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To understand general aspects of stability and folding of c-type cytochromes, we have studied the folding characteristics of cytochrome c553 from Desulfovibrio vulgaris (Hildenborough). This cytochrome is structurally similar but lacks sequence homology to other heme proteins; moreover, it has an abnormally low reduction potential. Unfolding of oxidized and reduced cytochrome c553 by guanidine hydrochloride (GuHCl) was monitored by circular dichroism (CD) and Soret absorption; the same unfolding curves were obtained with both methods supporting that cytochrome c553 unfolds by an apparent two-state process. Reduced cytochrome c553 is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c553 is 100(20) mV more negative than that of the folded protein. In contrast to many other unfolded cytochrome c proteins, upon unfolding at pH 7.0 both oxidized and reduced heme in cytochrome c553 become high-spin. The lack of heme misligation in unfolded cytochrome c553 implies that its unfolded structure is less constrained than those of cytochromes c with low-spin, misligated hemes.

The unfolding of the CuA domain of cytochrome oxidase from the thermophilic bacterium Thermus thermophilus, induced by guanidine hydrochloride (GuHCl)1 at different temperatures, has been monitored by CD as well by electronic absorption (with the oxidized protein) and by fluorescence (with the reduced protein). The same unfolding curves were obtained with the different methods, providing evidence for a two-state model for the unfolding equilibrium. This was also supported by the shape of the unfolding equilibrium curves and by the observed refolding of the unfolded, oxidized protein on dilution of the denaturant. The oxidized protein cannot be unfolded by GuHCl at room temperature, and it was found to be thermally very stable as well, since, even in the presence of 7 M GuHCl, it is not fully unfolded until above 80 degrees C. For the reduced protein at room temperature, the unfolding equilibrium curve yielded a folding free energy of -65 kJ/mol. The corresponding value for the oxidized protein (-85 kJ/mol) could be estimated indirectly from a thermodynamic cycle connecting the folded and unfolded forms in both oxidation states and the known reduction potentials of the metal site in the folded and unfolded states; the potential is increased on unfolding, consistent with the higher folding stability of the oxidized form. The difference in folding stability between the oxidized and reduced proteins (20 kJ/mol) is exceptionally high, and this is ascribed to the unique structure of the dinuclear CuA site. The unfolded, reduced protein was found to refold partially on oxidation with ferricyanide.

The unfolding by guanidine hydrochloride (GuHCl) and the refolding on dilution of zinc and apoazurin have been monitored by far-UV circular dichroism (CD). With the native protein, the unfolding was followed by CD and optical absorption in the visible spectral region. With the zinc protein, the reversible unfolding has also been followed by tryptophan fluorescence and NMR. The zinc and Cu2+ metal ions remain associated with the protein in the unfolded state. When the unfolding of the native protein is followed by CD, the initial, reversible transition due to unfolding is followed by a slow change associated with the reduction of Cu2+ by the thiol group of the ligand Cys112. The unfolding of apoazurin displays two CD transitions, which evidence suggests represent different folding domains, the least stable one including the metal-binding site and the other one the rest of the beta-sheet structure. Both occur at a lower GuHCl concentration than the unfolding of the native protein. The CD titrations also demonstrate that zinc azurin has a lower stability than the copper protein. Unfolding of zinc azurin followed by tryptophan fluorescence occurs at a much lower GuHCl concentration than the CD changes, and NMR spectra show that there is no loss of secondary and tertiary structure at this concentration, whereas the CD-detected loss of secondary structure correlates with the NMR changes. Thus, the fluorescence change is ascribed to a small local perturbation of the structure around the single tryptophan residue. The differences in stability of the three forms of azurin are discussed in terms of the rack mechanism. A bound metal ion stabilizes the native fold, and this stabilization is larger for Cu(II) than for Zn(II), reflecting the higher affinity of the protein for Cu(II).

Experimental data for the unfolding of cytochrome c and azurin by guanidinium chloride (GuHCl) are used to construct free-energy diagrams for the folding of the oxidized and reduced proteins. With cytochrome c , the driving force for folding the reduced protein is larger than that for the oxidized form. Both the oxidized and the reduced folded forms of yeast cytochrome c are less stable than the corresponding states of the horse protein. Due to the covalent attachment of the heme and its fixed tetragonal coordination geometry, cytochrome c folding can be described by a two-state model. A thermodynamic cycle leads to an expression for the difference in self-exchange reorganization energies for the folded and unfolded proteins. The reorganization energy for electron exchange in the folded protein is approximately 0.5 eV smaller than that for a heme in aqueous solution. The finding that reduced azurin unfolds at lower GuHCl concentrations than the oxidized protein suggests that the coordination structure of copper is different in oxidized and reduced unfolded states: it is likely that the geometry of Cu I in the unfolded protein is linear or trigonal, whereas Cu II prefers to be tetragonal. The evidence indicates that protein folding lowers the azurin reorganization energy by roughly 1.7 eV relative to an aqueous Cu(1, 10-phenanthroline) 2 2+/+ reference system.

