Protein Conformational Changes Research Articles

Prognostic effect of RAS/BRAF mutations in patients (pts) with metastatic colorectal cancer (mCRC).

191 Background: Somatic alterations in KRAS and BRAF have prognostic as well as predictive impact in pts with mCRC; however, the differential impact of various somatic alterations in these genes need further characterization. We analyzed the prognostic impact of specific somatic mutations in K RAS and BRAF in mCRC pts. Methods: We retrospectively reviewed the electronic medical records of pts with mCRC at our institution who underwent comprehensive genomic profiling (CGP) utilizing the Foundation One assay. Prevalence of genetic alterations was estimated using proportions and compared between groups using a chi-squared test. Patients were followed for survival from metastatic diagnosis until death or last follow-up, with left truncation at the time of CGP. Kaplan-Meier estimates were used to estimate overall survival, and groups were compared using a Cox-regression based likelihood ratio test. Results: 192 pts were identified - median age at diagnosis was 55 years, 62% (119/192) presented with metachronous metastatic disease, and 28% (54/192) had a rectal primary. Somatic mutations in KRAS were found in 49% (95/192) pts, and 53% (50/95) had a left sided primary (p = 0.3). Majority of the KRAS mutations localized to codon 12 (72/95 -76%), KRAS G12C comprised 12% (11/95). Median Overall Survival (mOS) of KRAS mutated pts was 3.0 years compared to 3.5 years for KRAS wild type (WT) pts (p = 0.5). Median OS of pts with different KRAS mutations were as follows: codon 12 mutations (excluding G12C) - 2.7 years; KRAS G12C – 5.2 years; non-codon 12 KRAS mutations - 4.8 years. BRAF mutations were identified in 7.8% (15/192) pts, and 67% (10/15) had a right sided primary (p = 0.062). BRAF V600E represented the most common alteration in BRAF – 87% (13/15). Patients with BRAF mutation had a mOS of 1.8 years compared to 3.1 years for BRAF WT pts (p = 0.2). Median OS of pts with different BRAF mutations were as follows: BRAF V600E – 1.8 years and BRAF non V600E - 2.1 years (p = 0.4). Conclusions: The numerically higher mOS in pts with KRAS G12C and non-codon 12 KRAS mutations merit further biologic characterization with functional assays. Individualized therapeutic strategies must be conceptualized for mCRC pts with specific RAS/BRAF mutations, considering their widely disparate prognosis and putative downstream signaling mechanisms. Dynamic molecular simulation to understand conformational changes in proteins associated with specific mutations will be pivotal to optimizing precision therapeutic strategies.

Java application for calculating protein-induced bilayer deformation energies using an analytical solution

The local deformation of a bilayer due to protein-bilayer hydrophobic mismatch has an associated energetic cost, the bilayer deformation energy (ΔGdef), which contributes to the total energetic cost of membrane protein conformational changes (ΔGtotal). We present an analytical solution to the continuum elastic model of protein-induced bilayer deformations (Nielsen et al., 1998, 2000), which allows us to calculate ΔGdef for a cylindrical membrane inclusion, together with a user-friendly Java implementation that calculates ΔGdef for two different states (different hydrophobic lengths) of an inclusion.

Association mechanism of remimazolam-serum albumin nano-drug for potential clinical application

Bovine serum albumin (BSA) was selected to act as a stabilizer for the growth of remimazolam-BSA particles using thermal denaturation precipitation. The remimazolam-BSA NPs showed a sphere shape, with a ζ-potential of −11.3 mV and an average diameter of 84.3 nm under electron microscopy. The drug release study of remimazolam in vitro showed a sustained and controlled release pattern. The common ways of spectroscopy like circular dichroism (CD), UV–vis, and steady-state fluorescence were all used to investigate the physicochemical association properties. A moderate binding affinity between BSA and remimazolam with a stoichiometry of 1:1 was revealed by fluorescence results (binding constant: 1.87 × 104 M−1 at body temperature). The participation of hydrophobic forces in the formation of the complex has been confirmed by analyzing the thermodynamic results of the binding data (ΔS○ = 110 J mol-1 K-1 and ΔH○ = 8.63 × 103 J mol-1). The prior remimazolam binding site is located in site II of BSA with slight protein conformational changes and probable energy transfer.

