Cooperativity of complex salt bridges

The energetic contribution of complex salt bridges, in which one charged residue (anchor residue) forms salt bridges with two or more residues simultaneously, has been suggested to have importance for protein stability. Detailed analysis of the net energetics of complex salt bridge formation using double- and triple-mutant cycle analysis revealed conflicting results. In two cases, it was shown that complex salt bridge formation is cooperative, i.e., the net strength of the complex salt bridge is more than the sum of the energies of individual pairs. In one case, it was reported that complex salt bridge formation is anti-cooperative. To resolve these different findings, we performed analysis of the geometries of salt bridges in a representative set of structures from the PDB and found that over 87% of all complex salt bridges anchored by Arg/Lys have a geometry such that the angle formed by their Calpha atoms, Theta, is <90 degrees . This preferred geometry is observed in the two reported instances when the energetics of complex salt bridge formation is cooperative, while in the reported anti-cooperative complex salt bridge, Theta is close to 160 degrees . Based on these observations, we hypothesized that complex salt bridges are cooperative for Theta < 90 degrees and anti-cooperative for 90 degrees < Theta < 180 degrees . To provide a further experimental test for this hypothesis, we engineered a complex salt bridge with Theta = 150 degrees into a model protein, the activation domain of human procarboxypeptidase A2 (ADA2h). Experimentally derived stabilities of the ADA2h variants allowed us to show that the complex salt bridge in ADA2h is anti-cooperative.     

Threading a peptide through a peptide: protein loops, rotaxanes, and knots

Proteins adopt complex folds in nature that typically avoid conformations that are knotted or “threaded” through closed loops. Is this the result of fundamental barriers to folding, or have proteins simply evolved to avoid threaded conformations? Organic synthesis has been used in supramolecular chemistry to install topological links in small molecules. By following these principles, we now show that it is possible to assemble a topologically linked protein complex by threading a linear protein through a cyclic protein to form a [2]pseudo‐rotaxane. Subsequent ring closure using native chemical ligation cyclizes the linear protein, forming a [2]heterocatenane. Although the kinetics of protein threading are slower than the folding kinetics of the native protein, threading appears to be a highly efficient process.     

Solvation of the folding-transition state in Pseudomonas aeruginosa azurin is modulated by metal: Solvation of azurin's folding nucleus

The role of water in protein folding, specifically its presence or not in the transition-state structure, is an unsolved question. There are two common classes of folding-transition states: diffuse transition states, in which almost all side chains have similar, rather low phi (phi) values, and polarized transition states, which instead display distinct substructures with very high phi-values. Apo-and zinc-forms of Pseudomonas aeruginosa azurin both fold in two-state equilibrium and kinetic reactions; while the apo-form exhibits a polarized transition state, the zinc form entails a diffuse, moving transition state. To examine the presence of water in these two types of folding-transition states, we probed the equilibrium and kinetic consequences of replacing core valines with isosteric threonines at six positions in azurin. In contrast to regular hydrophobic-to-alanine phi-value analysis, valine-to-threonine mutations do not disrupt the core packing but stabilize the unfolded state and can be used to assess the degree of solvation in the folding-transition state upon combination with regular phi-values. We find that the transition state for folding of apo-azurin appears completely dry, while that for zinc-azurin involves partially formed interactions that engage water molecules. This distinct difference between the apo-and holo-folding nuclei can be rationalized in terms of the shape of the free-energy barrier.     

Differential scanning calorimetric,circular dichroism,and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans

The thermal unfolding of xylanase A from Streptomyces lividans, and of its isolated substrate binding and catalytic domains, was studied by differential scanning calorimetry and Fourier transform infrared and circular dichroism spectroscopy. Our calorimetric studies show that the thermal denaturation of the intact enzyme is a complex process consisting of two endothermic events centered near 57 and 64 degrees C and an exothermic event centered near 75 degrees C, all of which overlap slightly on the temperature scale. A comparison of the data obtained with the intact enzyme and isolated substrate binding and catalytic domains indicate that the lower- and higher-temperature endothermic events are attributable to the thermal unfolding of the xylan binding and catalytic domains, respectively, whereas the higher-temperature exothermic event arises from the aggregation and precipitation of the denatured catalytic domain. Moreover, the thermal unfolding of the two domains of the native enzyme are thermodynamically independent and differentially sensitive to pH. The unfolding of the substrate binding domain is a reversible two-state process and, under appropriate conditions, the refolding of this domain to its native conformation can occur. In contrast, the unfolding of the catalytic domain is a more complex process in which two subdomains unfold independently over a similar temperature range. Also, the unfolding of the catalytic domain leads to aggregation and precipitation, which effectively precludes the refolding of the protein to its native conformation. These observations are compatible with the results of our spectroscopic studies, which show that the catalytic and substrate binding domains of the enzyme are structurally dissimilar and that their native conformations are unaffected by their association in the intact enzyme. Thus, the calorimetric and spectroscopic data demonstrate that the S. lividans xylanase A consists of structurally dissimilar catalytic and substrate binding domains that, although covalently linked, undergo essentially independent thermal denaturation. These observations provide valuable new insights into the structure and thermal stability of this enzyme and should assist our efforts at engineering xylanases that are more thermally robust and otherwise better suited for industrial applications.     

Understanding the relevance of local conformational stability and dynamics to the aggregation propensity of an IgG1 and IgG2 monoclonal antibodies

Aggregation of monoclonal antibodies is often a multi‐step process involving structural alterations in monomeric proteins and subsequent formation of soluble or insoluble oligomers. The role of local conformational stability and dynamics of native and/or partially altered structures in determining the aggregation propensity of monoclonal antibodies, however, is not well understood. Here, we investigate the role of conformational stability and dynamics of regions with distinct solvent exposure in determining the aggregation propensity of an IgG1 and IgG2 monoclonal antibody. The temperatures employed span the pre‐unfolding range (10–40°C) and the onset temperatures (T_onset) for exposure of apolar residues (～50°C), alterations in secondary structures (～60°C) and initiation of visible aggregate formation (～60°C). Solvent‐exposed regions were found to precede solvent‐shielded regions in an initiation of aggregation for both proteins. Such a process was observed upon alterations in overall tertiary structure while retaining the secondary structures in both the proteins. In addition, a greater dynamic nature of solvent‐shielded regions in potential intermediates of IgG1 and the improved conformational stability increased its resistance to aggregation when compared to IgG2. These results suggest that local conformational stability and fluctuations of partially altered structures can influence the aggregation propensity of immunoglobulins.     

The interrelationship between ligand binding and thermal unfolding of the folate binding protein. The role of self-association and pH

The present study utilized a combination of DLS (dynamic light scattering) and DSC (differential scanning calorimetry) to address thermostability of high-affinity folate binding protein (FBP), a transport protein and cellular receptor for the vitamin folate. At pH 7.4 (pI = 7–8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3 μM holo-FBP showed Tm (76 °C) and molar enthalpy (146 kcal M^− 1) values increasing to 78 °C and 163 kcal M^− 1 at 10 μM holo-FBP, while those of apo-FBP were 55 °C and 105 kcal M^− 1. Besides ligand binding, intermolecular forces involved in concentration-dependent multimerization thus contribute to the thermostability of holo-FBP. Hence, thermal unfolding and dissociation of holo-FBP multimers occur simultaneously consistent with a gradual decrease from octameric to monomeric holo-FBP (10 μM) in DLS after a step-wise rise in temperature to 78 °C ≈ Tm. Stable holo-FBP multimers may protect naturally occurring labile folates against decomposition or bacterial utilization. DSC established an interrelationship between diminished folate binding at pH 5, especially in NaCl-free buffers, and low thermostability. Positively charged apo-FBP was almost completely unfolded and aggregated at pH 5 (Tm 38 °C) and holo-FBP, albeit more thermostable, was labile with aggregation tendency. Addition of 0.15 M NaCl increased thermostability of apo-FBP drastically, and even more so that of holo-FBP. Electrostatic forces thus seem to contribute to a diminished thermostability at low pH. Fluorescence spectroscopy after irreversible thermal unfolding of FBP revealed a weak-affinity folate binding.     