Proteins fold and function inside cell compartments that are full of other proteins, membranes and DNA. The crowded environment results in increased viscosity, excluded-volume effects and amplified opportunity for specific and non-specific inter-molecular interactions. These environmental factors are not accounted for in the mechanistic studies of protein folding and function that have been executed during the last decades. The question thus arises as for how these effects - present when polypeptides normally fold in vivo - modulate protein biophysical parameters? To take a step closer to understanding the in vivo scenario, we assess how crowded environments affect protein stability, structure and folding reactions in vitro. For this we use synthetic macromolecular crowding agents, which take up significant volume but do not interact with the target proteins, in combination with strategically selected proteins and a range of biophysical/spectroscopic methods. We have found that in the presence of macromolecular crowding in vitro, proteins become more thermodynamically stable (magnitude depends inversely on protein stability in buffer) and, protein-folded states may change both secondary structure content and overall shape. Finally, excluded volume effects may speed up folding kinetics and decrease the ruggedness of the folding energy landscape. Our findings demonstrate that excluded volume effects tune protein biophysical parameters: this is of mechanistic relevance since proteins have evolved to fold and function in crowded environments.

Protein folding is an essential requirement for most biological functions. Therefore, if folding of proteins can be specifically modulated, we have the power to tune/inhibit protein function, disease progression, and create novel analytical tools. This is an inter-disciplinary research project that aims to find small molecules that selectively modulate protein-folding reactions. The small molecules used in the study are bicyclic 2-pyridone derivatives . These compounds were originally designed to inhibit the assembly of bacterial pili, and are peptidomimetics that were directed to block the periplasmic chaperone PapD required for pilus biogenesis. A subgroup of these compounds are designed to inhibit the formation of curlin-based bacterial biofilms. Curlicides and pilicides thus are potential new antibiotics to fight bacterial infections. The curlicides bind the major curlin protein CsgA inhibiting its oligomerization into amyloid fibrils Moreover, some of these compounds have been found to inhibit β-amyloid peptide fibrillization. During the project we studied the interactions of some selected compounds with Pseudomas aureginosa azurin (beta-sheet model protein), Borrelia burgdorferi Vlse and human alpha-synuclein. We have found that two of the compounds we have studied, namely FNO75 and its fluorescently labelled analogue CB84 are interacting with VlsE directly, while binding to azurin occurs upon refolding of the protein after heat denaturation. Moreover, weve tested the effect of the compounds on alpha synuclein fibrillization. Surprisingly, it was found that FNO75, which is known to inhibit curli and beta-fibrillization was speeding up the aggregation of alpha synuclein.

Folding processes of simple proteins have been studied for years in test tubes. However, many proteins in the cells are more complex: some bind cofactors and others interact with other polypeptides in order to form their functional units. To obtain mechanistic information of the folding reactions of such proteins, we combine protein engineering in strategic model systems (e.g., a/b flavodoxin, b-barrel azurin, all-a VlsE) with a range of biophysical methods (e.g., circular dichroism, stopped-flow mixing, calorimetry). To take a step closer to the in vivo scenario, we assess how the crowdedness of the cell milieu affects protein biophysical parameters using synthetic macromolecular crowding agents (e.g., Ficoll, dextran) that take up significant volume but do not interact with the targets or have interfering spectroscopic signals. We have found that in the presence of macromolecular crowding in vitro, proteins can fold faster, become more thermodynamically stable and, surprisingly, the folded forms may change in terms of both secondary structure content and overall shape. Our discoveries imply that Nature may use excluded volume effects as a tool to tune protein biophysical parameters, and thereby function, in vivo.

