Excited States of apo-Guanidine-III Riboswitch Contribute to Guanidinium Binding through Both Conformational and Induced-Fit Mechanisms.

Riboswitches are mRNA segments that regulate gene expression through conformational changes driven by their cognate ligand binding. The ykkC motif forms a riboswitch class that selectively senses a guanidinium ion (Gdm+) and regulates the downstream expression of proteins which aid in the efflux of excess Gdm+ from the cells. The aptamer domain (AD) of the guanidine-III riboswitch forms an H-type pseudoknot with a triple helical domain that binds a Gdm+. We studied the binding of Gdm+ to the AD of the guanidine (ykkC)-III riboswitch using computer simulations to probe the specificity of the riboswitch to Gdm+ binding. We show that Gdm+ binding is a fast process occurring on the nanosecond time scale, with minimal conformational changes to the AD. Using machine learning and Markov-state models, we identified the excited conformational states of the AD, which have a high Gdm+ binding propensity, making the Gdm+ binding landscape complex exhibiting both conformational selection and induced-fit mechanisms. The proposed apo-AD excited states and their role in the ligand-sensing mechanism are amenable to experimental verification. Further, targeting these excited-state conformations in discovering new antibiotics can be explored.

Surgical outcomes of common arterial trunk repair beyond infancy.

Background: The aim of this study is to analyze the clinical outcomes of common arterial trunk repair beyond infancy in terms of both early- and long-term outcomes. Methods: Between January 2003 and December 2019, 56 patients underwent repair for common arterial trunk beyond infancy at our institute. Median age was 34.5 months, 51.8% were females, and 48.2% were males. Results: 48.2% were type 1, 46.4% were type 2, and 5.4% were type 3. 17.9% patients underwent direct connection technique for right ventricular outflow tract reconstruction; remaining received a conduit. The most common type of truncal valve anatomy was tricuspid (82.1%). Early mortality was 7%. Univariable analysis identified age (p = 0.003), weight (p = 0.04), duration of ventilation (p = 0.036), and pulmonary hypertensive crisis (p ≤ 0.001) as factors affecting early mortality. In our overall cohort of beyond infancy repair for common arterial trunk, at 10 years, the survival, freedom from reintervention for right ventricular outflow tract reconstruction, freedom from ≥ moderate conduit obstruction, freedom from impaired right ventricle function, and freedom from ≥ moderate truncal valve regurgitation were 76.7%, 89.7%, 74%, 88.6%, and 66.3%, respectively. Conclusion: Repair for common arterial trunk in patients presenting beyond 1 year of age is challenging; however, it can be done with satisfactory early and late outcomes in terms of mortality and reintervention.

Mechanism of Fluoride Ion Encapsulation by Magnesium Ions in a Bacterial Riboswitch.

Riboswitches sense various ions in bacteria and activate gene expression to synthesize proteins that help maintain ion homeostasis. The crystal structure of the aptamer domain (AD) of the fluoride riboswitch shows that the F- ion is encapsulated by three Mg2+ ions bound to the ligand-binding domain (LBD) located at the core of the AD. The assembly mechanism of this intricate structure is unknown. To this end, we performed computer simulations using coarse-grained and all-atom RNA models to bridge multiple time scales involved in riboswitch folding and ion binding. We show that F- encapsulation by the Mg2+ ions bound to the riboswitch involves multiple sequential steps. Broadly, two Mg2+ ions initially interact with the phosphate groups of the LBD using water-mediated outer-shell coordination and transition to a direct inner-shell interaction through dehydration to strengthen their interaction with the LBD. We propose that the efficient binding mode of the third Mg2+ and F- is that they form a water-mediated ion pair and bind to the LBD simultaneously to minimize the electrostatic repulsion between three Mg2+ bound to the LBD. The tertiary stacking interactions among the LBD nucleobases alone are insufficient to stabilize the alignment of the phosphate groups to facilitate Mg2+ binding. We show that the stability of the whole assembly is an intricate balance of the interactions among the five phosphate groups, three Mg2+, and the encapsulated F- ion aided by the Mg2+ solvated water. These insights are helpful in the rational design of RNA-based ion sensors and fast-switching logic gates.

Allosteric Determinants in High Temperature Requirement A Enzymes Are Conserved and Regulate the Population of Active Conformations.

