Increased Solvent Accessibility Research Articles

Structural flexibility and heterogeneity of recombinant human glial fibrillary acidic protein (GFAP).

Glial fibrillary acidic protein (GFAP) is a promising biomarker for brain and spinal cord disorders. Recent studies have highlighted the differences in the reliability of GFAP measurements in different biological matrices. The reason for these discrepancies is poorly understood as our knowledge of the protein's 3-dimensional conformation, proteoforms, and aggregation remains limited. Here, we investigate the structural properties of GFAP under different conditions. For this, we characterized recombinant GFAP proteins from various suppliers and applied hydrogen-deuterium exchange mass spectrometry (HDX-MS) to provide a snapshot of the conformational dynamics of GFAP in artificial cerebrospinal fluid (aCSF) compared to the phosphate buffer. Our findings indicate that recombinant GFAP exists in various conformational species. Furthermore, we show that GFAP dimers remained intact under denaturing conditions. HDX-MS experiments show an overall decrease in H-bonding and an increase in solvent accessibility of GFAP in aCSF compared to the phosphate buffer, with clear indications of mixed EX2 and EX1 kinetics. To understand possible structural interface regions and the evolutionary conservation profiles, we combined HDX-MS results with the predicted GFAP-dimer structure by AlphaFold-Multimer. We found that deprotected regions with high structural flexibility in aCSF overlap with predicted conserved dimeric 1B and 2B domain interfaces. Structural property predictions combined with the HDX data show an overall deprotection and signatures of aggregation in aCSF. We anticipate that the outcomes of this research will contribute to a deeper understanding of the structural flexibility of GFAP and ultimately shed light on its behavior in different biological matrices.

Myoglobin and Hemoglobin: Discoloration, Lipid Oxidation, and Solvent Access to the Heme Pocket

Conversion of the heme iron in myoglobin (Mb) and hemoglobin (Hb) from Fe2+ to Fe3+ is a critical step that causes quality deterioration—such as discoloration and generation of oxidative species, including dissociated heme, that oxidize lipids and proteins—in muscle foods. Increased solvent access to the heme pocket has been proposed to cause oxidation of the heme iron and decrease heme affinity for the globin, although empirical results are lacking. This review introduces plasma-induced modification of biomolecules (PLIMB) as an approach to modify amino acids of Mb and Hb and thereby assess solvent access to the heme pocket. After PLIMB, liquid chromatography tandem mass spectrometry peptide analysis and a user-friendly, software platform are used to quantify modified amino acid side chains of the heme proteins. Our findings indicate that PLIMB➔liquid chromatography-tandem mass spectrometry provides a platform to measure solvent access to portions of the heme pocket environment. Evaluation of PLIMB under additional conditions(e.g., different pH values) can differentiate the role of solvent access to the heme pocket relative to the“outer-sphere”mechanism of heme protein oxidation and the ability of hydrogen bonding to stabilize heme within metHb. Some aspects of heme protein-mediated lipid oxidation that occur at low O2partial pressures are discussed in relationship to solvent access to the heme pocket. Other approaches to study mechanisms of discoloration and lipid oxidation related to Mb/Hb oxidation and heme loss from metHb are also discussed.

Molecular basis of polyglutamine-modulated ELF3 aggregation in Arabidopsis temperature response.