The co-chaperonin protein 10 (cpn10) assists cpn60 in the folding of nonnative polypeptides in a wide range of organisms. All known cpn10 molecules are heptamers of seven identical subunits that are linked together by β-strand interactions at a large and flexible interface. Unfolding of human mitochondrial cpn10 in urea results in an unfolded heptameric state whereas GuHCl additions result in unfolded monomers. To address the role of specific interface residues in the assembly of cpn10 we prepared two point-mutated variants, in each case removing a hydrophobic residue positioned at the subunit-subunit interface. Replacing valine-100 with a glycine (Val100Gly cpn10) results in a wild-type-like protein with seven-fold symmetry although the thermodynamic stability is decreased and the unfolding processes in urea and GuHCl both result in unfolded monomers. In sharp contrast, replacing phenylalanine-8 with a glycine (Phe8Gly cpn10) results in a protein that has lost the ability to assemble. Instead, this protein exists mostly as unfolded monomers. We conclude that valine-100 is a residue important to adopt an oligomeric unfolded state but it does not affect the ability to assemble in the folded state. In contrast, phenylalanine-8 is required for both heptamer assembly and monomer folding and therefore this mutation results in unfolded monomers at physiological conditions. Despite the plasticity and large size of the cpn10 interface, our observations show that isolated interface residues can be crucial for both the retention of a heptameric unfolded structure and for subunit folding.

Background Pigment epithelium-derived factor (PEDF) has binding affinity for cell-surface receptors in retinoblastoma cells and for glycosaminoglycans. We investigated the effects of glycosaminoglycans on PEDF-receptor interactions.

Various genome projects are identifying thousands of new protein sequences every year, which has led to an expanded interest in protein folding. There are two parts to the protein folding problem: the first is predicting the three-dimensional structure of a protein from its amino-acid sequence; and the second — the focus of this Web alert — is to understand how proteins fold. Increased interest in protein folding has been fuelled by the belief that many human diseases (Alzheimer’s and Parkinson’s, for example) are caused by aggregation or misfolding of proteins. The FASEB Society has a valuable essay (aimed at the general public) that describes known diseases related to protein (mis)folding (www.faseb.org/opar/protfold/protein.html). It is possible that the selective pressures that act during evolution function to preserve amino-acid sequences that not only form structures that adequately perform specific biological functions, but also to preserve those that fold quickly enough to avoid aggregation or misfolded states.Although a search for ‘protein folding’ using a search engine like AltaVista has more than 13,600 hits, the number of useful protein-folding resources is limited. A good place to start, to get acquainted with the people in the field, is the comprehensive list of protein-folding groups compiled by Heinrich Roder’s laboratory at Fox Chase Cancer Center (http://www.fccc.edu/research/labs/roder/folding_groups.html), which contains links to more than 80 research teams working on various aspects of protein folding. Several of the scientists listed here have their own web pages, some of which are very creative, and include links to other useful sites.Protein folding is quite different from small-molecule chemical reactions (in which covalent bonds are formed or broken in well-defined transition states), because of the many degrees of freedom allowed a polypeptide chain. In order to understand the complexity of protein-folding processes at the microscopic level, a statistical approach is required. The current theoretical and computational frontline, with respect to protein-folding dynamics, is well described in the Pittsburgh Supercomputing Center’s interactive collection of articles from 1998 (http://www.psc.edu/science/lifeproc.html). In particular, the papers by C.L. Brooks III and P. Kollman are admirable. Vijay Pande’s website (http://www.stanford.edu/group/pandegroup/#research) is also excellent, and includes movies of all-atom molecular-dynamics simulations of protein folding.The Database of Macromolecular Movements (http://bioinfo.mbb.yale.edu/MolMovDB/) is another amusing site to visit. It contains short videos of motions in proteins (including a few examples of folding and unfolding). This site also includes free software tools for protein geometric analyses.In order to place protein folding in its realistic in vivo context, there are additional factors to address. Around 5% of all proteins made in cells (often larger, multidomain proteins) require help from ‘chaperonin’ proteins to fold properly. To learn more about these reactions, visit the Chaperonin Home Page (http://bioc09.uthscsa.edu/∼seale/Chap/chap.html) located at the University of Texas Health Science Center. This web page links to researchers in the field and lists thermodynamic data for a large number of mutants of the most famous chaperonin pair GroEL/GroES. The site also includes a protein-structure gallery and a recent paper alert.20–30% of all proteins function within cellular membranes. It has not yet been resolved (from either a thermodynamic or a kinetic viewpoint) how such proteins are synthesized and then inserted into the membranes. An excellent resource for this topic is the web pages of Stephen White’s laboratory at UC Irvine (http://blanco.biomol.uci.edu/mp_assembly.html). The general principles of membrane-protein structure and stability, the group’s own research approach, a wealth of interesting links and downloadable key research papers are all available here.In order to perform their biological functions, many proteins coordinate small cofactors such as metal ions. The recently created PROMISE database (http://bioinf.leeds.ac.uk/promise/) provides a wealth of information on these types of proteins (although there is not much on the proteins’ folding properties yet). In addition, this site links to an impressive number of bioinorganic web resources.The field of protein folding is growing at a fast pace, as is the number of researchers that need to keep up with the current literature and news. A perfect way to do this is to attend the Johns Hopkins Protein Folding Meeting, which has taken place each spring for the past five years. This meeting attracts the finest people in protein folding, and the conference covers all aspects of this fascinating field. The program for the 5th Meeting (taking place this month) can be viewed at http://www.jhu.edu/∼ipmb/protein.html.Protein folding is a rapidly expanding field that involves researchers from many disciplines. We expect new web resources to be developed that continue to facilitate discussion and collaboration.