High temperature requirement A (HtrA) are allosterically regulated enzymes wherein effector binding to the PDZ domain triggers proteolytic activity. Yet, it remains unclear if the inter-residue network governing allostery is conserved across HtrA enzymes. Here, we investigated and identified the inter-residue interaction networks by molecular dynamics simulations on representative HtrA proteases, Escherichia coli DegS and Mycobacterium tuberculosis PepD, in effector-bound and free forms. This information was used to engineer mutations that could potentially perturb allostery and conformational sampling in a different homologue, M. tuberculosis HtrA. Mutations in HtrA perturbed allosteric regulation─a finding consistent with the hypothesis that the inter-residue interaction network is conserved across HtrA enzymes. Electron density from data collected on cryo-protected HtrA crystals revealed that mutations altered the topology of the active site. Ensemble models fitted into electron density calculated from room-temperature diffraction data showed that only a fraction of these models had a catalytically competent active site conformation alongside a functional oxyanion hole thus providing experimental evidence that these mutations influenced conformational sampling. Mutations at analogous positions in the catalytic domain of DegS perturbed the coupling between effector binding and proteolytic activity, thus confirming the role of these residues in the allosteric response. The finding that a perturbation in the conserved inter-residue network alters conformational sampling and the allosteric response suggests that an ensemble allosteric model best describes regulated proteolysis in HtrA enzymes.

pH Regulates Ligand Binding to an Enzyme Active Site by Modulating Intermediate Populations.

Understanding the mechanism of ligands binding to their protein targets and the influence of various factors governing the binding thermodynamics is essential for rational drug design. The solution pH is one of the critical factors that can influence ligand binding to a protein cavity, especially in enzymes whose function is sensitive to the pH. Using computer simulations, we studied the pH effect on the binding of a guanidinium ion (Gdm+) to the active site of hen egg-white lysozyme (HEWL). HEWL serves as a model system for enzymes with two acidic residues in the active site and ligands with Gdm+ moieties, which can bind to the active sites of such enzymes and are present in several approved drugs treating various disorders. The computed free energy surface (FES) shows that Gdm+ binds to the HEWL active site using two dominant binding pathways populating multiple intermediates. We show that the residues close to the active site that can anchor the ligand could play a critical role in ligand binding. Using a Markov state model, we quantified the lifetimes and kinetic pathways connecting the different states in the FES. The protonation and deprotonation of the acidic residues in the active site in response to the pH change strongly influence the Gdm+ binding. There is a sharp jump in the ligand-binding rate constant when the pH approaches the largest pKa of the acidic residue present in the active site. The simulations reveal that, at most, three Gdm+ can bind at the active site, with the Gdm+ bound in the cavity of the active site acting as a scaffold for the other two Gdm+ ions binding. These results can aid in providing greater insights into designing novel molecules containing Gdm+ moieties that can have high binding affinities to inhibit the function of enzymes with acidic residues in their active site.

pH Induced Switch in the Conformational Ensemble of Intrinsically Disordered Protein Prothymosin-α and Its Implications for Amyloid Fibril Formation.

Aggregation of intrinsically disordered proteins (IDPs) can lead to neurodegenerative diseases. Although there is experimental evidence that acidic pH promotes IDP monomer compaction leading to aggregation, the general mechanism is unclear. We studied the pH effect on the conformational ensemble of prothymosin-α (proTα), which is involved in multiple essential functions, and probed its role in aggregation using computer simulations. We show that compaction in the proTα dimension at low pH is due to the protein's collapse in the intermediate region (E41-D80) rich in glutamic acid residues, enhancing its ß-sheet content. We observed by performing dimer simulations that the conformations with high ß-sheet content could act as aggregation-prone (N*) states and nucleate the aggregation process. The simulations initiated using N* states form dimers within a microsecond time scale, whereas the non-N* states do not form dimers within this time scale. This study contributes to understanding the general principles of pH-induced IDP aggregation.

Salt-Induced Transitions in the Conformational Ensembles of Intrinsically Disordered Proteins.