Some plants, like Arabidopsis thaliana, possess a molecular mechanism that allows them to sense changes in temperature and respond by modifying growth rate, photoperiod length and timing of flowering. Underlying this mechanism is a tripartite protein complex called the Evening Complex (EC) that functions as a DNA transcription repressor targeting growth-related genes. ELF3, a large disordered scaffolding protein, is the primary component of the EC. At low temperature ELF3 sits over DNA targets occluding transcription machinery and halting growth. Rising temperature promote ELF3 condensate formation as it becomes sequestered in large reversible nuclear condensates allowing DNA transcription and plant growth to proceed. A C-terminal prion-like domain (PrD) has been observed to be sufficient for ELF3 aggregation. Within this PrD region lies a poly-glutamine repeat of variable length, the size of which has been found to modulate the thermal responsiveness as measured by hypocotyl (stem) elongation and condensate formation. We use a polymer chain growth approach to build large ensembles and characterize monomeric ELF3-PrD at a range of polyQ lengths and temperatures. We then explore temperature-dependent dynamics of wild-type ELF3-PrD and a mutant, F527A, using chain growth structures as initial conformations for replica exchange (REST2) simulations. We find increased solvent accessibility of expanded polyQ tracts, promotion of temperature-sensitive helices adjacent to polyQ tracts, and exposure of a cluster of aromatic residues at increased temperature, all three of which promote inter-protein interaction. Protein condensate formation is a ubiquitous but poorly understood mechanism implicated in a range of biological functions and malfunction. Our computational approach to understanding the molecular basis for the temperature sensitivity of this system has wider implications on the understanding of phenomena regulating condensate formation more generally.

Origin of Increased Solvent Accessibility of Peptide Bonds in Mutual Synergetic Folding Proteins.

Mutual Synergetic Folding (MSF) proteins belong to a recently discovered class of proteins. These proteins are disordered in their monomeric but ordered in their oligomeric forms. Their amino acid composition is more similar to globular proteins than to disordered ones. Our preceding work shed light on important structural aspects of the structural organization of these proteins, but the background of this behavior is still unknown. We suggest that solvent accessibility is an important factor, especially solvent accessibility of the peptide bonds can be accounted for this phenomenon. The side chains of the amino acids which form a peptide bond have a high local contribution to the shielding of the peptide bond from the solvent. During the oligomerization step, other non-local residues contribute to the shielding. We investigated these local and non-local effects of shielding based on Shannon information entropy calculations. We found that MSF and globular homodimeric proteins have different local contributions resulting from different amino acid pair frequencies. Their non-local distribution is also different because of distinctive inter-subunit contacts.

Pressure Unfolding of Proteins: New Insights into the Role of Bound Water.

High pressures can be detrimental for protein stability, resulting in unfolding and loss of function. This phenomenon occurs because the unfolding transition is accompanied by a decrease in volume, which is typically attributed to the elimination of cavities that are present within the native state as a result of packing defects. We present a novel computational approach that enables the study of pressure unfolding in atomistically detailed protein models in implicit solvent. We include the effect of pressure using a transfer free energy term that allows us to decouple the effect of protein residues and bound water molecules on the volume change upon unfolding. We discuss molecular dynamics simulations results using this protocol for two model proteins, Trp-cage and staphylococcal nuclease (SNase). We find that the volume reduction of bound water is the key energetic term that drives protein denaturation under the effect of pressure, for both Trp-cage and SNase. However, we note differences in unfolding mechanisms between the smaller Trp-cage and the larger SNase protein. Indeed, the unfolding of SNase, but not Trp-cage, is seen to be further accompanied by a reduction in the volume of internal cavities. Our results indicate that, for small peptides, like Trp-cage, pressure denaturation is driven by the increase in solvent accessibility upon unfolding, and the subsequent increase in the number of bound water molecules. For larger proteins, like SNase, the cavities within the native fold act as weak spots, determining the overall resistance to pressure denaturation. Our simulations display a striking agreement with the pressure-unfolding profile experimentally obtained for SNase and represent a promising approach for a computationally efficient and accurate exploration of pressure-induced denaturation of proteins.

Role of CNTNAP2 in autism manifestation outlines the regulation of signaling between neurons at the synapse