Impact of high moisture contents on the structure and functional properties of pea protein isolate during extrusion

A twin-screw extruder was used to extrude pea protein isolate (PPI) hydrated with different moisture content (30, 40, 50, and 60%); natural PPI with a moisture content of 6.84% was used as a control. The impacts of extrusion on PPI's functional properties and structure were then investigated. The results showed that extrusion significantly reduced (P < 0.05) PPI's solubility and foaming capacity. Foam stability of PPI extrudates decreased with increasing moisture content. Compared with natural PPI, the emulsifying activity and emulsifying stability index increased from 18.32 m2/g and 25.98 min to 28.67 m2/g and 39.69 min, respectively. The particle size of all PPI extrudates increased significantly (P < 0.05) with increasing moisture content. Fourier transform infrared spectroscopy (FTIR) indicated the conversion of β-sheet and α-helix structures to β-turn and random coil structures. PPI's maximum fluorescence emission wavelength (λmax) was red-shifted in the fluorescence spectrum, demonstrating a change in protein conformation. In summary, extrusion can improve the functional characteristics of PPI.

The breakdown of protein hydrogen bonding networks facilitates biotransformation of protein wastewaters during anaerobic digestion methanogenesis: Focus on protein structure and conformation

The low methanogenic efficiency of protein wastewater during anaerobic digestion can be attributed to the hydrolysis rate-limiting caused by the complex native structure of protein. In this study, the characterization of secondary structure alterations of protein molecules under acid-base stress was investigated and the effect of structure and conformation alterations on the methanogenic efficiency of protein wastewater biotransformation was analyzed. The optimal methane yields were obtained for protein wastewater pretreated with acid and base at pH = 3 and pH = 12, which was 29.4% and 35.7% higher than that of the control group (without pretreatment), reaching 142.6 ± 4.0 mL/g protein and 149.6 ± 16.1 mL/g protein, respectively. The time economy evaluation showed that 6 h pretreatment time was scientific and reasonable whether pH = 3 or pH = 12, since the methane gain effect reached 74.4% and 82.2% longing with the anaerobic digestion proceeded to 120 h, respectively. Endogenous fluorescence characteristics illustrated that the microenvironment of protein molecules has changed regardless of acid or alkali pretreatment. The circular dichroism (CD) analysis revealed that only the content of α-helix in the secondary structure of the protein at pH = 12 decreased by 46.3%, while the contents of β-sheet, β-turn and unordered structure were 29.5 ± 0.8%, 18.9 ± 0.6% and 32.2 ± 1.3%, respectively. The increase in the composition of the unordered structure demonstrated an irreversible damage to the hydrogen bonding network in the protein. FTIR spectroscopy further confirmed that the stretching vibrations of CO in amide I led to the destruction of the hydrogen bonding network and the unfolding of the protein structure. Thus, the above work provides new insights into the anaerobic digestion of protein wastewater for methanogenic processes from the perspective of protein structure and conformational changes.

Protein-enabled detection of ibuprofen and sulfamethoxazole using solid-state nanopores.

Enabled by proteins, we present an all-electrical method for rapid detection of small pharmaceuticals (ibuprofen and sulfamethoxazole [SMZ]) in aqueous media using silicon nitride pores. Specifically, we use carrier proteins, bovine serum albumin (BSA), and take advantage of their interactions with two small drug molecules to form BSA-drug complexes which can be detected by nm-diameter pores, thereby confirming the presence of small pharmaceuticals. We demonstrate detection of ibuprofen and SMZ at concentrations down to 100nM (∼21μg/L) and 48.5nM (12μg/L), respectively. We observe changes in electrical signal characteristics (reflected in event durations, rates, current magnitudes, and estimated particle diameters) of BSA-drug complexes compared to BSA-only, and differences between these two small pharmaceuticals, possibly paving a path toward developing selective sensors by identifying "electrical fingerprints" of these molecules in the future. These distinct electrical signals are likely a combined result of diffusion, electrophoretic and electroosmotic effects, interactions between the pore and particles, which depend on pore diameters, pH, and the resulting surface charges. The use of single-molecule-counting nanopores allows sensing of small pharmaceuticals, studies of protein conformational changes, and may aid in efforts to evaluate the impact of small drug molecules on aquatic and human life.

Orthogonal Activation of Metabotropic Glutamate Receptor Using Coordination Chemogenetics.

