Protein Conformational Stability Research Articles

Controllable synthesis of hydrogen-bonded organic framework encapsulated enzyme for continuous production of chiral hydroxybutyric acid in a two-stage cascade microreactor

Constructing a framework carrier to stabilize protein conformation, induce high embedding efficiency, and acquire low mass-transfer resistance is an urgent issue in the development of immobilized enzymes. Hydrogen-bonded organic frameworks (HOFs) have promising application potential for embedding enzymes. In fact, no metal involvement is required, and HOFs exhibit superior biocompatibility, and free access to substrates in mesoporous channels. Herein, a facile in situ growth approach was proposed for the self-assembly of alcohol dehydrogenase encapsulated in HOF. The micron-scale bio-catalytic composite was rapidly synthesized under mild conditions (aqueous phase and ambient temperature) with a controllable embedding rate. The high crystallinity and periodic arrangement channels of HOF were preserved at a high enzyme encapsulation efficiency of 59%. This bio-composite improved the tolerance of the enzyme to the acid-base environment and retained 81% of its initial activity after five cycles of batch hydrogenation involving NADH coenzyme. Based on this controllably synthesized bio-catalytic material and a common lipase, we further developed a two-stage cascade microchemical system and achieved the continuous production of chiral hydroxybutyric acid (R-3-HBA).

Hemoglobin-PEG Interactions Probed by Small-Angle X-ray Scattering: Insights for Crystallization and Diagnostics Applications.

Protein-protein interactions, controlling protein aggregation in the solution phase, are crucial for the formulation of protein therapeutics and the use of proteins in diagnostic applications. Additives in the solution phase are factors that may enhance the protein's conformational stability or induce crystallization. Protein-PEG interactions do not always stabilize the native protein structure. Structural information is needed to validate excipients for protein stabilization in the development of protein therapeutics or use proteins in diagnostic assays. The present study investigates the impact of polyethylene glycol (PEG) molecular weight and concentration on the spatial structure of human hemoglobin (Hb) at neutral pH. Small-angle X-ray scattering (SAXS) in combination with size-exclusion chromatography is employed to characterize the Hb structure in solution without and with the addition of PEG. Our results evidence that human hemoglobin maintains a tetrameric conformation at neutral pH. The dummy atom model, reconstructed from the SAXS data, aligns closely with the known crystallographic structure of methemoglobin (metHb) from the Protein Data Bank. We established that the addition of short-chain PEG600, at concentrations of up to 10% (w/v), acts as a stabilizer for hemoglobin, preserving its spatial structure without significant alterations. By contrast, 5% (w/v) PEG with higher molecular weights of 2000 and 4000 leads to a slight reduction in the maximum particle dimension (Dmax), while the radius of gyration (Rg) remains essentially unchanged. This implies a reduced hydration shell around the protein due to the dehydrating effect of longer PEG chains. At a concentration of 10% (w/v), PEG2000 interacts with Hb to form a complex that does not distort the protein's spatial configuration. The obtained results provide a deeper understanding of PEG's influence on the Hb structure in solution and broader knowledge regarding protein-PEG interactions.

Relationship between protein conformational stability and its immunogenicity when administering antigens to mice using adjuvants-Analysis employed the CH2 domain in human antibodies.

Antigen-presenting cells (APCs) play a crucial role in the immune system by breaking down antigens into peptide fragments that subsequently bind to major histocompatibility complex (MHC) molecules. Previous studies indicate that stable proteins can impede CD4+ T cell stimulation by hindering antigen processing and presentation. Conversely, certain proteins require stabilization in order to activate the immune response. Several factors, including the characteristics of the protein and the utilization of different adjuvants in animal experiments, may contribute to this disparity. In this study, we investigated the impact of adjuvants on antigen administration in mice, specifically focusing on the stability of the CH2 domain. Consequently, the CH2 domain induced a stronger IgG response in comparison to the stabilized one when using Alum and PBS (without adjuvant). On the other hand, animal experiment using Freund's adjuvant showed the opposite results. These findings indicate the significance of considering the intrinsic conformational stability of a protein when eliciting its immunogenicity, particularly within the context of vaccine development.

Molecular Insights into the Conformational and Binding Behaviors of Human Serum Albumin Induced by Surface-Active Ionic Liquids.