BackgroundAutism is characterized by high heritability and a complex genetic mutational landscape with restricted social behavior and impaired social communication. Whole-exome sequencing is a reliable tool to pinpoint variants for unraveling the disease pathophysiology. The present meta-analysis was performed using 222 whole-exome sequences deposited by Simons Simplex Collection (SSC) at the European Nucleotide Archive. This sample cohort was used to identify causal mutations in autism-specific genes to create a mutational landscape focusing on the CNTNAP2 gene.ResultsThe authors account for the identification of 15 high confidence genes with 24 variants for autism with Simons Foundation Autism Research Initiative (SFARI) gene scoring. These genes encompass critical autism pathways such as neuron development, synapse complexity, cytoskeleton, and microtubule activation. Among these 15 genes, overlapping variants were present across multiple samples: KMT2C in 167 cases, CNTNAP2 in 192 samples, CACNA1C in 152 cases, and SHANK3 in 124 cases. Pathway analysis identifies clustering and interplay of autism genes—WDFY3, SHANK2, CNTNAP2, HOMER1, SYNGAP1, and ANK2 with CNTNAP2. These genes coincide across autism-relevant pathways, namely abnormal social behavior and intellectual and cognitive impairment. Based on multiple layers of selection criteria, CNTNAP2 was chosen as the master gene for the study. It is an essential gene for autism with speech-language delays, a typical phenotype in most cases under study. It showcases nine variants across multiple samples with one damaging variant, T589P, with a GERP rank score range of 0.065–0.95. This unique variant was present across 86.5% of the samples impairing the epithelial growth factor (EGF) domain. Established microRNA (miRNA) genes hsa-mir-548aq and hsa-mir-548f were mutated within the CNTNAP2 region, adding to the severity. The mutated protein showed reduced stability by 0.25, increased solvent accessibility by 9%, and reduced depth by 0.2, which rendered the protein non-functional. Secondary physical interactors of CNTNAP2 through CNTN2 proteins were mutated in the samples, further intensifying the severity.ConclusionCNTNAP2 has been identified as a master gene in autism manifestation responsible for speech-language delay by impairing the EGF protein domain and downstream cascade. The decrease in EGF is correlated with vital autism symptoms, especially language disabilities.

Effect of Y50H and S187G substitutions on thermostability and exonuclease activity of TK1646 from Thermococcus kodakarensis

TK1646 is a highly thermostable single strand specific 3′-5′ exonuclease. Exonucleases play important role in maintaining the genome integrity at elevated temperatures. Therefore, it is important to examine the factors contributing to thermostability of these exonucleases. In this study we report on production, purification and characterization of S187G and Y50H mutants of TK1646, focusing on the factors leading to thermostability of TK1646. Characterization of the recombinant proteins indicated that these substitutions did not drastically affect the catalysis of single stranded DNA. However, both of these substitutions reduced the thermostability of the recombinant proteins. Half-lives of Y50H and S187G mutants were 95 and 155 min, respectively, at 100 °C in comparison to 180 min of the wild type. Bioinformatics analysis indicated an increase in solvent accessibility of the mutated residues and disruption of hydrogens bonds. Molecular modelling and superimposition of the 3D structures of the mutants and the wild type demonstrated that one of the active site residues, Glu145, was shifted away from the metal ion in both the mutants which may be responsible for the decrease in catalytic activity. Compact secondary structure, hydrophobicity and hydrogen bonding might be the major factors contributing to the thermostability of TK1646.

Structural impact due to PPQEE deletion in multiple cancer associated protein - Integrin αV: An In silico exploration

A heterodimeric receptor subunit, Integrin αV, often complexed with Integrin β3 plays a vital role in cell signaling to regulate angiogenesis and promote cancer progression. The paramount β-turn formed from pentapeptide residues (PPQEE) in the cytoplasmic domain of Integrin αV was previously reported as crucial for cell signaling and its deletion was proved deleterious for protein's cell membrane adhesion and ligand binding properties. This study revealed conformational changes in the Integrin αV subunit upon deletion of PPQEE residues through in silico structural modelling approach followed by analysis of alteration of binding sites. Human Protein Atlas database helped to identify the association of Integrin αV to the unfavourable prognosis of three gastrointestinal cancers: stomach, liver and pancreatic cancers. Molecular modelling and docking techniques were carried out for the necessary complex formations (wild-type and mutant-type). Further comparison was performed for the complexes. The changes in protein's conformation and stability due to PPQEE deletion were observed in both independent subunit and heterodimer. The most noteworthy conformational shift was the disruption of a transmembrane helix into coil, which accounted for protein's impaired cell membrane adhesion, increased solvent accessibility and decreased stability. The deletion also caused a reduction of beta-turn regions, which disrupted ligand binding in the cytoplasmic domain of Integrin αV subunit. This study emphasized on structural basis of how the deletion of PPQEE residues alters stability, ligand binding and signaling activity of Integrin αV subunit highlighting the importance of these residues in maintenance of protein's native structure.

