Wilfredo Colón

Cytochrome c on Silica Nanoparticles: Influence of Nanoparticle Size on Protein Structure, Stability, and Activity

The structure, thermodynamic and kinetic stability, and activity of cytochrome c (cyt c) on silica nanoparticles (SNPs) of different sizes have been studied. Adsorption of cyt c onto larger SNPs results in both greater disruption of the cyt c global structure and more significant changes of the local heme microenvironment than upon adsorption onto smaller SNPs. The disruption of the heme microenvironment leads to a more solvent-accessible protein active site, as suggested by Soret circular dichroism spectroscopy and through an increase in peroxidase activity as a function of increased SNP size. Similarly, the stability of cyt c decreases more dramatically upon adsorption onto larger SNPs. These results are consistent with changes in protein-nanoparticle interactions that depend on the size or surface curvature of the supporting nanostructure. This study provides further fundamental insights into the effects of nanoscale surfaces on adsorbed protein structure and function.

Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS

Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfide-reduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chemically and structurally diverse ALS-associated mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quantitative thermodynamic stability analyses. In the absence of ALS-associated mutations, reduced apo SOD1 is marginally stable but predominantly folded. Mutations generally result in slight decreases to substantial increases in the fraction of unfolded protein. Calorimetry, ultracentrifugation, and light scattering show that all mutations enhance aggregation propensity, with the effects varying widely, from subtle increases in most cases, to pronounced formation of 40–100 nm soluble aggregates by A4V, a mutation that is associated with particularly short disease duration. Interestingly, although there is a correlation between observed aggregation and stability, there is minimal to no correlation between observed aggregation, predicted aggregation propensity, and disease characteristics. These findings suggest that reduced apo SOD1 does not play a dominant role in modulating disease. Rather, additional and/or multiple forms of SOD1 and additional biophysical and biological factors are needed to account for the toxicity of mutant SOD1 in ALS.

Identifying the subproteome of kinetically stable proteins via diagonal 2D SDS/PAGE.

Most proteins are in equilibrium with partially and globally unfolded conformations. In contrast, kinetically stable proteins (KSPs) are trapped by an energy barrier in a specific state, unable to transiently sample other conformations. Among many potential roles, it appears that kinetic stability (KS) is a feature used by nature to allow proteins to maintain activity under harsh conditions and to preserve the structure of proteins that are prone to misfolding. The biological and pathological significance of KS remains poorly understood because of the lack of simple experimental methods to identify this property and its infrequent occurrence in proteins. Based on our previous correlation between KS and a proteins resistance to the denaturing detergent SDS, we show here the application of a diagonal 2D (D2D) SDS/PAGE assay to identify KSPs in complex mixtures. We applied this method to the lysate of Escherichia coli and upon proteomics analysis have identified 50 nonredundant proteins that were SDS-resistant (i.e., kinetically stable). Structural and functional analyses of a subset (44) of these proteins with known 3D structure revealed some potential structural and functional biases toward and against KS. This simple D2D SDS/PAGE assay will allow the widespread investigation of KS, including the proteomics-level identification of KSPs in different systems, potentially leading to a better understanding of the biological and pathological significance of this intriguing property of proteins.

Murine apolipoprotein serum amyloid A in solution forms a hexamer containing a central channel

Serum amyloid A (SAA) is a small apolipoprotein that binds to high-density lipoproteins in the serum. Although SAA seems to play a role in host defense and lipid transport and metabolism, its specific functions have not been defined. Despite the growing implications that SAA plays a role in the pathology of various diseases, a high-resolution structure of SAA is lacking because of limited solubility in the high-density lipoprotein-free form. In this study, complementary methods including glutaraldehyde cross-linking, size-exclusion chromatography, and sedimentation-velocity analytical ultracentrifugation were used to show that murine SAA2.2 in aqueous solution exists in a monomer–hexamer equilibrium. Electron microscopy of hexameric SAA2.2 revealed that the subunits are arranged in a ring forming a putative central channel. Limited trypsin proteolysis and mass spectrometry analysis identified a significantly protease-resistant SAA2.2 region comprising residues 39–86. The isolated 39–86 SAA2.2 fragment did not hexamerize, suggesting that part of the N terminus is involved in SAA2.2 hexamer formation. Circular-dichroism spectrum deconvolution and secondary-structure prediction suggest that SAA2.2 contains ≈50% of its residues in α-helical conformation and <10% in β-structure. These findings are consistent with the recent discovery that human SAA1.1 forms a membrane channel and have important implications for understanding the 3D structure, multiple functions, and pathological roles of this highly conserved protein.