Salts modulate the behavior of intrinsically disordered proteins (IDPs) and influence the formation of membraneless organelles through liquid-liquid phase separation (LLPS). In low ionic strength solutions, IDP conformations are perturbed by the screening of electrostatic interactions, independent of the salt identity. In this regime, insight into the IDP behavior can be obtained using the theory for salt-induced transitions in charged polymers. However, salt-specific interactions with the charged and uncharged residues, known as the Hofmeister effect, influence IDP behavior in high ionic strength solutions. There is a lack of reliable theoretical models in high salt concentration regimes to predict the salt effect on IDPs. We propose a simulation methodology using a coarse-grained IDP model and experimentally measured water to salt solution transfer free energies of various chemical groups that allowed us to study the salt-specific transitions induced in the IDPs conformational ensemble. We probed the effect of three different monovalent salts on five IDPs belonging to various polymer classes based on charged residue content. We demonstrate that all of the IDPs of different polymer classes behave as self-avoiding walks (SAWs) at physiological salt concentration. In high salt concentrations, the transitions observed in the IDP conformational ensembles are dependent on the salt used and the IDP sequence and composition. Changing the anion with the cation fixed can result in the IDP transition from a SAW-like behavior to a collapsed globule. An important implication of these results is that a suitable salt can be identified to induce condensation of an IDP through LLPS.

TPP Riboswitch Populates Holo-Form-like Structure Even in the Absence of Cognate Ligand at High Mg2+ Concentration.

Riboswitches are noncoding RNA that regulate gene expression by folding into specific three-dimensional structures (holo-form) upon binding by their cognate ligand in the presence of Mg2+. Riboswitch functioning is also hypothesized to be under kinetic control requiring large cognate ligand concentrations. We ask the question under thermodynamic conditions, can the riboswitches populate structures similar to the holo-form only in the presence of Mg2+ and absence of cognate ligand binding. We addressed this question using thiamine pyrophosphate (TPP) riboswitch as a model system and computer simulations using a coarse-grained model for RNA. The folding free energy surface (FES) shows that with the initial increase in Mg2+ concentration ([Mg2+]), the aptamer domain (AD) of TPP riboswitch undergoes a barrierless collapse in its dimensions. On further increase in [Mg2+], intermediates separated by barriers appear on the FES, and one of the intermediates has a TPP ligand-binding competent structure. We show that site-specific binding of the Mg2+ aids in the formation of tertiary contacts. For [Mg2+] greater than physiological concentration, AD folds into a structure similar to the crystal structure of the TPP holo-form even in the absence of the TPP ligand. The folding kinetics shows that TPP AD populates an intermediate due to the misalignment of two arms present in the structure, which acts as a kinetic trap, leading to larger folding timescales. The predictions of the intermediate structures from the simulations are amenable for experimental verification.

Energy Landscape of Ubiquitin Is Weakly Multidimensional.

Single molecule pulling experiments report time-dependent changes in the extension (X) of a biomolecule as a function of the applied force (f). By fitting the data to one-dimensional analytical models of the energy landscape, we can extract the hopping rates between the folded and unfolded states in two-state folders as well as the height and the location of the transition state (TS). Although this approach is remarkably insightful, there are cases for which the energy landscape is multidimensional (catch bonds being the most prominent). To assess if the unfolding energy landscape in small single domain proteins could be one-dimensional, we simulated force-induced unfolding of ubiquitin (Ub) using the coarse-grained self-organized polymer-side chain (SOP-SC) model. Brownian dynamics simulations using the SOP-SC model reveal that the Ub energy landscape is weakly multidimensional (WMD), governed predominantly by a single barrier. The unfolding pathway is confined to a narrow reaction pathway that could be described as diffusion in a quasi-1D X-dependent free energy profile. However, a granular analysis using the Pfold analysis, which does not assume any form for the reaction coordinate, shows that X alone does not account for the height and, more importantly, the location of the TS. The f-dependent TS location moves toward the folded state as f increases, in accord with the Hammond postulate. Our study shows that, in addition to analyzing the f-dependent hopping rates, the transition state ensemble must also be determined without resorting to X as a reaction coordinate to describe the unfolding energy landscapes of single domain proteins, especially if they are only WMD.

Asymmetry in histone rotation in forced unwrapping and force quench rewrapping in a nucleosome.

Single molecule pulling experiments have shown that DNA in the nucleosomes unwraps in two stages from the histone protein core (HPC). The first stage, attributed to the rupture of the outer DNA turn, occurs between 3 and 5 pNs, and is reversible. The inner DNA turn ruptures irreversibly at forces between 9 and 15 pNs (or higher) in the second stage. Molecular simulations using the Self-Organized Polymer model capture the experimental findings. The unwrapping of the outer DNA turn is independent of the pulling direction. The rupture of the DNA inner turn depends on the pulling direction and involves overcoming substantial energetic (most likely electrostatic in origin) and kinetic barriers. They arise because the mechanical force has to generate sufficient torque to rotate the HPC by 180°. On the other hand, during the rewrapping process, HPC rotation is stochastic, with force playing no role. The assembly of the outer DNA wrap upon force quench nearly coincides with the unwrapping process, confirming the reversibility of the outer turn rupture. The asymmetry in HPC rotation during unwrapping and rewrapping explains the observed hysteresis in the stretch-release cycles in experiments. We propose experiments to test the prediction that HPC rotation produces kinetic barriers in the unwrapping process.