Interaction of Alpha-Synuclein and Its Mutants with Rigid Lipid Vesicle Mimics of Varying Surface Curvature.

Abnormal aggregation of alpha-synuclein (α-syn), an intrinsically disordered neuronal protein, is strongly implicated in the development of Parkinson's disease. Efforts to better understand α-syn's native function and its pathogenic role in neurodegeneration have revealed that the protein interacts with anionic lipid vesicles via adoption of an amphipathic α-helical structure; however, the ability of α-syn to remodel lipid membranes has made it difficult to decipher the role of vesicle surface curvature in protein binding behavior. In this study, sodium dodecyl sulfate (SDS)-coated gold nanoparticles (AuNPs), which mimic bilayer vesicle architecture, were synthesized in order to conduct a systematic investigation into the binding interaction of α-syn and two of its mutants (A30P and E46K) with rigid lipid vesicle mimics of defined surface curvature. By incorporating a rigid AuNP core (∼10-100 nm), the ability of α-syn to remodel the vesicle mimics was removed and their surface curvature could be fixed. Proteomics studies showed that, upon binding of free α-syn to the surface of SDS-AuNPs, the N-terminus of α-syn became less solvent accessible, whereas its C-terminus became more accessible. Interestingly, α-syn's non-amyloid-β component (NAC) region also exhibited increased solvent accessibility, suggesting that α-syn bound to rigid vesicle-like structures could possess heightened aggregation propensity and therefore pathogenicity. Additionally, both the A30P and E46K mutations were found to adopt distinct binding modes on the mimics' surface. In contrast with previous reports, similar binding affinities were observed for WT, A30P, and E46K α-syn toward SDS-AuNPs of all sizes, indicating the potential importance of vesicle deformability in determining α-syn binding behavior.

Molecular dynamics investigation of a redox switch in the anti-HIV protein SAMHD1.

HIV-1 is restricted in macrophages and certain quiescent myeloid cells due to a "Scorched Earth" dNTP starvation strategy attributed to the sterile alpha motif and HD domain protein-SAMHD1. Active SAMHD1 tetramers are assembled by GTP-Mg+2-dNTP cross bridges and cleave the triphosphate groups of dNTPs at a K m of ~10 μM, which is consistent with dNTP concentrations in cycling cells, but far higher than the equivalent concentration in quiescent cells. Given the substantial disparity between the dNTP concentrations required to activate SAMHD1 tetramers (~10 μM) and the dNTP concentrations in noncycling cells (~10 nM), the possibility of alternate enzymatically active forms of SAMHD1, including monomers remains open. In particular, the possibility of redox regulation of such monomers is also an open question. There have been experimental studies on the regulation of SAMHD1 by Glutathione driven redox reactions recently. Therefore, in this work, we have performed all-atom molecular dynamics simulations to study the dynamics of monomeric SAMHD1 constructs in the context of the three redox-susceptible Cysteine residues and compared them to monomers assembled within a tetramer. Our results indicate that assembly into a tetramer causes ordering of the catalytic core and increased solvent accessibility of the Catalytic Site. We have also found that glutathionylation of surface exposed C522 causes long range allosteric disruptions extending into the protein core. Finally, we see evidence suggesting a transient interaction between C522 and C341. Such a disulfide linkage has been hypothesized by experimental models, but has never been observed in crystal structures before.

A quercetin-based flavanoid (rutin) reverses amyloid fibrillation in β-lactoglobulin at pH 2.0 and 358 K.