Modification of cysteine 111 in Cu/Zn superoxide dismutase results in altered spectroscopic and biophysical properties

Cu/Zn superoxide dismutase (SOD) mutations are involved in about 20% of all cases of familial amyotrophic lateral sclerosis (FALS). Recently, it has been proposed that aberrant copper activity may be occurring within SOD at an alternative binding, and cysteine 111 has been identified as a potential copper ligand. Using a commercial source of human SOD isolated from erythrocytes, an anomalous absorbance at 325 nm was identified. This unusual property, which does not compromise SOD activity, had previously been shown to be consistent with a sulfhydryl modification at a cysteine residue. Here, we utilized limited trypsin proteolysis and mass spectrometry to show that the modification has a mass of 32 daltons and is located at cysteine 111. The reaction of SOD with sodium sulfide, which can react with cysteine to form a persulfide group, and with potassium cyanide, which can selectively remove persulfide bonds, confirmed the addition of a persulfide group at cysteine 111. Gel electrophoresis and glutaraldehyde cross‐linking revealed that this modification makes the acid‐induced denaturation of SOD fully irreversible. Furthermore, the modified protein exhibits a slower acid‐induced unfolding, and is more resistant to oxidation‐induced aggregation caused by copper and hydrogen peroxide. Thus, these results suggest that cysteine 111 can have a biochemical and biophysical impact on SOD, and suggest that it can interact with copper, potentially mediating the copper‐induced oxidative damage of SOD. It will be of interest to study the role of cysteine 111 in the oxidative damage and aggregation of toxic SOD mutants.

Biochemical Characterization of Major Bone-Matrix Proteins Using Nanoscale-Size Bone Samples and Proteomics Methodology

There is growing evidence supporting the need for a broad scale investigation of the proteins and protein modifications in the organic matrix of bone and the use of these measures to predict fragility fractures. However, limitations in sample availability and high heterogeneity of bone tissue cause unique experimental and/or diagnostic problems. We addressed these by an innovative combination of laser capture microscopy with our newly developed liquid chromatography separation methods, followed by gel electrophoresis and mass spectrometry analysis. Our strategy allows in-depth analysis of very limited amounts of bone material, and thus, can be important to medical sciences, biology, forensic, anthropology, and archaeology. The developed strategy permitted unprecedented biochemical analyses of bone-matrix proteins, including collagen modifications, using nearly nanoscale amounts of exceptionally homogenous bone tissue. Dissection of fully mineralized bone-tissue at such degree of homogeneity has not been achieved before. Application of our strategy established that: (1) collagen in older interstitial bone contains higher levels of an advanced glycation end product pentosidine then younger osteonal tissue, an observation contrary to the published data; (2) the levels of two enzymatic crosslinks (pyridinoline and deoxypiridinoline) were higher in osteonal than interstitial tissue and agreed with data reported by others; (3) younger osteonal bone has higher amount of osteopontin and osteocalcin then older interstitial bone and this has not been shown before. Taken together, these data show that the level of fluorescent crosslinks in collagen and the amount of two major noncollagenous bone matrix proteins differ at the level of osteonal and interstitial tissue. We propose that this may have important implications for bone remodeling processes and bone microdamage formation.

Distinctive interactions in the holoenzyme formation for two isoforms of glutamate decarboxylase

The interactions between glutamate decarboxylase (GAD) and its cofactor pyridoxal phosphate (PLP) play a key role in the regulation of GAD activity. The enzyme has two isoforms, GAD65 and GAD67. A comparison of binding constants, rate constants, and kinetic profiles for the formation of holoenzyme (holoGAD65 and holoGAD67) revealed that the two isoforms interact distinctively with the cofactor. GAD67 exhibits a higher binding constant for PLP binding, making it more difficult to dissociate PLP from holoGAD67 than holoGAD65. Meanwhile, PLP binding occurs at a much slower rate for GAD67 than GAD65, as evidenced by lower rate constants and a slower initial rate of the holoenzyme formation. Jobs plots revealed a stoichiometry of 1:1 for PLP binding to GAD65 before and after the saturation level of PLP, while 1:2 for PLP binding to GAD67 prior to the saturation of PLP and 1:1 at the saturation level of PLP. These results suggested that the two binding sites of GAD65 exhibit similar affinities for PLP. In contrast, one binding site of GAD67 exhibits a significantly higher affinity for PLP than the other binding site. Based on these findings, it was proposed that a slower PLP binding to GAD67 than GAD65 and a less ease to dissociate PLP from holoGAD67 than holoGAD65 are important underlying factors. This attributes to GAD67 being more highly saturated by PLP and GAD65 being less saturated by PLP. A larger conformation change constant for GAD67 than GAD65 supported a significant conformational change induced by the initial PLP binding to GAD67, which affects the other binding site affinity of GAD67. The present studies provided valuable insights into distinctive properties between the two isoforms of GAD.