Extensive research has been carried out to investigate the stability and function of human serum albumin (HSA) when exposed to surface-active ionic liquids (SAILs) with different head groups (imidazolium, morpholinium, and pyridinium) and alkyl chain lengths (ranging from decyl to tetradecyl). Analysis of the protein fluorescence spectra indicates noticeable changes in the secondary structure of HSA with varying concentrations of all SAILs tested. Helicity calculations based on the Fourier transform infrared (FTIR) data show that HSA becomes more organized at the micellar concentration of SAILs, leading to an increased protein activity at this level. Small-angle neutron scattering (SANS) data confirm the formation of a bead-necklace structure between the SAILs and HSA. Atomistic molecular dynamics (MD) simulation results identify several hotspots on the protein surface for interaction with SAIL, which results in the modulation of protein conformational fluctuation and stability. Furthermore, fluorescence resonance energy transfer (FRET) experiments with the intramolecular charge transfer (ICT) probe trans-ethyl p-(dimethylamino) cinnamate (EDAC) demonstrate that higher alkyl chain lengths and SAIL concentrations result in a significantly increased energy transfer efficiency. The findings of this study provide a detailed molecular-level understanding of how the protein structure and function are affected by the presence of SAILs, with potential implications for a wide range of applications involving protein-SAIL composite systems.

Molecular dynamics simulations of betaine-based deep eutectic solvents with varying iso-alcohols chain lengths

Betaine-based (BET) deep eutectic solvents (DESs) made with isoalcohols of varying hydrocarbon chain lengths, namely triethylene glycol (TEG), 1, 2-ethanediol (ETD), 1, 3-propanediol (PPD) and 1, 4-butanediol (BTD) were modelled. Their effects on the formation of aqueous biphasic system (ABS) with 2 M dipotassium phosphate salt solution (K2HPO4) as well as the extraction of common protein molecules, bovine serum albumin (BSA), were studied using molecular dynamics simulation with Gromacs. Qualitative and quantitative data were used to analyse the simulation results, which includes visualised systems in equilibrium, root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration, radial distribution function (RDF) and hydrogen bond analysis. The results and literature research validated the role of inorganic salt solute concentration on the phase forming ability as well as the possibility of protein denaturation. More investigations and experimental works are to be carried out regarding the type of inorganic salt and its concentration for aqueous biphasic systems (ABSs) with alcohol based DESs and salt solution for protein extraction purposes. This is essential to understand the salting-out effect and to isolate the effects of hydrocarbon chain length from other influencing factors. Additionally, inorganic salt concentrations, presence of ether linkage as well as hydrocarbon chain length is shown to play a role in the dynamics between molecules in the system as well as protein conformational changes. Overall, ABS incorporating PPD and BTD with hydrocarbon chain lengths not exceeding C4 demonstrated superior performance in terms of protein conformational stability and interaction, showcasing their efficacy in protein extraction process.

Effect of surfactants on lactate dehydrogenase aqueous solutions: A comparative study of poloxamer 188, polysorbate 20 and 80

The effect of three commonly used surfactants, poloxamer 188 (P188), polysorbate 20 and 80 (PS20 and PS80), on the stability of a model protein, lactate dehydrogenase (LDH), was compared in aqueous solutions. In the absence of a surfactant, protein solution revealed a gradual decrease in surface tension as a function of time. The addition of surfactant resulted in a rapid decrease in the surface tension. This suggested that the surface behavior was dictated by the surfactant. PS20 and PS80 were more effective than P188 in preventing LDH adsorption on the solution surface. The advantage of polysorbates over P188 was also evident from the higher LDH tetramer recovery after shaking (room temperature, 30 h), especially when the surfactants were used at concentrations ≤ 0.01% w/v. However, PS20 and PS80 accelerated protein unfolding during quiescent storage at 40 °C. Based on circular dichroism results, polysorbates perturbed the tertiary structure of LDH but not the secondary structure, while P188 did not impact the protein structure and stability. Polysorbates were more effective in stabilizing LDH against mechanical stress (shaking), but their adverse effects on protein conformational stability need to be carefully evaluated.

Analysis of the effect of cations on protein conformational stability using solid-state nanopores.

The conformation of proteins is closely related to their biological functions, and it is affected by many factors, including the type of cations in solution. However, it is difficult to detect the conformational changes of a protein in situ. As a single-molecule sensing technology, nanopores can convert molecular structural information into analyzable current signals within a reasonable time range. Herein, we detect and analyze the effects of two different types of monovalent cations (Na+ and Li+) on a model protein bovine serum albumin (BSA) conformation using SiNx nanopores with different diameters. The quantitative analysis results show that the excluded volume of BSA in LiCl salt solutions is larger than the value in NaCl solution, indicating that Li+ is more prone to unfolding the proteins and making them unstable. This study demonstrated that nanopores enable the in situ detection of the structure of proteins at the single-molecule level and provide a new approach for the quantitative analysis of proteins.

