Unveiling Metastable Ensembles of GRB2 and the Relevance of Interdomain Communication during Folding.

The folding process of multidomain proteins is a highly intricate phenomenon involving the assembly of distinct domains into a functional three-dimensional structure. During this process, each domain may fold independently while interacting with others. The folding of multidomain proteins can be influenced by various factors, including their composition, the structure of each domain, or the presence of disordered regions, as well as the surrounding environment. Misfolding of multidomain proteins can lead to the formation of nonfunctional structures associated with a range of diseases, including cancers or neurodegenerative disorders. Understanding this process is an important step for many biophysical analyses such as stability, interaction, malfunctioning, and rational drug design. One such multidomain protein is growth factor receptor-bound protein 2 (GRB2), an adaptor protein that is essential in regulating cell survival. GRB2 consists of one central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. The SH2 domain interacts with phosphotyrosine regions in other proteins, while the SH3 domains recognize proline-rich regions on protein partners during cell signaling. Here, we combined computational and experimental techniques to investigate the folding process of GRB2. Through computational simulations, we sampled the conformational space and mapped the mechanisms involved by the free energy profiles, which may indicate possible intermediate states. From the molecular dynamics trajectories, we used the energy landscape visualization method (ELViM), which allowed us to visualize a three-dimensional (3D) representation of the overall energy surface. We identified two possible parallel folding routes that cannot be seen in a one-dimensional analysis, with one occurring more frequently during folding. Supporting these results, we used differential scanning calorimetry (DSC) and fluorescence spectroscopy techniques to confirm these intermediate states in vitro. Finally, we analyzed the deletion of domains to compare our model outputs to previously published results, supporting the presence of interdomain modulation. Overall, our study highlights the significance of interdomain communication within the GRB2 protein and its impact on the formation, stability, and structural plasticity of the protein, which are crucial for its interaction with other proteins in key signaling pathways.

The influence of pH on the structure and stability of the Grb2 dimer reveals changes in the inter-domain and molecular interaction: Could it be a modulation mechanism?

Cancer cells present an increased replicative potential as a hallmark. The increased replication leads to a higher intracellular pH. Grb2, an adapter protein, is mainly involved in several types of cancers due to its role in signaling pathways responsible for cell growth and proliferation. At pH 7, we observed a more compact structure, as seen by DLS and 1H NMR relaxation experiments, with high cooperativity within domains. On the other hand, we observed an increase in disordered structures at pH 8, with relative independence between domains characterized by higher melting temperatures and enthalpy of unfolding. CD and DLS corroborate with these observations at pH 8, conferring more flexibility among the domains, followed by lower unfolding cooperativity and increased hydrodynamic diameter at higher pH. In addition, 15N-HSQC chemical shift perturbations experiments showed significant differences in the positions of several amino acids spread on the Grb2 structure when pH was changed, which agrees with the previous results. Finally, the molecular dynamic analysis demonstrates that Grb2 presents a movement pattern where both SH3 domains move toward the center of the protein at pH 7. On the contrary, the pattern changes its direction at pH 8, where domains move outside the center of the protein, conferring a more elongated structure at higher pH. So, Grb2 presents significant structural and dynamic changes modulated by pH. If considering the role of Grb2 in cell signaling upstream, these conformational changes could be a critical mechanistic behavior of this protein, preventing/disrupting the stability of the cell signaling pathways related to cancer.

Impact of the COVID-19 Pandemic on Cancer Staging: An Analysis of Patients With Breast Cancer From a Community Practice in Brazil.