Double Domain Swapping in Human γC and γD Crystallin Drives Early Stages of Aggregation.

Human γD (HγD) and γC (HγC) are two-domain crystallin (Crys) proteins expressed in the nucleus of the eye lens. Structural perturbations in the protein often trigger aggregation, which eventually leads to cataract. To decipher the underlying molecular mechanism, it is important to characterize the partially unfolded conformations, which are aggregation-prone. Using a coarse grained protein model and molecular dynamics simulations, we studied the role of on-pathway folding intermediates in the early stages of aggregation. The multidimensional free energy surface revealed at least three different folding pathways with the population of partially structured intermediates. The two dominant pathways confirm sequential folding of the N-terminal [Ntd] and the C-terminal domains [Ctd], while the third, least favored, pathway involves intermediates where both the domains are partially folded. A native-like intermediate (I*), featuring the folded domains and disrupted interdomain contacts, gets populated in all three pathways. I* forms domain swapped dimers by swapping the entire Ntds and Ctds with other monomers. Population of such oligomers can explain the increased resistance to unfolding resulting in hysteresis observed in the folding experiments of HγD Crys. An ensemble of double domain swapped dimers are also formed during refolding, where intermediates consisting of partially folded Ntds and Ctds swap secondary structures with other monomers. The double domain swapping model presented in our study provides structural insights into the early events of aggregation in Crys proteins and identifies the key secondary structural swapping elements, where introducing mutations will aid in regulating the overall aggregation propensity.

Mg2+ Sensing by an RNA Fragment: Role of Mg2+-Coordinated Water Molecules.

RNA molecules selectively bind to specific metal ions to populate their functional active states, making it important to understand their source of ion selectivity. In large RNA systems, metal ions interact with the RNA at multiple locations, making it difficult to decipher the precise role of ions in folding. To overcome this complexity, we studied the role of different metal ions (Mg2+, Ca2+, and K+) in the folding of a small RNA hairpin motif (5'-ucCAAAga-3') using unbiased all-atom molecular dynamics simulations. The advantage of studying this system is that it requires specific binding of a single metal ion to fold to its native state. We find that even for this small RNA, the folding free energy surface (FES) is multidimensional as different metal ions present in the solution can simultaneously facilitate folding. The FES shows that specific binding of a metal ion is indispensable for its folding. We further show that in addition to the negatively charged phosphate groups, the spatial organization of electronegative nucleobase atoms drives the site-specific binding of the metal ions. Even though the binding site cannot discriminate between different metal ions, RNA folds efficiently only in a Mg2+ solution. We show that the rigid network of Mg2+-coordinated water molecules facilitates the formation of important interactions in the transition state. The other metal ions such as K+ and Ca2+ cannot facilitate the formation of such interactions. These results allow us to hypothesize possible metal-sensing mechanisms in large metalloriboswitches and also provide useful insights into the design of appropriate collective variables for studying large RNA molecules using enhanced sampling methods.

Mutual Diffusivity of an n-Hexane-2,2-Dimethyl Butane Binary Mixture Confined to Zeolite Y.

A molecular dynamic study of a mixture of n-hexane and 2,2-dimethyl butane (22DMB) confined to zeolite NaY has been carried out to understand the distinct diffusivity and mutual diffusivity. Results have been compared with the bulk mixture. For each of these mixtures, eight different runs were employed to compute distinct and mutual diffusivity. From the velocity auto- and cross-correlation functions between n-hexane and n-hexane, n-hexane and 22DMB, 22DMB and 22DMB, the self- and distinct diffusivity of the mixture has been computed. The thermodynamic factor and mutual diffusivity have been calculated. The ratio of D11 to Ds is seen to be 1.11 and 0.75 for the confined mixture, while they are 1.21 and 0.79 for the bulk mixture at 200 and 300 K, respectively.

Cosolvent effects on the growth of amyloid fibrils.