The osmolyte betaine promotes protein misfolding and disruption of protein aggregates

In this work the effect of betaine on the structure and aggregation of the GST-GFP fluorescent fusion protein was studied by different complementary techniques, including electron microscopy, dynamic light scattering, circular dichroism, and FTIR spectroscopy. Although osmolytes are known to be protein stabilizers in vivo, the effect of betaine on the structure and aggregation of our model protein was found to be strictly concentration dependent. We demonstrated that, by changing betaine concentration, it was possible to tune the formation of protein soluble assemblies and insoluble aggregates, as well as to disaggregate preformed aggregates. In particular, at a critical concentration of betaine between 5 and 7.5 mM, the protein precipitated into macroscopic prefibrillar structures, rich in intermolecular beta-sheets, which were found to bind thioflavine T and to be inaccessible to protease. Instead, at higher betaine concentration (10-20 mM) the misfolded protein lost its fluorescence, but formed soluble assemblies with hydrodynamic radius of about 16 nm. These structures displayed a reduced propensity to further aggregate under thermal treatment. In addition, betaine at this high concentration was also found to disrupt large preformed aggregates, obtained under different conditions, into protein soluble assemblies. It is the first time that a disaggregation process has been described for a chemical chaperone. A mechanism for the betaine concentration-dependent effect on protein misfolding, aggregation, and disaggregation is proposed and its possible physiological implications are discussed.     

Cumulative deamidations of the major lens protein γS‐crystallin increase its aggregation during unfolding and oxidation

Age‐related lens cataract is the major cause of blindness worldwide. The mechanisms whereby crystallins, the predominant lens proteins, assemble into large aggregates that scatter light within the lens, and cause cataract, are poorly understood. Due to the lack of protein turnover in the lens, crystallins are long‐lived. A major crystallin, γS, is heavily modified by deamidation, in particular at surface‐exposed N14, N76, and N143 to introduce negative charges. In this present study, deamidated γS was mimicked by mutation with aspartate at these sites and the effect on biophysical properties of γS was assessed via dynamic light scattering, chemical and thermal denaturation, hydrogen‐deuterium exchange, and susceptibility to disulfide cross‐linking. Compared with wild type γS, a small population of each deamidated mutant aggregated rapidly into large, light‐scattering species that contributed significantly to the total scattering. Under partially denaturing conditions in guanidine hydrochloride or elevated temperature, deamidation led to more rapid unfolding and aggregation and increased susceptibility to oxidation. The triple mutant was further destabilized, suggesting that the effects of deamidation were cumulative. Molecular dynamics simulations predicted that deamidation augments the conformational dynamics of γS. We suggest that these perturbations disrupt the native disulfide arrangement of γS and promote the formation of disulfide‐linked aggregates. The lens‐specific chaperone αA‐crystallin was poor at preventing the aggregation of the triple mutant. It is concluded that surface deamidations cause minimal structural disruption individually, but cumulatively they progressively destabilize γS‐crystallin leading to unfolding and aggregation, as occurs in aged and cataractous lenses.     

Early events in protein aggregation: molecular flexibility and hydrophobicity/charge interaction in amyloid peptides as studied by molecular dynamics simulations

In a previous article (Zbilut et al., Biophys J 2003;85:3544-3557), we demonstrated how an aggregation versus folding choice could be approached considering hydrophobicity distribution and charge. In this work, our aim is highlighting the mutual interaction of charge and hydrophobicity distribution in the aggregation process. Use was made of two different peptides, both derived from a transmembrane protein (amyloid precursor protein; APP), namely, Abeta(1-28) and Abeta(1-40). Abeta(1-28) has a much lower aggregation propensity than Abeta(1-40). The results obtained by means of molecular dynamics simulations show that, when submitted to the most "aggregation-prone" environment, corresponding to the isoelectric point and consequently to zero net charge, both peptides acquire their maximum flexibility, but Abeta(1-40) has a definitely higher conformational mobility than Abeta(1-28). The absence of a hydrophobic "tail," which is the most mobile part of the molecule in Abeta(1-40), is the element lacking in Abeta(1-28) for obtaining a "fully aggregating" phenotype. Our results suggest that conformational flexibility, determined by both hydrophobicity and charge effect, is the main mechanistic determinant of aggregation propensity.     