Cell-surface receptors play a pivotal role as transducers of extracellular input. Although different cell types express the same receptor, the physiological roles of the receptor are highly dependent on cell type. To understand each role, tactics for cell-specific activation of the target receptor are in high demand. Herein, we developed an orthogonal activation method targeting metabotropic glutamate receptor 1 (mGlu1), a G-protein coupled receptor. In this method, direct activation via coordination-based chemogenetics (dA-CBC) was adopted, where activation of mGlu1 was artificially induced by a protein conformational change in response to the coordination of a metal ion or metal-ion complex. Our structure-based protein design and screening approach identified mGlu1 mutants that were directly activated by the coordination of Cu2+ or Zn2+, in addition to our previous Pd-complex-sensitive mGlu1 mutant. Notably, the activation of the mutants was mutually orthogonal, resulting in cell-type selective activation in a model system using HEK293 cells.

A systematic review on active sites and functions of PIM-1 protein.

The Proviral Integration of Molony murine leukemia virus (PIM)-1 protein contributes to the solid cancers and hematologic malignancies, cell growth, proliferation, differentiation, migration, and other life activities. Many studies have related these functions to its molecular structure, subcellular localization and expression level. However, recognition of specific active sites and their effects on the activity of this constitutively active kinase is still a challenge. Based on the close relationship between its molecular structure and functional activity, this review covers the specific residues involved in the binding of ATP and different substrates in its catalytic domain. This review then elaborates on the relevant changes in protein conformation and cell functions after PIM-1 binds to different substrates. Therefore, this intensive study can improve the understanding of PIM-1-regulated signaling pathways by facilitating the discovery of its potential phosphorylation substrates.

FTIR spectroscopy confirms age-related changes in protein conformation in a new independent dataset of human plasma samples

The loss of proteostasis is a key hallmark of aging and has been well documented using several model systems. However, in biofluids such as blood plasma, little is known about how the loss of proteostasis affects protein conformation during aging. Our previous work has demonstrated age-related changes in protein conformation in human plasma samples using FTIR spectroscopy and conformation-specific antibodies. To prove the potential of spectroscopy to detect slight changes in protein secondary structure during aging, we have applied the same methodology to a new and independent set of 24 human plasma samples from donors with ages ranging from 40 to 75 years. The results clearly show that from middle to old age there is a decrease in antiparallel β-sheet structures and an increase in intermolecular β-sheets, indicative of an increase in aggregation-prone proteins in human plasma over time. This confirms the potential of FTIR spectroscopy as a technique to evaluate protein conformational changes related to health and disease and as a suitable method to be used in a clinical setting to produce quick and reliable results.

Direct, Rapid, and Simple Evaluation of the Expression and Conformation of Beta-Amyloid in Bacterial Cells by FTIR Spectroscopy.

The expression and conformation of bacterial proteins and peptides can be monitored in situ by Fourier transform infrared spectroscopy (FTIR), provided that the concentration of the protein of interest is sufficient. Here, we describe a simple protocol to analyze the conformation adopted by a specific amyloid protein in Escherichia coli cells, the pleiotropic regulator Hfq.E. coli cells expressing Hfq under an inducible promoter are analyzed. The change in protein conformation is analyzed by comparing the different populations versus controls (i.e., Δhfq cells, totally devoid of the Hfq protein) by difference spectroscopy, second derivation, curve-fitting, and principal component analysis. All the analyses were performed in the free, open-source software Quasar. We describe the detailed protocol for analyzing the data in Quasar. We show that the specific absorption of the β-amyloid conformation can be easily detected in the WT-Hfq, with bands at 1624cm-1 and 1693cm-1 indicating the presence of both parallel and antiparallel β-sheets. Furthermore, we show that FTIR spectroscopy is sensitive enough to probe the conformation of an amyloid protein backbone in vivo and to analyze its conformation in situ, directly in bacterial cells, without the need for protein purification.

Mechanisms of long-distance allosteric couplings in proton-binding membrane transporters.

Membrane transporters that use proton binding and proton transfer for function couple local protonation change with changes in protein conformation and water dynamics. Changes of protein conformation might be required to allow transient formation of hydrogen-bond networks that bridge proton donor and acceptor pairs separated by long distances. Inter-helical hydrogen-bond networks adjust rapidly to protonation change, and ensure rapid response of the protein structure and dynamics. Membrane transporters with known three-dimensional structures and proton-binding groups inform on general principles of protonation-coupled protein conformational dynamics. Inter-helical hydrogen bond motifs between proton-binding carboxylate groups and a polar sidechain are observed in unrelated membrane transporters, suggesting common principles of coupling protonation change with protein conformational dynamics.