PURPOSE: A nationwide lockdown was enforced in Brazil starting in March 2020 because of the COVID-19 pandemic when cancer screening activities were reduced. In this study, we evaluated the impact of the COVID-19 pandemic on breast cancer (BC) diagnosis. METHODS: We extracted data from the medical records of patients age older than 18 years who were diagnosed with BC and started treatment or follow-up in private oncology institutions in Brazil between 2018 and 2021. The primary objective was to compare the stage distribution during the COVID-19 pandemic (2020-2021) with a historical prepandemic control cohort (2018-2019). Early BC was defined as stage I-II and advanced disease as stage IV. RESULTS: We collected data for 11,753 patients with an initial diagnosis of BC, with 6,493 patients in the pandemic (2020-2021) and 5,260 patients in the prepandemic period (2018-2019). We observed a lower prevalence of early-stage BC (63.6% v 68.4%) and a higher prevalence of advanced-stage BC (16.9 v 12.7%), after the onset of the pandemic (both P < .01). This pattern was similar for both estrogen receptor-positive/human epidermal growth factor receptor 2-negative and human epidermal growth factor receptor 2-positive tumors: significantly decreased in the early stage from 69% to 67% and 68% to 58%, respectively, and a considerable increase in advanced-stage disease from 13% to 15% and 13% to 20%, respectively. For triple-negative BC, there was a significantly higher percentage of patients with advanced-stage disease during the pandemic (17% v 11%). Overall, age 50 years or older and postmenopausal status were associated with a greater risk of advanced stage at diagnosis during the pandemic period. CONCLUSION: We observed a substantial increase in the number of cases of advanced-stage BC in Brazil during the COVID-19 pandemic.

Aqueous mixtures of cornstarch and Pluronic® F127 studied by experimental and computational techniques.

The possibility of interaction between cornstarch (CS) and amphiphilic molecules, such as the micelle-forming triblock copolymer Pluronic® F127 (F127), also known by Poloxamer 407, indicates that CS-F127 aqueous mixtures can regulate either the starch solubility or the copolymer micellization. Herein experimental and computational techniques were used to investigate CS-F127 aqueous mixtures aiming to highlight the role of these compounds on the molecular complexation. Dynamic light scattering results show that CS in water is highly polydisperse, while the F127 concentration and temperature influence the micellization process and the interaction with CS. Circular dichroism data of CS supernatants indicate the existence of small helical-like granules (Dh ≈ 800 nm) in the CS-F127 mixed aqueous solutions at 25 °C. UV-Vis spectrophotometry shows a small absorption band around 267 or 275 nm characteristic of micelles, granules, or molecular complexes, while FTIR and X-ray diffractometry indicate negligible structural changes. Lugol iodine tests at 25 °C show that both the precipitate and supernatant in the mixtures undergo some structural changes also indicating molecular complexation. Molecular dynamic simulations show the formation of stabilized inclusion complexes (V-amylose), where the propylene oxide segment of the copolymer inside the amylose helix and the ethylene oxide branches facing the aqueous media. These results together reveal weak CS-F127 interactions, evidencing a small solubility of CS both in the absence and presence of F127 as a solubilizing agent. Furthermore, moderate CS amounts do not change the F127 micelle structure.

On the roles of AA15 lytic polysaccharide monooxygenases derived from the termite Coptotermes gestroi.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which catalyze the oxidative cleavage of polysaccharides. LPMOs belonging to family 15 in the Auxiliary Activity (AA) class from the Carbohydrate-Active Enzyme database are found widespread across the Tree of Life, including viruses, algae, oomycetes and animals. Recently, two AA15s from the firebrat Thermobia domestica were reported to have oxidative activity, one towards cellulose or chitin and the other towards chitin, signalling that AA15 LPMOs from insects potentially have different biochemical functions. Herein, we report the identification and characterization of two family AA15 members from the lower termite Coptotermes gestroi. Addition of Cu(II) to CgAA15a or CgAA15b had a thermostabilizing effect on both. Using ascorbate and O2 as co-substrates, CgAA15a and CgAA15b were able to oxidize chitin, but showed no activity on celluloses, xylan, xyloglucan and starch. Structural models indicate that the LPMOs from C. gestroi (CgAA15a/CgAA15b) have a similar fold but exhibit key differences in the catalytic site residues when compared to the cellulose/chitin-active LPMO from T. domestica (TdAA15a), especially the presence of a non-coordinating phenylalanine nearby the Cu ion in CgAA15a/b, which appears as a tyrosine in the active site of TdAA15a. Despite the overall similarity in protein folds, however, mutation of the active site phenylalanine in CgAA15a to a tyrosine did not expanded the enzymatic specificity from chitin to cellulose. Our data show that CgAA15a/b enzymes are likely not involved in lignocellulose digestion but might play a role in termite developmental processes as well as on chitin and nitrogen metabolisms.

A novel mechanism of ß-glucosidase stimulation through a monosaccharide binding-induced conformational change.