β-lactoglobulin (BLG) is a well characterized milk protein and a model for folding and aggregation studies. Rutin is a quercetin based-flavanoid and a famous dietary supplement. It is a potential protector from coronary heart disease, cancers, and inflammatory bowel disease. In this study, amyloid fibrillation is reported in BLG at pH 2.0 and temperature 358 K. It is inhibited to some extent by rutin with a rate of 99.3 h-1 M-1. Amyloid fibrillation started taking place after 10 h of incubation and completed near 40 h at a rate of 16.6 × 10-3 h-1, with a plateau during 40-108 h. Disruption of tertiary structure of BLG and increased solvent accessibility of hydrophobic core seem to trigger intermolecular assembly. Increase in 7% β-sheet structure at the cost of 10% α-helical structures and the electron micrograph of BLG fibrils at 108 h further support the formation of amyloid. Although it could not block amyloidosis completely, and even the time required to reach plateau remains the same, a decrease of growth rate from 16.6 × 10-3 to 13.5 × 10-3 h-1 was observed in the presence of 30.0 μM rutin. Rutin seems to block solvent accessibility of the hydrophobic core of BLG. A decrease in the fibril population was observed in electron micrographs, with the increase in rutin concentration. All evidences indicate reversal of fibrillation in BLG in the presence of rutin.

Sphingobacterium sp. T2 Manganese Superoxide Dismutase Catalyzes the Oxidative Demethylation of Polymeric Lignin via Generation of Hydroxyl Radical.

Sphingobacterium sp. T2 contains two extracellular manganese superoxide dismutase enzymes which exhibit unprecedented activity for lignin oxidation but via an unknown mechanism. Enzymatic treatment of lignin model compounds gave products whose structures were indicative of aryl-Cα oxidative cleavage and demethylation, as well as alkene dihydroxylation and alcohol oxidation. 18O labeling studies on the SpMnSOD-catalyzed oxidation of lignin model compound guiaiacylglycerol-β-guaiacyl ether indicated that the an oxygen atom inserted by the enzyme is derived from superoxide or peroxide. Analysis of an alkali lignin treated by SpMnSOD1 by quantitative 31P NMR spectroscopy demonstrated 20-40% increases in phenolic and aliphatic OH content, consistent with lignin demethylation and some internal oxidative cleavage reactions. Assay for hydroxyl radical generation using a fluorometric hydroxyphenylfluorescein assay revealed the release of 4.1 molar equivalents of hydroxyl radical by SpMnSOD1. Four amino acid replacements in SpMnSOD1 were investigated, and A31H or Y27H site-directed mutant enzymes were found to show no lignin demethylation activity according to 31P NMR analysis. Structure determination of the A31H and Y27H mutant enzymes reveals the repositioning of an N-terminal protein loop, leading to widening of a solvent channel at the dimer interface, which would provide increased solvent access to the Mn center for hydroxyl radical generation.

The Gelsolin Pathogenic D187N Mutant Exhibits Altered Conformational Stability and Forms Amyloidogenic Oligomers.

Gelsolin is an actin-severing protein that attains an open functional conformation in the presence of Ca2+ or low pH. Mutations (D187N/Y) in the second domain of gelsolin trigger the proteolytic pathway producing amyloidogenic fragments that form the pathological hallmark of gelsolin amyloidosis and lattice corneal dystrophy type 2 (LCD2). Here, we show that the D187N mutant gelsolin in a Ca2+ depleted, low pH-activated, open conformation could assemble into amyloidogenic oligomers without necessarily undergoing the specific proteolytic step. Although both wild-type (WT) and mutant proteins exhibit closely overlapping globular shapes at physiological conditions, the latter exhibits subjugated actin depolymerization, loss of thermodynamic stability, and folding cooperativity. Mutant gelsolin displayed aberrant conformational unwinding and formed structural conformers with high associative properties at low pH conditions. A SAXS intensity profile and Guinier analysis of these conformers showed the formation of unusual, higher order aggregates. Extended incubation at low pH resulted in the formation of thioflavin T and Congo red positive, β-sheet rich aggregates with a fibrillar, amyloid-like morphology visible under electron and atomic force microscopy. Mass spectrometric analysis of disaggregated end-stage fibrils displayed peptide fragments encompassing the entire protein sequence, indicating the involvement of full length mutant gelsolin in fibril formation. Atomistic and REMD simulations indicated a larger increase in solvent accessibility and loss of fold architecture in mutant gelsolin at low pH as compared to WT gelsolin. Our findings support the existence of a secondary oligomerization-dependent aggregation pathway associated with gelsolin amyloidosis and can pave the way for better therapeutic strategies.