Here we show that increased amount of secondary structure is acquired in the folded states of two structurally-different proteins (alpha-helical VlsE and alpha/beta flavodoxin) in the presence of macromolecular crowding agents. The structural content of flavodoxin and VlsE is enhanced by 33% and 70%, respectively, in 400 mg/ml Ficoll 70 (pH 7, 20 degrees C) and correlates with higher protein-thermal stability. In the same Ficoll range, there are only small effects on the unfolded-state structures of the proteins. This is the first in vitro assessment of crowding effects on the native-state structures at physiological conditions. Our findings imply that for proteins with low intrinsic stability, the functional structures in vivo may differ from those observed in dilute buffers.

Proteins fold and function inside cells that are crowded with macromolecules. Here, we address the role of the resulting excluded volume effects by in vitro spectroscopic studies of Pseudomonas aeruginosa apoazurin stability (thermal and chemical perturbations) and folding kinetics (chemical perturbation) as a function of increasing levels of crowding agents dextran (sizes 20, 40, and 70 kDa) and Ficoll 70. We find that excluded volume theory derived by Minton quantitatively captures the experimental effects when crowding agents are modeled as arrays of rods. This finding demonstrates that synthetic crowding agents are useful for studies of excluded volume effects. Moreover, thermal and chemical perturbations result in free energy effects by the presence of crowding agents that are identical, which shows that the unfolded state is energetically the same regardless of method of unfolding. This also underscores the two-state approximation for apoazurin’s unfolding reaction and suggests that thermal and chemical unfolding experiments can be used in an interchangeable way. Finally, we observe increased folding speed and invariant unfolding speed for apoazurin in the presence of macromolecular crowding agents, a result that points to unfolded-state perturbations. Although the absolute magnitude of excluded volume effects on apoazurin is only on the order of 1–3 kJ/mol, differences of this scale may be biologically significant.

Pseudomonas aeruginosa azurin is a blue-copper protein with a beta-barrel fold. Here we report that, at conditions where thermal unfolding of apo-azurin is reversible, the reaction occurs in a single step with a transition midpoint (T(m)) of 69 degrees C (pH 7). The active-site mutation His117Gly creates a cavity in the beta-barrel near the surface but does not perturb the overall fold (T(m) of 64 degrees C, pH 7). Oxidation of the active-site cysteine (Cysteine-112) in wild-type azurin, which occurs readily at higher temperatures, results in a modified protein that cannot adopt a native-like structure. In sharp contrast, Cysteine-112 oxidation in His117Gly azurin yields a modified apo-azurin that appears folded and displays cooperative, reversible unfolding (T(m) approximately 55 degrees C, pH 7). We conclude that azurin's beta-barrel is a rigid structural element that constrains the structure of its surface; a bulky modification can only be accommodated if complementary space is provided.

Metal coordination is required for function of many proteins. For biosynthesis of proteins coordinating a metal, the question arises if the metal binds before, during or after folding of the polypeptide. Moreover, when the metal is bound to the protein, how does its coordination affect biophysical properties such as stability and dynamics? Understanding how metals are utilized by proteins in cells on a molecular level requires accurate descriptions of the thermodynamic and kinetic parameters involved in protein–metal complexes. Copper is one of the essential transition metals found in the active sites of many key proteins. To avoid toxicity of free copper ions, living systems have developed elaborate copper-transport systems that involve dedicated proteins that facilitate efficient and specific delivery of copper to target proteins. This review describes in vitro and in silico biophysical work assessing the role of copper in folding and stability of copper-binding proteins. Examples of proteins discussed are: a blue-copper protein (Pseudomonas aeruginosa azurin), members of copper-transport systems (bacterial CopZ, human Atox1 and ATP7B domains) and multi-copper ferroxidases (yeast Fet3p and human ceruloplasmin). The consequences of interactions between copper proteins and platinum-complexes are also discussed. This article is part of a Special Issue entitled: Cell Biology of Metals.