It is urgent the transition from a fossil fuel-based economy to a sustainable bioeconomy based on bioconversion technologies using renewable plant biomass feedstocks to produce high chemicals, bioplastics, and biofuels. ß-Glucosidases are key enzymes responsible for degrading the plant cell wall polymers, as they cleave glucan-based oligo- and polysaccharides to generate glucose. Monosaccharide-tolerant or -stimulated ß-glucosidases have been reported in the past decade. Here, we describe a novel mechanism of ß-glucosidase stimulation by glucose and xylose. The glycoside hydrolase 1 family ß-glucosidase from Thermotoga petrophila (TpBgl1) displays a typical glucose stimulation mechanism based on an increased Vmax and decreased Km in response to glucose. Through molecular docking and dynamics analyses, we mapped putative monosaccharide binding regions (BRs) on the surface of TpBgl1. Our results indicate that after interaction with glucose or xylose at BR1 site, an adjacent loop region assumes an extended conformation, which increases the entrance to the TpBgl1 active site, improving product formation. Biochemical assays with TpBgl1 BR1 mutants, TpBgl1D49A/Y410A and TpBgl1D49K/Y410H, resulted in decreasing and abolishing monosaccharide stimulation, respectively. These mutations also impaired the BR1 looping extension responsible for monosaccharide stimulation. This study provides a molecular basis for the rational design of ß-glucosidases for biotechnological applications.

NTL9 Folding at Constant pH: The Importance of Electrostatic Interaction and pH Dependence.

The folding process of the N-terminal domain of ribosomal protein L9 (NTL9) was investigated at constant-pH computer simulations. Evaluation of the role of electrostatic interaction during folding was carried out by including a Debye-Hückel potential into a Cα structure-based model (SBM). In this study, the charges of the ionizable residues and the electrostatic potential are susceptible to the solution conditions, such as pH and ionic strength, as well as to the presence of charged groups. Simulations were performed under different pHs, and the results were validated by comparing them with experimental values of pKa and with denaturation experiment data. Also, the free energy profiles, Φ-values, and folding routes were calculated for each condition. It was shown how charges vary along the folding under different pH, which is subject to different scenarios. This study reveals how simplified models can capture essential physical features, reproducing experimental results, and presenting the role of electrostatic interactions before, during, and after the transition state.

Information and redundancy in the burial folding code of globular proteins within a wide range of shapes and sizes.

Recent ab initio folding simulations for a limited number of small proteins have corroborated a previous suggestion that atomic burial information obtainable from sequence could be sufficient for tertiary structure determination when combined to sequence-independent geometrical constraints. Here, we use simulations parameterized by native burials to investigate the required amount of information in a diverse set of globular proteins comprising different structural classes and a wide size range. Burial information is provided by a potential term pushing each atom towards one among a small number L of equiprobable concentric layers. An upper bound for the required information is provided by the minimal number of layers L(min) still compatible with correct folding behavior. We obtain L(min) between 3 and 5 for seven small to medium proteins with 50 ≤ Nr ≤ 110 residues while for a larger protein with Nr = 141 we find that L ≥ 6 is required to maintain native stability. We additionally estimate the usable redundancy for a given L ≥ L(min) from the burial entropy associated to the largest folding-compatible fraction of "superfluous" atoms, for which the burial term can be turned off or target layers can be chosen randomly. The estimated redundancy for small proteins with L = 4 is close to 0.8. Our results are consistent with the above-average quality of burial predictions used in previous simulations and indicate that the fraction of approachable proteins could increase significantly with even a mild, plausible, improvement on sequence-dependent burial prediction or on sequence-independent constraints that augment the detectable redundancy during simulations.

Oligomeric state and structural stability of two hyperthermophilic ß-glucosidases from Thermotoga petrophila.