Substrate-Wrapped, Single-Walled Carbon Nanotube Probes for Hydrolytic Enzyme Characterization.

Hydrolytic enzymes are a topic of continual study and improvement due to their industrial impact and biological implications; however, the ability to measure the activity of these enzymes, especially in high-throughput assays, is limited to an established, few enzymes and often involves the measurement of secondary byproducts or the design of a complex degradation probe. Herein, a versatile single-walled carbon nanotube (SWNT)-based biosensor that is straightforward to produce and measure is described. The hydrolytic enzyme substrate is rendered as an amphiphilic polymer, which is then used to solubilize the hydrophobic nanotubes. When the target enzyme degrades the wrapping, the SWNT fluorescent signal is quenched due to increased solvent accessibility and aggregation, allowing quantitative measurement of hydrolytic enzyme activity. Using (6,5) chiral SWNT suspended with polypeptides and polysaccharides, turnover frequencies are estimated for cellulase, pectinase, and bacterial protease. Responses are recorded for concentrations as low as 5 fM using a well-characterized protease, Proteinase K. An established trypsin-based plate reader assay is used to compare this nanotube probe assay with standard techniques. Furthermore, the effect of freeze-thaw cycles and elevated temperature on enzyme activity is measured, suggesting freezing to have minimal impact even after 10 cycles and heating to be detrimental above 60 °C. Finally, rapid optimization of enzyme operating conditions is demonstrated by generating a response surface of cellulase activity with respect to temperature and pH to determine optimal conditions within 2 h of serial scans.

Conformational rearrangement of 1,2-d(GG) intrastrand cis-diammineplatinum crosslinked DNA is driven by counter-ion penetration within the minor groove of the modified site.

The major structural aberrations of DNA induced by a cis-diammineplatinum (II) 1,2-d(GG) intrastrand cross-link (CPT) have been known for decades. To gain deeper insights into the structural dynamics of the sequence-dependent DNA distortions adjacent to the CPT adduct, we employed molecular modeling and molecular dynamics (MD) simulations. The structural dynamics of native (N-DNA) and cisPt 1,2-d(GG) crosslinked (CPT-DNA) in the form of symmetric 36nt d(G2T15G*G*T15G2)●C2A15CCA15C2) oligonucleotide duplexes is compared. The selected sequence context enabled tracking of the origin of the DNA axis curvature at the YpR flexible points (N-DNA), the enhancement of axis bending, and further distortions due to steric/electrostatic perturbations arising from the CPT-crosslink. In addition to the known structural distortions of CPT-DNA: helix bend towards the major groove; local helix unwinding; high roll angle between cross-linked guanine bases; and adoption of A-form DNA on the 5'-side of the CPT-crosslink (TpG junction); our results show the existence of a singular irreversible and reproducible conformational rearrangement, not previously observed, resulting in two stable CPT-DNA1 and CPT-DNA2 conformers. The CPT-DNA2 conformation presents an enhanced DNA axis bend and a wider and shallower minor grove with increased solvent accessibility within the modified site. It is concluded that the polymorphous (unstable) DNA environment near the cisPt 1,2-d(GG) unit in synergy with specific dynamic events, such as prolonged minor groove retention of particular Na+ ions and water redistribution within the d(TG*G*T) site, together with the formation of extra and more stable H-bonds between Pt(NH3)2 amines and neighboring nucleotides, are cooperatively responsible for the initiation of the conformational rearrangement leading to the CPT-DNA2 conformer, which, surprisingly, closely resembles the HMGB1-bound CPT-DNA structure. Graphical abstract Superimposed averaged structures of normal (N-DNA, green) and cisplatin intrastrand cross-linked (CPT-DNA, orange). Global DNA axes: N-DNA (blue beads); CPT-DNA (red beads); PT (yellow sphere); crosslinked dGs viewed from the minor groove (bold).