In vitro stability of various enzymes by proline from H2O2 mediated oxidative damage

Plants under stress need to favour certain pathways so as to survive the stress period. Protection of specific enzymes by proline and other osmolytes could be one such mechanism to favour some pathways/processes. Therefore, the influence of osmolyte proline on conformational changes of various proteins caused by hydrogen peroxide (H2O2) was studied by intrinsic and extrinsic fluorescence emissions. H2O2 caused conformational change in proteins. Results indicated that for Alcohol dehydrogenase (AD) and Glutamate dehydrogenase (GD) enzymes, H2O2 induced conformational change was high and that for Glucose 6-phosphate dehydrogenase (G6PDH) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was low. Fluorescence and far-UV, CD measurements of catalase demonstrated that the H2O2 stabilized the protein secondary structure at low concentrations but destabilized it at higher concentrations. Intrinsic and ANS fluorescence results showed that proline at a concentration of 1.0 M prompted a reduction in the H2O2-induced exposed hydrophobic surfaces of studied enzymes, to different degrees which suggests its differential protective effect. Furthermore, SDS-PAGE studies revealed that proline was not able to reduce or inhibit the H2O2 mediated aggregation of GAPDH.

Modeling of Protein Conformational Changes With Rosetta Guided by Limited Experimental Data

Modeling of Protein Conformational Changes With Rosetta Guided by Limited Experimental Data

Deep learning-based molecular dynamics simulation for structure-based drug design against SARS-CoV-2

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has led to a global pandemic. Deep learning (DL) technology and molecular dynamics (MD) simulation are two mainstream computational approaches to investigate the geometric, chemical and structural features of protein and guide the relevant drug design. Despite a large amount of research papers focusing on drug design for SARS-COV-2 using DL architectures, it remains unclear how the binding energy of the protein-protein/ligand complex dynamically evolves which is also vital for drug development. In addition, traditional deep neural networks usually have obvious deficiencies in predicting the interaction sites as protein conformation changes. In this review, we introduce the latest progresses of the DL and DL-based MD simulation approaches in structure-based drug design (SBDD) for SARS-CoV-2 which could address the problems of protein structure and binding prediction, drug virtual screening, molecular docking and complex evolution. Furthermore, the current challenges and future directions of DL-based MD simulation for SBDD are also discussed.

Insights into the enzymatic catalytic mechanism of bCinS: the importance of protein conformational change

The detailed reaction mechanism of bCinS catalyzing the substrate NPP was explored by multiscale simulations. The enzyme experiences the “closed-A” state to generate (S)-terpineol, and then changes to the “closed-B” state for 1,8-cineole production.

Monitoring protein conformational changes using fluorescent nanoantennas.

Understanding the relationship between protein structural dynamics and function is crucial for both basic research and biotechnology. However, methods for studying the fast dynamics of structural changes are limited. Here, we introduce fluorescent nanoantennas as a spectroscopic technique to sense and report protein conformational changes through noncovalent dye-protein interactions. Using experiments and molecular simulations, we detect and characterize five distinct conformational states of intestinal alkaline phosphatase, including the transient enzyme-substrate complex. We also explored the universality of the nanoantenna strategy with another model protein, Protein G and its interaction with antibodies, and demonstrated a rapid screening strategy to identify efficient nanoantennas. These versatile nanoantennas can be used with diverse dyes to monitor small and large conformational changes, suggesting that they could be used to characterize diverse protein movements or in high-throughput screening applications.

Ligand-Induced Conformational and Dynamical Changes in a GT-B Glycosyltransferase: Molecular Dynamics Simulations of Heptosyltransferase I Complexes.