Cells are equipped with cosolvents that modulate protein folding and aggregation to withstand water stress. The effect of cosolvents on the aggregation rates depends on whether the polypeptide sequence is an intrinsically disordered protein (IDP) or can fold into a specific native structure. Cosolvents, which act as denaturants generally slow down aggregation in IDPs, while expediting it in globular proteins. In contrast, protecting osmolytes facilitate aggregation in IDPs, while slowing it down in globular proteins. In this review we highlight the recent computational approaches to gain insight into the role of cosolvents on the aggregation mechanism of IDPs and globular proteins. Computer simulations using the molecular transfer model, which implements the cosolvent effects in coarse-grained protein models in conjunction with enhanced sampling techniques played an important role in elucidating the effect of cosolvents on the growth of amyloid fibrils.

Shared hydrogen bonds: water in aluminated faujasite.

Water confined in faujasite, a zeolite, with aluminium content, exhibits properties different from those of bulk water as well as water confined in siliceous faujasite. The RDF between oxygen of water (OW) and oxygen of aluminium (OAl) shows a prominent first peak near to 2.9 Å similar to any oxygen-oxygen RDF seen in bulk water and unlike water confined in siliceous faujasite. Further, HW-OAl shows a peak near 1.9 Å suggesting hydrogen bonding between hydrogen of water and OAl. The water satisfies the hydrogen bond criteria with both O1Al and O2Al indicating that it is participating in a shared hydrogen bond. The hydrogen bond exchange between such a water forming a shared hydrogen bond to OAl and another water molecule H2Ob is investigated through the changes in the distances and appropriate angles. The O-Al-O angle of the zeolite increases by about 7 degrees on the formation of the shared hydrogen bond. The jump dynamics of the shared hydrogen bond when the two bonds break simultaneously has been obtained and this is reported. This jump reorientation dynamics is different compared to normal hydrogen bonding reported by Laage and Hynes: it has a short lifetime, around 50-100 fs computed from SHB(t). The intermittent and continuous hydrogen bond correlation functions are also reported.

A Transient Intermediate Populated in Prion Folding Leads to Domain Swapping.

Aggregation of misfolded prion proteins causes fatal neurodegenerative disorders in both humans and animals. There is an extensive effort to identify the elusive aggregation-prone conformations (N*) of prions, which are early stage precursors to aggregation. We studied temperature- and force-induced unfolding of the structured C-terminal domain of mouse (moPrP) and human prion proteins (hPrP) using molecular dynamics simulations and coarse-grained protein models. We find that these proteins sparsely populate intermediate states bearing the features of N* and readily undergo domain-swapped dimerization by swapping the short ß-strands present at the beginning of the C-terminal domain. The structure of the N* state is similar for both moPrP and hPrP, indicating a common pathogenic precursor across different species. Interestingly, disease-resistant hPrP (G127V) showed a drastic reduction in the population of the N* state further hinting a pathogenic connection to these partially denatured conformations. This study proposes a plausible runaway domain-swapping mechanism to describe the onset of prion aggregation.

Role of Guanidinium-Carboxylate Ion Interaction in Enzyme Inhibition with Implications for Drug Design.

Guanidinium cation (Gdm+) interacts strongly with amino acids of different polarities modulating protein structure and function. Using density functional theory calculations and molecular dynamics simulations, we studied the interaction of Gdm+ with carboxylate ions mimicking its interaction with acidic amino acids and explored its effect in enzymatic folding and activity. We show that, in low concentrations, Gdm+ stabilizes carboxylate ion dimers by acting as a bridge between them, thereby reducing the electrostatic repulsion. We further show that this carboxylate-Gdm+-carboxylate interaction can have an effect on the structure-activity relationship in enzymes with active sites containing two acidic residues. Using five enzymes (hen egg white lysozyme, T4 lysozyme, HIV-1 protease, pepsin, and creatine kinase), which have two acidic amino acids in their active sites, we show that, in low concentrations (<0.5 M), Gdm+ strongly binds to the enzyme active site, thereby potentially inhibiting its activity without unfolding it. This can lead to misleading conclusions in experiments, which infer the extent of enzyme unfolding from activity measurements. However, the carboxylate-Gdm+-carboxylate specific interaction can be exploited in drug discovery as drugs based on guanidinium derivatives are already being used to treat various maladies related to muscle weakness, cancer, diabetes etc. Guanidinium derivatives can be designed as potential drug molecules to inhibit activity or functioning of enzymes, which have binding pockets with two acidic residues in close vicinity.

Universal Nature of Collapsibility in the Context of Protein Folding and Evolution.

