is often achieved by proper modification of the target protein during and/or after protein synthesis in the host cells. Such co-translational or post-translational processing of recombinant proteins is typically enabled by co-expressing the required enzymes, folding chaperones, co-factors and/or processing enzymes in the host. Various applications of the co-expression technology in protein production are discussed in this review with representative examples described.     

Crystal structure of the Atx1 metallochaperone protein at 1.02 A resolution.

BACKGROUND: Metallochaperone proteins function in the trafficking and delivery of essential, yet potentially toxic, metal ions to distinct locations and particular proteins in eukaryotic cells. The Atx1 protein shuttles copper to the transport ATPase Ccc2 in yeast cells. Molecular mechanisms for copper delivery by Atx1 and similar human chaperones have been proposed, but detailed structural characterization is necessary to elucidate how Atx1 binds metal ions and how it might interact with Ccc2 to facilitate metal ion transfer. RESULTS: The 1.02 A resolution X-ray structure of the Hg(II) form of Atx1 (HgAtx1) reveals the overall secondary structure, the location of the metal-binding site, the detailed coordination geometry for Hg(II), and specific amino acid residues that may be important in interactions with Ccc2. Metal ion transfer experiments establish that HgAtx1 is a functional model for the Cu(I) form of Atx1 (CuAtx1). The metal-binding loop is flexible, changing conformation to form a disulfide bond in the oxidized apo form, the structure of which has been solved to 1.20 A resolution. CONCLUSIONS: The Atx1 structure represents the first structure of a metallochaperone protein, and is one of the largest unknown structures solved by direct methods. The structural features of the metal-binding site support the proposed Atx1 mechanism in which facile metal ion transfer occurs between metal-binding sites of the diffusible copper-donor and membrane-tethered copper-acceptor proteins. The Atx1 structural motif represents a prototypical metal ion trafficking unit that is likely to be employed in a variety of organisms for different metal ions.     

Determinants for Simultaneous Binding of Copper and Platinum to Human Chaperone Atox1: Hitchhiking not Hijacking

Cisplatin (CisPt) is an anticancer agent that has been used for decades to treat a variety of cancers. CisPt treatment causes many side effects due to interactions with proteins that detoxify the drug before reaching the DNA. One key player in CisPt resistance is the cellular copper-transport system involving the uptake protein Ctr1, the cytoplasmic chaperone Atox1 and the secretory path ATP7A/B proteins. CisPt has been shown to bind to ATP7B, resulting in vesicle sequestering of the drug. In addition, we and others showed that the apo-form of Atox1 could interact with CisPt in vitro and in vivo. Since the function of Atox1 is to transport copper (Cu) ions, it is important to assess how CisPt binding depends on Cu-loading of Atox1. Surprisingly, we recently found that CisPt interacted with Cu-loaded Atox1 in vitro at a position near the Cu site such that unique spectroscopic features appeared. Here, we identify the binding site for CisPt in the Cu-loaded form of Atox1 using strategic variants and a combination of spectroscopic and chromatographic methods. We directly prove that both metals can bind simultaneously and that the unique spectroscopic signals originate from an Atox1 monomer species. Both Cys in the Cu-site (Cys12, Cys15) are needed to form the di-metal complex, but not Cys41. Removing Met10 in the conserved metal-binding motif makes the loop more floppy and, despite metal binding, there are no metal-metal electronic transitions. In silico geometry minimizations provide an energetically favorable model of a tentative ternary Cu-Pt-Atox1 complex. Finally, we demonstrate that Atox1 can deliver CisPt to the fourth metal binding domain 4 of ATP7B (WD4), indicative of a possible drug detoxification mechanism.     

Assessing the causes and consequences of co-polymerization in amyloid formation

How, and why, different proteins form amyloid fibrils is most often studied in vitro using a single purified protein sequence. However, many amyloid diseases involve co-aggregation of different protein species, including proteins with/without post-translational modifications (e.g., different strains of PrP), proteins of different length (e.g., β₂-microglobulin and ΔN6, Aβ40, and Aβ42), sequence variants (e.g., Aβ and Aβ_ARC), and proteins from different organisms (e.g., bovine PrP and human PrP). The consequences of co-aggregation of different proteins upon the structure, stability, species transmission and toxicity of the resulting amyloid aggregates is discussed here, including the role of co-aggregation in expanding the repertoire of oligomeric and fibrillar structures and how this can affect their biological and biophysical properties.