Proscan: a structure-based proline design web server.

The ability to control protein conformations and dynamics through structure-based design has been useful in various scenarios, including engineering of viral antigens for vaccines. One effective design strategy is the substitution of residues to proline amino acids, which due to its unique cyclic side chain can favor and rigidify key backbone conformations. To provide the community with a means to readily identify and explore proline designs for target proteins of interest, we developed the Proscan web server. Proscan provides assessment of backbone angles, energetic and deep learning-based favorability scores, and other parameters for proline substitutions at each position of an input structure, along with interactive visualization of backbone angles and candidate substitution sites on structures. It identifies known favorable proline substitutions for viral antigens, and was benchmarked against datasets of proline substitution stability effects from deep mutational scanning and thermodynamic measurements. This tool can enable researchers to identify and prioritize designs for prospective vaccine antigen targets, or other designs to favor stability of key protein conformations. Proscan is available at: https://proscan.ibbr.umd.edu.

Modulation of Aggregation and Immunogenicity of a Protein: Based on the Study of Hen Lysozyme

This study focuses on the modulation of protein aggregation and immunogenicity. As a starting point for investigating long-range interactions within a non-native protein, the effects of perturbing denatured protein states on their aggregation, including the formation of amyloid fibrils, were evaluated. The effects of adducts, sugar modifications, and stabilization on protein aggregation were then examined. We also investigated how protein immunogenicity was affected by enhancing protein conformational stability and other factors.

Predicted deleterious variants in ABCA1, LPL, LPA and KIF6 are associated with statin response and adverse events in patients with familial hypercholesterolemia and disturb protein structure and stability.

This study explored the association of deleterious variants in pharmacodynamics (PD) genes with statin response and adverse effects in patients with familial hypercholesterolemia (FH) and analyzed their potential effects on protein structure and stability. Clinical and laboratory data were obtained from 144 adult FH patients treated with statins. A panel of 32 PD genes was analyzed by exon-targeted gene sequencing. Deleterious variants were identified using prediction algorithms and their structural effects were analyzed by molecular modeling studies. A total of 102 variants were predicted as deleterious (83 missense, 8 stop-gain, 4 frameshift, 1 indel, 6 splicing). The variants ABCA1 rs769705621 (indel), LPA rs41267807 (p.Tyr2023Cys) and KIF6 rs20455 (p.Trp719Arg) were associated with reduced low-density lipoprotein cholesterol (LDLc) response to statins, and the LPL rs1801177 (p.Asp36Asn) with increased LDLc response (P < 0.05). LPA rs3124784 (p.Arg2016Cys) was predicted to increase statin response (P = 0.022), and ABCA1 rs769705621 to increase the risk of statin-related adverse events (SRAE) (P = 0.027). LPA p.Arg2016Cys and LPL p.Asn36Asp maintained interactions with solvent, LPA p.Tyr2023Cys reduced intramolecular interaction with Gln1987, and KIF6 p.Trp719Arg did not affect intramolecular interactions. DDMut analysis showed that LPA p.Arg2016Cys and p.Tyr2023Cys and LPL p.Asp36Asn caused energetically favorable changes, and KIF6 p.Trp719Arg resulted in unfavorable energetic changes, affecting protein stability. Deleterious variants in ABCA1, LPA, LPL and KIF6 are associated with variability in LDLc response to statins, and ABCA1 rs769705621 is associated with SRAE risk in FH patients. Molecular modeling studies suggest that LPA p.Tyr2023Cys and KIF6 p.Trp719Arg disturb protein conformational structure and stability.

Impact of Confinement within a Hydrogel Mesh on Protein Thermodynamic Stability and Aggregation Kinetics.