The ß-glucosidases are enzymes essential for several industrial applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts. In a recent study, we have provided a biochemical characterization of two hyperthermostable ß-glucosidases from Thermotoga petrophila belonging to the families GH1 (TpBGL1) and GH3 (TpBGL3). Here, as part of a continuing investigation, the oligomeric state, the net charge, and the structural stability, at acidic pH, of the TpBGL1 and TpBGL3 were characterized and compared. Enzymatic activity is directly related to the balance between protonation and conformational changes. Interestingly, our results indicated that there were no significant changes in the secondary, tertiary and quaternary structures of the ß-glucosidases at temperatures below 80 °C. Furthermore, the results indicated that both the enzymes are stable homodimers in solution. Therefore, the observed changes in the enzymatic activities are due to variations in pH that modify protonation of the enzymes residues and the net charge, directly affecting the interactions with ligands. Finally, the results showed that the two ß-glucosidases displayed different pH dependence of thermostability at temperatures above 80 °C. TpBGL1 showed higher stability at pH 6 than at pH 4, while TpBGL3 showed similar stability at both pH values. This study provides a useful comparison of the structural stability, at acidic pH, of two different hyperthermostable ß-glucosidases and how it correlates with the activity of the enzymes. The information described here can be useful for biotechnological applications in the biofuel and food industries.

Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance.

Endo-ß-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features.

Comparative analysis of three hyperthermophilic GH1 and GH3 family members with industrial potential.

Beta-glucosidases (BGLs) are enzymes of great potential for several industrial processes, since they catalyze the cleavage of glucosidic bonds in cellobiose and other short cellooligosaccharides. However, features such as good stability to temperature, pH, ions and chemicals are required characteristics for industrial applications. This work aimed to provide a comparative biochemical analysis of three thermostable BGLs from Pyrococcus furiosus and Thermotoga petrophila. The genes PfBgl1 (GH1 from P. furiosus), TpBgl1 (GH1 from T. petrophila) and TpBgl3 (GH3 from T. petrophila) were cloned and proteins were expressed in Escherichia coli. The purified enzymes are hyperthermophilic, showing highest activity at temperatures above 80°C at acidic (TpBgl3 and PfBgl1) and neutral (TpBgl1) pHs. The BGLs showed greatest stability to temperature mainly at pH 6.0. Activities using a set of different substrates suggested that TpBgl3 (GH3) is more specific than GH1 family members. In addition, the influence of six monosaccharides on BGL catalysis was assayed. While PfBgl1 and TpBgl3 seemed to be weakly inhibited by monosaccharides, TpBgl1 was activated, with xylose showing the strongest activation. Under the conditions tested, TpBgl1 showed the highest inhibition constant (Ki=1100.00mM) when compared with several BGLs previously characterized. The BGLs studied have potential for industrial use, specifically the enzymes belonging to the GH1 family, due to its broad substrate specificity and weak inhibition by glucose and other saccharides.

Understanding the function of conserved variations in the catalytic loops of fungal glycoside hydrolase family 12.

Enzymes that cleave the xyloglucan backbone at unbranched glucose residues have been identified in GH families 5, 7, 12, 16, 44, and 74. Fungi produce enzymes that populate 20 of 22 families that are considered critical for plant biomass deconstruction. We searched for GH12-encoding genes in 27 Eurotiomycetes genomes. After analyzing 50 GH12-related sequences, the conserved variations of the amino acid sequences were examined. Compared to the endoglucanases, the endo-xyloglucanase-associated YSG deletion at the negative subsites of the catalytic cleft with a SST insertion at the reducing end of the substrate-binding crevice is highly conserved. In addition, a highly conserved alanine residue was identified in all xyloglucan-specific enzymes, and this residue is substituted by arginine in more promiscuous glucanases. To understand the basis for the xyloglucan specificity displayed by certain GH12 enzymes, two fungal GH12 endoglucanases were chosen for mutagenesis and functional studies: an endo-xyloglucanase from Aspergillus clavatus (AclaXegA) and an endoglucanase from A. terreus (AtEglD). Comprehensive molecular docking studies and biochemical analyses were performed, revealing that mutations at the entrance of the catalytic cleft in AtEglD result in a wider binding cleft and the alteration of the substrate-cleavage pattern, implying that a trio of residues coordinates the interactions and binding to linear glycans. The loop insertion at the crevice-reducing end of AclaXegA is critical for catalytic efficiency to hydrolyze xyloglucan. The understanding of the structural elements governing endo-xyloglucanase activity on linear and branched glucans will facilitate future enzyme modifications with potential applications in industrial biotechnology.

Deciphering the synergism of endogenous glycoside hydrolase families 1 and 9 from Coptotermes gestroi.