Understanding the dynamical motions and ligand recognition motifs of heptosyltransferase I (HepI) can be critical to discerning the behavior of other glycosyltransferase (GT) enzymes. Prior studies in our lab have demonstrated that GTs in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conserved structural fold and dynamical motions, despite low sequence homology, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through molecular dynamics simulations and ligand binding free energy analysis of HepI in the apo and bound complexes (for all kinetically relevant combinations of the native substrates/products), we have determined the energetically favored enzymatic pathway for ligand binding and release. Our principal component, dynamic cross correlation, and network analyses of the simulations have revealed correlated motions involving residues within the N-terminal domain communicating with C-terminal domain residues via both proximal amino acid residues and also functional groups of the bound substrates. Analyses of the structural changes, energetics of substrate/product binding, and changes in pKa have elucidated a variety of inter and intradomain interactions that are critical for enzyme catalysis. These data corroborate our experimental observations of protein conformational changes observed in both presteady state kinetic and circular dichroism analyses of HepI. These simulations provided invaluable structural insights into the regions involved in HepI conformational rearrangement upon ligand binding. Understanding the specific interactions governing conformational changes is likely to enhance our efforts to develop novel dynamics disrupting inhibitors against GT-B structural enzymes in the future.

Crystal structure of CmABCB1 multi-drug exporter in lipidic mesophase revealed by LCP-SFX.

CmABCB1 is a Cyanidioschyzon merolae homolog of human ABCB1, a well known ATP-binding cassette (ABC) transporter responsible for multi-drug resistance in various cancers. Three-dimensional structures of ABCB1 homologs have revealed the snapshots of inward- and outward-facing states of the transporters in action. However, sufficient information to establish the sequential movements of the open-close cycles of the alternating-access model is still lacking. Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has proven its worth in determining novel structures and recording sequential conformational changes of proteins at room temperature, especially for medically important membrane proteins, but it has never been applied to ABC transporters. In this study, 7.7 mono-acyl-glycerol with cholesterol as the host lipid was used and obtained well diffracting microcrystals of the 130 kDa CmABCB1 dimer. Successful SFX experiments were performed by adjusting the viscosity of the crystal suspension of the sponge phase with hy-droxy-propyl methyl-cellulose and using the high-viscosity sample injector for data collection at the SACLA beamline. An outward-facing structure of CmABCB1 at a maximum resolution of 2.22 Å is reported, determined by SFX experiments with crystals formed in the lipidic cubic phase (LCP-SFX), which has never been applied to ABC transporters. In the type I crystal, CmABCB1 dimers interact with adjacent molecules via not only the nucleotide-binding domains but also the transmembrane domains (TMDs); such an interaction was not observed in the previous type II crystal. Although most parts of the structure are similar to those in the previous type II structure, the substrate-exit region of the TMD adopts a different configuration in the type I structure. This difference between the two types of structures reflects the flexibility of the substrate-exit region of CmABCB1, which might be essential for the smooth release of various substrates from the transporter.

Impact of high‐pressure treatments on enzyme activity of fruit‐based beverages: an overview

SummaryWith an ever‐increasing demand for clean label products, there is a greater need for efficient and environmentally friendly processes to compete the conventional thermal or chemical treatments. For instance, high‐pressure processing (HPP) has been widely studied in the fruit industry from the last two decades. HPP can inactivate or activate different enzymes in fruit juices, pulp, and purées. HPP treatment inactivates the enzymes by the alterating the conformation of the protein structure and the active site. Depending on the enzyme, pressure, pH, temperature and treatment time, HPP can increase enzyme activity due to the release of membrane‐bound enzymes and also due to changes in protein conformation and active site that facilitate interaction with the substrate. Furthermore, the combination of high pressure, temperature and reduced treatment times offered greater inactivation of enzymes in fruit beverages. This study aimed to investigate the inactivation kinetics of endogenous enzymes in fruit beverages.

CGRAP: A Web Server for Coarse-Grained Rigidity Analysis of Proteins

Elucidating protein rigidity offers insights about protein conformational changes. An understanding of protein motion can help speed drug development, and provide general insights into the dynamic behaviors of biomolecules. Existing rigidity analysis techniques employ fine-grained, all-atom modeling, which has a costly run-time, particularly for proteins made up of more than 500 residues. In this work, we introduce coarse-grained rigidity analysis, and showcase that it provides flexibility information about a protein that is similar in accuracy to an all-atom modeling approach. We assess the accuracy of the coarse-grained method relative to an all-atom approach via a comparison metric that reasons about the largest rigid clusters of the two methods. The apparent symmetry between the all-atom and coarse-grained methods yields very similar results, but the coarse-grained method routinely exhibits 40% reduced run-times. The CGRAP web server outputs rigid cluster information, and provides data visualization capabilities, including a interactive protein visualizer.