Relevance of weak flavin binding in human D‐amino acid oxidase

In the brain, the human flavoprotein D ‐amino acid oxidase (hDAAO) is involved in the degradation of the gliotransmitter D ‐serine, an important modulator of NMDA‐receptor‐mediated neurotransmission; an increase in hDAAO activity (that yields a decrease in D ‐serine concentration) was recently proposed to be among the molecular mechanisms leading to the onset of schizophrenia susceptibility. This human flavoenzyme is a stable homodimer (even in the apoprotein form) that distinguishes from known D ‐amino acid oxidases because it shows the weakest interaction with the flavin cofactor in the free form. Instead, cofactor binding is significantly tighter in the presence of an active site ligand. In order to understand how hDAAO activity is modulated, we investigated the FAD binding process to the apoprotein moiety and compared the folding and stability properties of the holoenzyme and the apoprotein forms. The apoprotein of hDAAO can be distinguished from the holoenzyme form by the more “open” tertiary structure, higher protein fluorescence, larger exposure of hydrophobic surfaces, and higher sensitivity to proteolysis. Interestingly, the FAD binding only slightly increases the stability of hDAAO to denaturation by urea or temperature. Taken together, these results indicate that the weak cofactor binding is not related to protein (de)stabilization or oligomerization (as instead observed for the homologous enzyme from yeast) but rather should represent a means of modulating the activity of hDAAO. We propose that the absence in vivo of an active site ligand/substrate weakens the cofactor binding, yielding the inactive apoprotein form and thus avoiding excessive D ‐serine degradation.     

DichroWeb,a website for calculating protein secondary structure from circular dichroism spectroscopic data

Circular dichroism (CD) spectroscopy is a widely‐used method for characterizing the secondary structures of proteins. The well‐established and highly used analysis website, DichroWeb (located at: http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) enables the facile quantitative determination of helix, sheet, and other secondary structure contents of proteins based on their CD spectra. DichroWeb includes a range of reference datasets and algorithms, plus graphical and quantitative methods for determining the quality of the analyses produced. This article describes the current website content, usage and accessibility, as well as the many upgraded features now present in this highly popular tool that was originally created nearly two decades ago.     

Conformational properties of intrinsically disordered proteins bound to the surface of silica nanoparticles

Background

Protein-nanoparticle (NP) interactions dictate properties of nanoconjugates relevant to bionanotechnology. Non-covalent adsorption generates a protein corona (PC) formed by an inner and an outer layer, the hard and soft corona (HC, SC). Intrinsically disordered proteins (IDPs) exist in solution as conformational ensembles, whose response to the presence of NPs is not known.

Sonication of proteins causes formation of aggregates that resemble amyloid

Despite the widespread use of sonication in medicine, industry, and research, the effects of sonication on proteins remain poorly characterized. We report that sonication of a range of structurally diverse proteins results in the formation of aggregates that have similarities to amyloid aggregates. The formation of amyloid is associated with, and has been implicated in, causing of a wide range of protein conformational disorders including Alzheimer's disease, Huntington's disease, Parkinson's disease, and prion diseases. The aggregates cause large enhancements in fluorescence of the dye thioflavin T, exhibit green-gold birefringence upon binding the dye Congo red, and cause a red-shift in the absorbance spectrum of Congo red. In addition, circular dichroism reveals that sonication-induced aggregates have high beta-content, and proteins with significant native alpha-helical structure show increased beta-structure in the aggregates. Ultrastructural analysis by electron microscopy reveals a range of morphologies for the sonication-induced aggregates, including fibrils with diameters of 5-20 nm. The addition of preformed aggregates to unsonicated protein solutions results in accelerated and enhanced formation of additional aggregates upon heating. The dye-binding and structural characteristics, as well as the ability of the sonication-induced aggregates to seed the formation of new aggregates are all similar to the properties of amyloid. These results have important implications for the use of sonication in food, biotechnological and medical applications, and for research on protein aggregation and conformational disorders.     

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26-42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).