In this study, we characterize the thermodynamics of hybridization, binding kinetics and conformations of four ribose-modified (2′-fluoro, 2′-O-propyl, 2′-O-methoxyethyl and 2′-O-aminopropyl) decameric mixed-sequence oligonucleotides. Hybridization to the complementary non-modified DNA or RNA decamer was probed by fluorescence and circular-dichroism spectroscopy and compared to the same duplex formed between two non-modified strands. The thermal melting points of DNA–DNA duplexes were increased by 1.8, 2.2, 0.3 and 1.3°C for each propyl, methoxyethyl, aminopropyl and fluoro modification, respectively. In the case of DNA–RNA duplexes, the melting points were increased by 3.1, 4.1 and 1.0°C for each propyl, methoxyethyl and aminopropyl modification, respectively. The high stability of the duplexes formed with propyl-, methoxyethyl- and fluoro-modified oligonucleotides correlated with high preorganization in these single-strands. Despite higher thermodynamic duplex stability, hybridization kinetics to complementary DNA or RNA was slower for propyl- and methoxyethyl-modified oligonucleotides than for the non-modified control. In contrast, the positively-charged aminopropyl-modified oligonucleotide showed rapid binding to the complementary DNA or RNA.

After Ctr1-mediated cell uptake, copper (Cu) is transported by the cytoplasmic Cu chaperone Atox1 to P1B type ATPases ATP7A and ATP7B in the Golgi network, for incorporation into Cudependent enzymes. Atox1 is a small 68-residue protein that binds Cu in a conserved CXXC motif; it delivers Cu to target domains in ATP7A/B via direct protein-protein interactions. Specific transcription factors regulating expression of the human Cu transport proteins have not been reported although Atox1 was recently suggested to have dual functionality such that it, in addition to its cytoplasmic chaperone function, acts as a transcription factor in the nucleus. To examine this hypothesis, here we investigated the localization of Atox1 in HeLa cells using fluorescence imaging in combination with in vitro binding experiments to fluorescently labeled DNA duplexes harboring the proposed promotor sequence. We found that whereas Atox1 is present in the nucleus in HeLa cells, it does not bind to DNA in vitro. It appears that Atox1 mediates transcriptional regulation via additional (unknown) proteins.

Amyloid formation of the protein α-synuclein promotes neurodegeneration in Parkinson's disease. The normal function of α-synuclein includes synaptic vesicle transport and fusion, and the protein binds strongly to negatively charged vesicles in vitro. Here, we demonstrate that nonresonant angle-resolved second-harmonic scattering detects α-synuclein binding to liposomes through changes in water orientational correlations and can thus be used as a high-accuracy and high-throughput label-free probe of protein-liposome interactions. The obtained results support a binding model in which the N-terminus of α-synuclein adopts an α-helical conformation that lies flat on the vesicle surface while the negatively charged C-terminus remains in solution.

In vivo, proteins fold and associate in highly crowded environments. Previousexperiments on several monomeric proteins have shown that macromolecular crowding stabilizes towards heat perturbation and also can modulate native-state structure. To assess the effects of macromolecular crowding on unfolding of an oligomeric protein, where denaturation involves both unfolding and dissociation, we here tested the effects of the synthetic crowding agent Ficoll 70 on cpn10, a heptameric protein consisting of seven identical β-barrel subunits assembling into a ring. The stability of the heptamer is dominated by subunit-subunit interactions and the individual subunits have low stability on their own. Using far-UV circular dichroism (CD), tyrosine fluorescence and cross-linking experiments, we investigated thermal and chemical stability without and with crowding agent. We find that cpn10 is thermally stabilized by about 5°C in 300 mg/ml Ficoll 70 as compared to in buffer. Whereas there is a large effect of Ficoll 70 on the urea-induced unfolding reaction (midpoint shifts by 2 M), a smaller effect is seen when GuHCl is used as the chemical denaturant (midpoint shifts by 0.6 M). Kinetic unfolding experiments show that the higher equilibrium stability found for cpn10 in crowded conditions can be explained in part by slower unfolding rates.

Parkinson's disease affects a growing number of the population and involves motor complications due to the death of dopamine neurons. Cytosolic inclusions containing amyloid fibrils of α-synuclein ...