Though protein stability and aggregation have been well characterized in dilute solutions, the influence of a confining environment that exists (e.g., in intercellular and tissue spaces and therapeutic formulations) on the protein structure is largely unknown. Herein, the effects of confinement on stability and aggregation were explored for proteins of different sizes, stability, and hydrophobicity when encapsulated in hydrophilic poly(ethylene glycol) hydrogels. Denaturation curves show linear correlations between confinement size (mesh size) and thermodynamic stability, i.e., unfolding free energy and surface area accessible for solvation (represented by m-value). Two clusters of protein types are identifiable from these correlations; the clusters are defined by differences in protein stability, surface area, and aggregation propensity. Proteins with higher stability, larger surface area, and lower aggregation propensity (e.g., lysozyme and myoglobin) are less affected by the confinement imposed by mesh size than proteins with lower stability, smaller surface area, and higher aggregation propensity (e.g., growth hormone and aldehyde dehydrogenase). According to aggregation kinetics measured by thioflavin T fluorescence, confinement in smaller mesh sizes resulted in slower aggregation rates than that in larger mesh sizes. Compared to that in buffer solution, the confinement of a hydrophobic protein (e.g., human insulin) in the hydrogels accelerates protein aggregation. Conversely, the confinement of a hydrophilic protein (e.g., human amylin) in the hydrogels decelerates or prevents aggregation, with the rates of aggregation inversely proportional to mesh size. These findings provide new insights into protein conformational stability in confined microenvironments relevant to various cellular, tissue, and therapeutics scenarios.

Deciphering the chemical constituents and antimicrobial activity of Prangos pabularia Lindl. using LC-MS/MS in combination with experimental evaluation and computational studies

This study meticulously explores the antimicrobial potential of Prangos pabularia Lindl.’s aerial parts through a comprehensive blend of in vitro and in silico analysis. Extracts with varying polarities underwent LC-MS/MS identification of active components, followed by in vitro and in silico assessments of antimicrobial efficacy against Escherichia coli, Bacillus cereus, Candida albicans, Candida glabrata, and Candida paropsilosis. The methanolic extract exhibited significant antimicrobial activity with a MIC value of 48 μg/mL against all tested strains. Molecular docking revealed the compound 9-(3-methylbut-2-enoxy)-furo-(3,2-g)-chromen-7-one’s highest binding affinity against the penicillin-binding protein (PBP) bacterial drug target molecule. Other compounds also displayed substantial interactions with key antimicrobial drug target proteins. Further, Molecular dynamics simulations affirmed the stability of protein and ligand conformations. Collectively, these results underscore Prangos pabularia Lindl.’s aerial parts as a promising botanical resource in combating diverse microbial infections. This comprehensive approach not only validates it’s in vitro antimicrobial properties but also provides molecular insights into interaction mechanisms, advancing our comprehension of the plant’s therapeutic potential.

Hydrogen-Deuterium Exchange Dynamics of NISTmAb Measured by Small Angle Neutron Scattering.

Understanding protein dynamics and conformational stability holds great significance in biopharmaceutical research. Hydrogen-deuterium exchange (HDX) is a quantitative methodology used to examine these fundamental properties of proteins. HDX involves measuring the exchange of solvent-accessible hydrogens with deuterium, which yields valuable insights into conformational fluctuations and conformational stability. While mass spectrometry is commonly used to measure HDX on the peptide level, we explore a different approach using small-angle neutron scattering (SANS). In this work, SANS is demonstrated as a complementary and noninvasive HDX method (HDX-SANS). By assessing subtle changes in the tertiary and quaternary structure during the exchange process in deuterated buffer, along with the influence of added electrolytes on protein stability, SANS is validated as a complementary HDX technique. The HDX of a model therapeutic antibody, NISTmAb, an IgG1κ, is monitored by HDX-SANS over many hours using several different formulations, including salts from the Hofmeister series of anions, such as sodium perchlorate, sodium thiocyanate, and sodium sulfate. The impact of these formulation conditions on the thermal stability of NISTmAb is probed by differential scanning calorimetry. The more destabilizing salts led to heightened conformational dynamics in mAb solutions even at temperatures significantly below the denaturation point. HDX-SANS is demonstrated as a sensitive and noninvasive technique for quantifying HDX kinetics directly in mAb solution, providing novel information about mAb conformational fluctuations. Therefore, HDX-SANS holds promise as a potential tool for assessing protein stability in formulation.