Structure of cellulose microfibrils in mature cotton fibres

The structure of cellulose microfibrils in mature cotton fibres from three varieties – Gossypium hirsutum, G. barbadense and G. arboreum – has been investigated by a multi-technique approach combining small angle scattering techniques with spectroscopy and diffraction. Cellulose microfibrils present a Iβ-rich crystalline structure with limited surface disorder. Small angle scattering (SAXS and SANS) data have been successfully fitted using a core-shell model and the results obtained indicate that the cellulose microfibrils, formed by the association of 2–3 elementary fibrils, are composed of a ca. 2nm impermeable crystalline core, surrounded by a partially hydrated paracrystalline shell, with overall cross-sections of ca. 3.6–4.7nm. Different low levels of cell wall matrix components have a strong impact on the microfibril architecture and enable moisture penetration upon hydration. Furthermore, the higher amounts of non-cellulosic components in G. barbadense result in a less dense packing of cellulose microfibrils and increased solvent accessibility.

Biological Semiconductors: Structural Control of Heme Redox Potentials in PpcA, a 3-Heme Cytochrome

In an effort to tune and optimize relative bandgap potentials found in biological nanowires that form from multiheme domains of members of the cytochrome c7 family, we developed and extensively characterized 12 point mutations of PpcA, a 3-heme member of the cytochrome c7 family native to Geobacter sulfurreducens. These mutations were engineered to influence the redox potential (Em) of the middle heme (heme III) in PpcA by using four different strategies: performing charge reversal mutations, decreasing solvent access to the heme plane with bulky residues, altering the native bis-histidine axial ligation of the heme, and by attempting to form hydrogen bonds with the propionates of the heme. The latter strategy is expected not only to increase Em but also to introduce a redox Bohr effect. Out of 12 mutants, 11 were successfully expressed in E.coli and show thermal stability in temperature-dependent CD experiments comparable to the wild-type protein (Tm > 90°C). HPLC-ESI-MS was used to confirm both the purity and the mass of the expressed mutants. Small-angle X-ray scattering confirmed that the mutant proteins formed the expected compact globular structures. In addition to ongoing attempts to obtain protein crystals for X-ray crystallography, we performed extensive all-atom molecular dynamics simulations for all mutants. Formation of propionate hydrogen bonds is supported by simulations for A19R and A23R, while increased solvent access is supported for A19I, A19R, A23R, H20M, and K60E. UV/Vis and NMR redox titrations were performed in order to measure the effect of the mutations on the electrochemical properties of all 3 hemes and to understand the underlying principles and viable approaches in tuning relative heme redox potentials. Successful development of this project may lead to biological semiconductors with much smaller footprints and selectively tunable bandgap properties.

SufE D74R Substitution Alters Active Site Loop Dynamics To Further Enhance SufE Interaction with the SufS Cysteine Desulfurase.

Many essential metalloproteins require iron-sulfur (Fe-S) cluster cofactors for their function. In vivo persulfide formation from l-cysteine is a key step in the biogenesis of Fe-S clusters in most organisms. In Escherichia coli, the SufS cysteine desulfurase mobilizes persulfide from l-cysteine via a PLP-dependent ping-pong reaction. SufS requires the SufE partner protein to transfer the persulfide to the SufB Fe-S cluster scaffold. Without SufE, the SufS enzyme fails to efficiently turn over and remains locked in the persulfide-bound state. Coordinated protein-protein interactions mediate sulfur transfer from SufS to SufE. Multiple studies have suggested that SufE must undergo a conformational change to extend its active site Cys loop during sulfur transfer from SufS. To test this putative model, we mutated SufE Asp74 to Arg (D74R) to increase the dynamics of the SufE Cys51 loop. Amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis of SufE D74R revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS. Our results indicate that the mutant protein has a stronger binding affinity for SufS than that of wild-type SufE. In addition, SufE D74R can still enhance SufS desulfurase activity and did not show saturation at higher SufE D74R concentrations, unlike wild-type SufE. These results show that dynamic changes may shift SufE to a sulfur-acceptor state that interacts more strongly with SufS.