Termites can degrade up to 90% of the lignocellulose they ingest using a repertoire of endogenous and symbiotic degrading enzymes. Termites have been shown to secrete two main glycoside hydrolases, which are GH1 (EC 3.2.1.21) and GH9 (EC 3.2.1.4) members. However, the molecular mechanism for lignocellulose degradation by these enzymes remains poorly understood. The present study was conducted to understand the synergistic relationship between GH9 (CgEG1) and GH1 (CgBG1) from Coptotermes gestroi, which is considered the major urban pest of São Paulo State in Brazil. The goal of this work was to decipher the mode of operation of CgEG1 and CgBG1 through a comprehensive biochemical analysis and molecular docking studies. There was outstanding degree of synergy in degrading glucose polymers for the production of glucose as a result of the endo-ß-1,4-glucosidase and exo-ß-1,4-glucosidase degradation capability of CgEG1 in concert with the high catalytic performance of CgBG1, which rapidly converts the oligomers into glucose. Our data not only provide an increased comprehension regarding the synergistic mechanism of these two enzymes for cellulose saccharification but also give insight about the role of these two enzymes in termite biology, which can provide the foundation for the development of a number of important applied research topics, such as the control of termites as pests as well as the development of technologies for lignocellulose-to-bioproduct applications.

Characterization of a hexameric exo-acting GH51 α-l-arabinofuranosidase from the mesophilic Bacillus subtilis.

α-l-Arabinofuranosidases (α-l-Abfases, EC 3.2.1.55) display a broad specificity against distinct glycosyl moieties in branched hemicellulose and recent studies have demonstrated their synergistic use with cellulases and xylanases for biotechnological processes involving plant biomass degradation. In this study, we examined the structural organization of the arabinofuranosidase (GH51 family) from the mesophilic Bacillus subtilis (AbfA) and its implications on function and stability. The recombinant AbfA showed to be active over a broad temperature range with the maximum activity between 35 and 50 °C, which is desirable for industrial applications. Functional studies demonstrated that AbfA preferentially cleaves debranched or linear arabinan and is an exo-acting enzyme producing arabinose from arabinoheptaose. The enzyme has a canonical circular dichroism spectrum of α/ß proteins and exhibits a hexameric quaternary structure in solution, as expected for GH51 members. Thermal denaturation experiments indicated a melting temperature of 53.5 °C, which is in agreement with the temperature­activity curves. The mechanisms associated with the unfolding process were investigated through molecular dynamics simulations evidencing an important contribution of the quaternary arrangement in the stabilization of the ß-sandwich accessory domain and other regions involved in the formation of the catalytic interface of hexameric Abfases belonging to GH51 family.

Assembling a xylanase-lichenase chimera through all-atom molecular dynamics simulations.

Multifunctional enzyme engineering can improve enzyme cocktails for emerging biofuel technology. Molecular dynamics through structure-based models (SB) is an effective tool for assessing the tridimensional arrangement of chimeric enzymes as well as for inferring the functional practicability before experimental validation. This study describes the computational design of a bifunctional xylanase-lichenase chimera (XylLich) using the xynA and bglS genes from Bacillus subtilis. In silico analysis of the average solvent accessible surface area (SAS) and the root mean square fluctuation (RMSF) predicted a fully functional chimera, with minor fluctuations and variations along the polypeptide chains. Afterwards, the chimeric enzyme was built by fusing the xynA and bglS genes. XylLich was evaluated through small-angle X-ray scattering (SAXS) experiments, resulting in scattering curves with a very accurate fit to the theoretical protein model. The chimera preserved the biochemical characteristics of the parental enzymes, with the exception of a slight variation in the temperature of operation and the catalytic efficiency (kcat/Km). The absence of substantial shifts in the catalytic mode of operation was also verified. Furthermore, the production of chimeric enzymes could be more profitable than producing a single enzyme separately, based on comparing the recombinant protein production yield and the hydrolytic activity achieved for XylLich with that of the parental enzymes.

Substrate-specific reorganization of the conformational ensemble of CSK implicates novel modes of kinase function.

Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk) is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell.

Macromolecular assembly of polycystin-2 intracytosolic C-terminal domain.