Cataract-causing mutations S78F and S78P of γD-crystallin decrease protein conformational stability and drive aggregation

Congenital cataract is the leading cause of childhood blindness, which primarily results from genetic factors. γD-crystallin is the most abundant γ-crystallin and is essential for maintaining lens transparency and refractivity. Numerous mutations in γD-crystallin have been reported with unclear pathogenic mechanism. Two different cataract-causing mutations Ser78Phe and Ser78Pro in γD-crystallin were previously identified at the same conserved Ser78 residue. In this work, firstly, we purified the mutants and characterized for the structural change using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and size-exclusion chromatography (SEC). Both mutants were prone to form insoluble precipitates when expressed in Escherichia coli strain BL21 (DE3) cells. Compared with wild-type (WT), both mutations caused structural disruption, increased hydrophobic exposure, decreased solubility, and reduced thermal stability. Next, we investigated the aggregation of the mutants at the cellular level. Overexpression the mutants in HLE-B3 and HEK 293T cells could induce aggresome formations. The environmental stresses (including heat, ultraviolet irradiation and oxidative stress) promoted the formation of aggregates. Moreover, the intracellular S78F and S78P aggregates could be reversed by lanosterol. Molecular dynamic simulation indicated that both mutations disrupted the structural integrity of Greek-key motif 2. Hence, our results reveal the vital role of conserved Ser78 in maintaining the structural stability, which can offer new insights into the mechanism of cataract formation.

Enhanced Stability Differentiation of Therapeutic Polyclonal Antibodies with All Ion Unfolding-Ion Mobility-Mass Spectrometry.

Compared with monoclonal antibodies, polyclonal antibodies (pAbs) have rather significant characteristics, including lower cost, shorter production cycle, and higher affinity. Therefore, to facilitate their applications in clinic, it is equally critical to comprehensively characterize the conformational stabilities of pAb at the molecular weight-resolved scale, which is technically challenging due to the lack of an effective analytical tool capable of simultaneously providing both stability and molecular weight information within an acceptable error range. Ion mobility-mass spectrometry (IM-MS) has grown as an alternative to rapidly assess protein conformational stability with accurate molecular weight information maintained, especially when equipped with a collision-induced unfolding (CIU) regime. Dynamic and transient conformational intermediates can be captured with the CIU-IM-MS technique, adding to traditional static structural measurements with collisional cross section. Most CIU-IM-MS-centered protocols are focusing on the application to isolated, targeted protein ions, namely, analyzing one single charge state at one time, limiting its analytical throughput and speed. In this study, we employed an enhanced unfolding regime, all ion unfolding (AIU), capable of the simultaneous operation of numerous ions at a time during stepped unfolding processes to analyze pAb. Results show that AIU can quantitatively characterize the subtle differences in conformational stability among four structurally similar pAbs with improved resolving capability by around a 2-4-fold increment in both stability and structure differentiating parameters. Besides, AIU also benefits from considerably saved time cost and improved spectrum quality with an elevated signal-to-noise ratio.

Counteraction Effects of Ammonium-Based Ionic Liquids on Urea-Induced Denaturation of α-Lactalbumin: A Comprehensive Molecular Simulation Study.

Ionic liquids (ILs) are known to stabilize protein conformations in aqueous medium. Importantly, ILs can also act as refolding additives in urea-driven denaturation of proteins. However, despite the importance of the problem, detailed microscopic understanding of the counteraction effects of ILs on urea-induced protein denaturation remains elusive. In this work, atomistic molecular dynamics (MD) simulations of the protein α-lactalbumin have been carried out in pure aqueous medium, in 8 M binary urea-water solution and in ternary urea-IL-water solutions containing ammonium-based ethyl ammonium acetate (EAA) as the IL at different concentrations (1-4 M). Attempts have been made to quantify detailed molecular-level understanding of the origin behind the counteraction effects of the IL on urea-induced partial unfolding of the protein. The calculations revealed significant conformational changes of the protein with multiple free energy minima due to its partial unfolding in binary urea-water solution. The counteraction effect of the IL was evident from the enhanced structural rigidity of the protein with propensity to transform into a single native free energy minimum state in ternary urea-IL-water solutions. Such an effect has been found to be associated with preferential direct binding of the IL components with the protein and simultaneous expulsion of urea from the interface, thereby providing additional stabilization of the protein in ternary solutions. Most importantly, modified rearrangement of the hydrogen bond network at the interface due to the formation of stronger protein-cation (PC) and protein-anion (PA) hydrogen bonds by breaking relatively weaker protein-urea (PU) and protein-water (PW) hydrogen bonds has been recognized as the microscopic origin behind the counteraction effects of EAA on urea-induced partial unfolding of the protein.

Mechanistic insights into heat shock protein 27, a potential therapeutic target for cardiovascular diseases.