Pathogenic Serum Amyloid A 1.1 Shows a Long Oligomer-rich Fibrillation Lag Phase Contrary to the Highly Amyloidogenic Non-pathogenic SAA2.2

Background: Murine SAA1.1 is pathogenic and SAA2.2 is non-pathogenic in AA amyloidosis. Results: SAA1.1 and SAA2.2 exhibit different biophysical properties, including fibrillation kinetics and fibril morphology. Conclusion: The distinct biophysical properties of highly homologous SAA proteins may contribute to their different pathogenicity during chronic inflammation. Significance: Structural and kinetic factors, more than their intrinsic amyloidogenicity, may determine the diverse pathogenicity among nearly identical SAA isoforms. Serum amyloid A (SAA) is best known for being the main component of amyloid in the inflammation-related disease amyloid A (AA) amyloidosis. Despite the high sequence identity among different SAA isoforms, not all SAA proteins are pathogenic. In most mouse strains, the AA deposits mostly consist of SAA1.1. Conversely, the CE/J type mouse expresses a single non-pathogenic SAA2.2 protein that is 94% identical to SAA1.1. Here we show that SAA1.1 and SAA2.2 differ in their quaternary structure, fibrillation kinetics, prefibrillar oligomers, and fibril morphology. At 37 °C and inflammation-related SAA concentrations, SAA1.1 exhibits an oligomer-rich fibrillation lag phase of a few days, whereas SAA2.2 shows virtually no lag phase and forms small fibrils within a few hours. Deep UV resonance Raman, far UV-circular dichroism, atomic force microscopy, and fibrillation cross-seeding experiments suggest that SAA1.1 and SAA2.2 fibrils possess different morphology. Both the long-lived oligomers of pathogenic SAA1.1 and the fleeting prefibrillar oligomers of non-pathogenic SAA2.2, but not their respective amyloid fibrils, permeabilized synthetic bilayer membranes in vitro. This study represents the first comprehensive comparison between the biophysical properties of SAA isoforms with distinct pathogenicities, and the results suggest that structural and kinetic differences in the oligomerization-fibrillation of SAA1.1 and SAA2.2, more than their intrinsic amyloidogenicity, may contribute to their diverse pathogenicity.

Serum amyloid A 2.2 refolds into a octameric oligomer that slowly converts to a more stable hexamer.

Serum amyloid A (SAA) is an inflammatory protein predominantly bound to high-density lipoprotein in plasma and presumed to play various biological and pathological roles. We previously found that the murine isoform SAA2.2 exists in aqueous solution as a marginally stable hexamer at 4-20°C, but becomes an intrinsically disordered protein at 37°C. Here we show that when urea-denatured SAA2.2 is dialyzed into buffer (pH 8.0, 4°C), it refolds mostly into an octameric species. The octamer transitions to the hexameric structure upon incubation from days to weeks at 4°C, depending on the SAA2.2 concentration. Thermal denaturation of the octamer and hexamer monitored by circular dichroism showed that the octamer is ∼10°C less stable, with a denaturation mid point of ∼22°C. Thus, SAA2.2 becomes kinetically trapped by refolding into a less stable, but more kinetically accessible octameric species. The ability of SAA2.2 to form different oligomeric species in vitro along with its marginal stability, suggest that the structure of SAA might be modulated in vivo to form different biologically relevant species.

Characterization of Interactions between Heparin/Glycosaminoglycan and Adeno-Associated Virus

Adeno-associated virus (AAV) is a key candidate in the development of gene therapy. In this work, we used surface plasmon resonance spectroscopy to study the interaction between AAV and heparin and other glycosaminoglycans (GAGs). Surface plasmon resonance results revealed that heparin binds to AAV with an extremely high affinity. Solution competition studies showed that binding of AAV to heparin is chain length-dependent. AAV prefers to bind full chain heparin. All sulfo groups (especially N-sulfo and 6-O-sulfo groups) on heparin are important for the AAV-heparin interaction. Higher levels of sulfo group substitution in GAGs enhance their binding affinities. Atomic force microscopy was also performed to image AAV-2 in a complex with heparin.