Mutations in PKD2 are responsible for approximately 15% of the autosomal dominant polycystic kidney disease cases. This gene encodes polycystin-2, a calcium-permeable cation channel whose C-terminal intracytosolic tail (PC2t) plays an important role in its interaction with a number of different proteins. In the present study, we have comprehensively evaluated the macromolecular assembly of PC2t homooligomer using a series of biophysical and biochemical analyses. Our studies, based on a new delimitation of PC2t, have revealed that it is capable of assembling as a homotetramer independently of any other portion of the molecule. Our data support this tetrameric arrangement in the presence and absence of calcium. Molecular dynamics simulations performed with a modified all-atoms structure-based model supported the PC2t tetrameric assembly, as well as how different populations are disposed in solution. The simulations demonstrated, indeed, that the best-scored structures are the ones compatible with a fourfold oligomeric state. These findings clarify the structural properties of PC2t domain and strongly support a homotetramer assembly of PC2.

Proteins at work: a combined small angle X-RAY scattering and theoretical determination of the multiple structures involved on the protein kinase functional landscape.

C-terminal Src kinase (Csk) phosphorylates and down-regulates the Src family tyrosine kinases (SFKs). Crystallographic studies of Csk found an unusual arrangement of the SH2 and SH3 regulatory domains about the kinase core, forming a compact structure. However, recent structural studies of mutant Csk in the presence of an inhibitor indicate that the enzyme accesses an expanded structure. To investigate whether wt-Csk may also access open conformations we applied small angle x-ray scattering (SAXS). We find wt-Csk frequently occupies an extended conformation where the regulatory domains are removed from the kinase core. In addition, all-atom structure-based simulations indicate Csk occupies two free energy basins. These basins correspond to ensembles of distinct global conformations of Csk: a compact structure and an extended structure. The transitions between these structures are entropically driven and accessible via thermal fluctuations that break local interactions. We further characterized the ensemble by generating theoretical scattering curves for mixed populations of conformations from both basins and compared the predicted scattering curves to the experimental profile. This population-combination analysis is more consistent with the experimental data than any rigid model. It suggests that Csk adopts a broad ensemble of conformations in solution, populating extended conformations not observed in the crystal structure that may play an important role in the regulation of Csk. The methodology developed here is broadly applicable to biological macromolecules and will provide useful information about what ensembles of conformations are consistent with the experimental data as well as the ubiquitous dynamic reversible assembly processes inherent in biology.

Geometrical features of the protein folding mechanism are a robust property of the energy landscape: a detailed investigation of several reduced models.

The concept of a funneled energy landscape and the principle of minimal frustration are the theoretical foundation justifying the applicability of structure-based models. In simulations, a protein is commonly reduced to a C(alpha)-bead representation. These simulations are sufficient to predict the geometrical features of the folding mechanism observed experimentally utilizing a concise formulation of the Hamiltonian with low computational costs. Toward a better understanding of the interplay between energetic and geometrical features in folding, the side chain is now explicitly included in the simulations. The simplest choice is the addition of C(beta)-beads at the center-of-mass position of the side chains. While one varies the energetic parameters of the model, the geometric aspects of the folding mechanism remain robust for a broad range of parameters. Energetic properties like folding barriers and protein stability are sensitive to the details of simulations. This robustness to geometry and sensitivity to energetic properties provide flexibility in choosing different parameters to represent changes in sequences, environments, stability or folding rate effects. Therefore, minimal frustration and the funnel concept guarantee that the geometrical features are robust properties of the folding landscape, while mutations and/or changes in the environment easily influence energy-dependent properties like folding rates or stability.

Frustration and hydrophobicity interplay in protein folding and protein evolution.

A lattice model is used to study mutations and compacting effects on protein folding rates and folding temperature. In the context of protein evolution, we address the question regarding the best scenario for a polypeptide chain to fold: either a fast nonspecific collapse followed by a slow rearrangement to form the native structure or a specific collapse from the unfolded state with the simultaneous formation of the native state. This question is investigated for optimized sequences, whose native state has no frustrated contacts between monomers, and also for mutated sequences, whose native state has some degree of frustration. It is found that the best scenario for folding may depend on the amount of frustration of the native structure. The implication of this result on protein evolution is discussed.