Heat shock protein 27 (HSP27) is a small chaperone protein that is overexpressed in a variety of cellular stress states. It is involved in regulating proteostasis and protecting cells from multiple sources of stress injury by stabilizing protein conformation and promoting the refolding of misfolded proteins. Previous studies have confirmed that HSP27 is involved in the development of cardiovascular diseases and plays an important regulatory role in this process. Herein, we comprehensively and systematically summarize the involvement of HSP27 and its phosphorylated form in pathophysiological processes, including oxidative stress, inflammatory responses, and apoptosis, and further explore the potential mechanisms and possible roles of HSP27 in the diagnosis and treatment of cardiovascular diseases. Targeting HSP27 is a promising future strategy for the treatment of cardiovascular diseases.

Determining the Conformational Stability of a Protein from Urea and Thermal Unfolding Curves

This article contains protocols for determining the conformational stability of a globular protein from either urea or thermal unfolding curves. Circular dichroism is the optical spectroscopic technique most commonly used to monitor protein unfolding. These protocols describe how to analyze data from an unfolding curve to obtain the thermodynamic parameters necessary to calculate conformational stability, and how to determine differences in stability between protein variants. Curr. Protoc. Protein Sci. 71:28.4.1-28.4.14. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Determining protein conformational stability from urea-induced unfolding curves Support Protocol 1: Preparing a urea stock solution Support Protocol 2: Analyzing urea unfolding curves Basic Protocol 2: Determining the conformational stability of a protein from thermal unfolding curves Support Protocol 3: Analyzing thermal unfolding curves Support Protocol 4: Determining differences in conformational stability for protein variants.

Impact of an Ionic Liquid on Amino Acid Side Chains: A Perspective from Molecular Simulation Studies.

Ionic liquids (ILs) are known to modify the structural stability of proteins. The modification of the protein conformation is associated with the accumulation of ILs around the amino acid (AA) side chains and the nature of interactions between them. To understand the microscopic picture of the structural arrangements of ILs around the AA side chains, room temperature molecular dynamics (MD) simulations have been carried out in this work with a series of hydrophobic, polar and charged AAs in aqueous solutions containing the IL 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]) at 2 M concentration. The calculations revealed distinctly nonuniform distribution of the IL components around different AAs. In particular, it is demonstrated that the BMIM+ cations preferentially interact with the aromatic AAs through favorable stacking interactions between the cation imidazolium head groups and the aromatic AA side chains. This results in preferential parallel alignments and enhanced population of the cations around the aromatic AAs. The potential of mean force (PMF) calculations revealed that such favorable stacking interactions provide greater stability to the contact pairs (CPs) formed between the aromatic AAs and the IL cations as compared to the other AAs. It is further quantified that for most of the AAs (except the cationic ones), a favorable enthalpy contribution more than compensates for the entropy cost to form stable CPs with the IL cations. These findings are likely to provide valuable fundamental information toward understanding the effects of ILs on protein conformational stability.

Evaluation of exogenous therapeutic protein activity under confinement and crowding effects.

Dysfunction of intracellular proteins is frequently associated with various diseases, such as cancer. The exogenous proteins in cells are usually assembled with specific configurations due to physiological confinement/crowding to exhibit novel features in the protein structure, folding or conformational stability, distinguished with their behaviors in buffer solutions. Here, we synthesized exogenous proteins under confined/crowded conditions, to explore protein activity within cells. The findings suggested that the confinement and crowding effects on protein activity are heterogeneous; they showed an inhibitory effect on HRP by decreasing Km from ∼9.5- and ∼21.7-fold and Vmax from ∼6.8- and ∼20.2-fold lower than that of dilute solutions. Interestingly, the effects on Cyt C seem to be more complicated, and crowding exerts a positive effect by increasing Km ∼ 3.6-fold and Vmax ∼ 1.5-fold higher than that of dilute solutions; however, confinement exhibits a negative effect by decreasing Km ∼2.0 and Vmax ∼8.3 times. Additionally, in contrast to traditional nanoparticle-based confinement models, we synthesized a biodegradable nanoparticle to mimic the confined space, and the biggest advantage of this novel model is that the particles can be degraded and thus it can provide more intuitive observations of the properties of the target proteins under confinement and after release. Furthermore, we also evaluated protein activity in different cellular environments, indicating that the exogenous protein activity was closely related to the crowdedness of cellular environments, and the inhibition of protein activity in MDA-MB-231 cancer cells was more obvious than in HEK293 normal cells. Finally, SAXS analysis revealed the correlation between the protein conformation and the different environments. Our work will provide a unique method for precisely assessing whether the target cellular environments are native matrix in which specific exogenous protein drugs are delivered to function or whether they display a therapeutic role, which is of great significance for screening and development of new drugs.