Theory and simulations predicted that the sizes of the unfolded states of globular proteins should decrease as the denaturant concentration is reduced from a high to a low value. However, small angle X-ray scattering (SAXS) data were used to assert the opposite, while interpretation of single molecule Förster resonance energy transfer experiments (FRET) supported the theoretical predictions. The disagreement between the two experiments is the SAXS-FRET controversy. By harnessing recent advances in SAXS and FRET experiments and setting these findings in the context of a general theory and simulations, which do not rely on experimental data, we establish that compaction of unfolded states under native conditions is universal. The theory also predicts that proteins rich in ß-sheets are more collapsible than α-helical proteins. Because the extent of compaction is small, experiments have to be accurate and their interpretations should be as model-free as possible. Theory also suggests that collapsibility itself could be a physical restriction on the evolution of foldable sequences, and also provides a physical basis for the origin of multidomain proteins.

Role of Disulfide Bonds and Topological Frustration in the Kinetic Partitioning of Lysozyme Folding Pathways.

Disulfide bonds in proteins can strongly influence the folding pathways by constraining the conformational space. Lysozyme has four disulfide bonds and is widely studied for its antibacterial properties. Experiments on lysozyme infer that the protein folds through a fast and a slow pathway. However, the reasons for the kinetic partitioning in the folding pathways are not completely clear. Using a coarse-grained protein model and simulations, we show that two out of the four disulfide bonds, which are present in the α-domain of lysozyme, are responsible for the slow folding pathway. In this pathway, a kinetically trapped intermediate state, which is close to the native state, is populated. In this state, the orientations of α-helices present in the α-domain are misaligned relative to each other. The protein in this state has to partially unfold by breaking down the interhelical contacts between the misaligned helices to fold to the native state. However, the topological constraints due to the two disulfide bonds present in the α-domain make the protein less flexible, and it is trapped in this conformation for hundreds of milliseconds. On disabling these disulfide bonds, we find that the kinetically trapped intermediate state and the slow folding pathway disappear. Simulations mimicking the folding of protein without disulfide bonds under oxidative conditions show that the native disulfide bonds are formed as the protein folds, indicating that folding guides the formation of disulfide bonds. The sequence of formation of the disulfide bonds is Cys64-Cys80 → Cys76-Cys94 → Cys30-Cys115 → Cys6-Cys127. Any disulfide bond that forms before its precursor in the sequence has to break and follow the sequence for the protein to fold. These results show that lysozyme also serves as a very good model system to probe the role of disulfide bonds and topological frustration in protein folding. The predictions from the simulations can be verified by single-molecule fluorescence resonance energy transfer or single-molecule pulling experiments, which can probe heterogeneity in the folding pathways.

Cosolvent Effects on the Growth of Protein Aggregates Formed by a Single Domain Globular Protein and an Intrinsically Disordered Protein.

Cosolvents modulate the stability of protein conformations and exhibit contrasting effects on the kinetics of aggregation by globular proteins and intrinsically disordered proteins (IDPs). The growth of ordered protein aggregates after the initial nucleation step is believed to proceed through a dock-lock mechanism. We have studied the effect of two denaturants [guanidinium chloride (GdmCl) and urea] and four protective osmolytes (trimethylamine N-oxide (TMAO), sucrose, sarcosine, and sorbitol) on the free energy surface (FES) of the dock-lock growth step of protein aggregation using a coarse-grained protein model and metadynamics simulations. We have used the proteins cSrc-SH3 and Aß9-40 as model systems representing globular proteins and IDPs, respectively. The effect of cosolvents on protein conformations is taken into account using the molecular transfer model (MTM). The computed FES shows that protective osmolytes stabilize the compact aggregates, while denaturants destabilize them for both cSrc-SH3 and Aß9-40. However, protective osmolytes increase the effective energy barrier for the multistep domain-swapped dimerization of cSrc-SH3, which is critical to the growth of protein aggregates by globular proteins, thus slowing down the overall aggregation rate. Contrastingly, denaturants decrease the effective barrier height for cSrc-SH3 dimerization and hence enhance the aggregation rate in globular proteins. The simulations further show that cSrc-SH3 monomers unfold before dimerization and the barrier to monomer unfolding regulates the effective rate of aggregation. In the case of IDP, Aß9-40, protective osmolytes decrease and denaturants increase the effective barriers in the dock-lock mechanism of fibril growth, leading to faster and slower growth kinetics, respectively.