Systematic Perturbations of Binuclear Non-heme Iron Sites: Structure and Dioxygen Reactivity of de Novo Due Ferri Proteins.

DFsc (single-chain due ferri) proteins allow for modeling binuclear non-heme iron enzymes with a similar fold. Three 4A → 4G variants of DFsc were studied to investigate the effects of (1) increasing the size of the substrate/solvent access channel (G4DFsc), (2) including an additional His residue in the first coordination sphere along with three additional helix-stabilizing mutations [3His-G4DFsc(Mut3)], and (3) the three helix-stabilizing mutations alone [G4DFsc(Mut3)] on the biferrous structures and their O2 reactivities. Near-infrared circular dichroism and magnetic circular dichroism (MCD) spectroscopy show that the 4A → 4G mutations increase coordination of the diiron site from 4-coordinate/5-coordinate to 5-coordinate/5-coordinate, likely reflecting increased solvent accessibility. While the three helix-stabilizing mutations [G4DFsc(Mut3)] do not affect the coordination number, addition of the third active site His residue [3His-G4DFsc(Mut3)] results in a 5-coordinate/6-coordinate site. Although all 4A→ 4G variants have significantly slower pseudo-first-order rates when reacting with excess O2 than DFsc (∼2 s(-1)), G4DFsc and 3His-G4DFsc(Mut3) have rates (∼0.02 and ∼0.04 s(-1)) faster than that of G4DFsc(Mut3) (∼0.002 s(-1)). These trends in the rate of O2 reactivity correlate with exchange coupling between the Fe(II) sites and suggest that the two-electron reduction of O2 occurs through end-on binding at one Fe(II) rather than through a peroxy-bridged intermediate. UV-vis absorption and MCD spectroscopies indicate that an Fe(III)Fe(III)-OH species first forms in all three variants but converts into an Fe(III)-μ-OH-Fe(III) species only in the 2-His forms, a process inhibited by the additional active site His ligand that coordinatively saturates one of the iron centers in 3His-G4DFsc(Mut3).

Why are the truncated cyclin Es more effective CDK2 activators than the full-length isoforms?

Cell cycle regulating enzymes, CDKs, become activated upon association with their regulatory proteins, cyclins. The G1 cyclin, cyclin E, is overexpressed and present in low molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. In vivo and in vitro studies have shown that these LMW isoforms of cyclin E hyperactivate CDK2 and accelerate the G1-S phase of cell division. The molecular basis of CDK2 hyperactivation due to LMW cyclin E isoforms in cancer cells is, however, unknown. Here, we employ a computational approach, combining homology modeling, bioinformatics analyses, molecular dynamics (MD) simulations, and principal component analyses to unravel the key structural features of CDK2-bound full-length and LMW isoforms of cyclin E1 and correlate those features to their differential activity. Results suggest that the missing N- and C-terminal regions of the cyclin E LMW isoforms constitute the Nuclear Localization Sequence (NLS) and PEST domains and are intrinsically disordered. These regions, when present in the full-length cyclin E/CDK2 complex, weaken the cyclin-CDK interface packing due to the loss of a large number of key interface interactions. Such weakening is manifested in the decreased contact area and increased solvent accessibility at the interface and also by the absence of concerted motions between the two partner proteins in the full-length complex. More effective packing and interactions between CDK2 and LMW cyclin E isoforms, however, produce more efficient protein-protein complexes that accelerate the cell division processes in cancer cells, where these cyclin E isoforms are overexpressed.