Secondary Structure Conformation Research Articles

Modulating Protein-Protein Interactions by Cyclic and Macrocyclic Peptides. Prominent Strategies and Examples.

Cyclic and macrocyclic peptides constitute advanced molecules for modulating protein–protein interactions (PPIs). Although still peptide derivatives, they are metabolically more stable than linear counterparts, and should have a lower degree of flexibility, with more defined secondary structure conformations that can be adapted to imitate protein interfaces. In this review, we analyze recent progress on the main methods to access cyclic/macrocyclic peptide derivatives, with emphasis in a few selected examples designed to interfere within PPIs. These types of peptides can be from natural origin, or prepared by biochemical or synthetic methodologies, and their design could be aided by computational approaches. Some advances to facilitate the permeability of these quite big molecules by conjugation with cell penetrating peptides, and the incorporation of β-amino acid and peptoid structures to improve metabolic stability, are also commented. It is predicted that this field of research could have an important future mission, running in parallel to the discovery of new, relevant PPIs involved in pathological processes.

Interfacial behavior of Lactate Oxidase at Air-Subphase interface

This article investigates the main aspects of the surface chemistry properties of the lactate oxidase (LacOx) enzyme monolayer at the air-subphase interface. Surface chemistry study determined the important properties like the surface packing and stability of the formed layer, whereas the spectroscopic experiments provided information regarding its secondary structure conformation of the enzyme. We have demonstrated that the LacOx in the monolayer form remained active for extended time period. In accordance to the data obtained from the isotherm it was also found that LacOx forms a stable monolayer that does not aggregate at the air-subphase interface. The stability of the monolayer at the air-subphase interface was studied by using compression-decompression cycles which revealed the stability with no significant evidence of aggregates or irreversible domains. This was further confirmed by UV–vis absorption and fluorescence measurements. Spectra from circular dichroism (CD) showed that the LB film retains the characteristic of an α-helix conformation.

The Charged Linker Modulates the Conformations and Molecular Interactions of Hsp90.

The molecular chaperone Hsp90 supports the functional activity of specific substrate proteins (clients). For client processing, the Hsp90 dimer undergoes a series of ATP‐driven conformational rearrangements. Flexible linkers connecting the three domains of Hsp90 are crucial to enable dynamic arrangements. The long charged linker connecting the N‐terminal (NTD) and middle (MD) domains exhibits additional functions in vitro and in vivo. The structural basis for these functions remains unclear. Here, we characterize the conformation and dynamics of the linker and NTD−MD domain interactions by NMR spectroscopy. Our results reveal two regions in the linker that are dynamic and exhibit secondary structure conformation. We show that these regions mediate transient interactions with strand β8 of the NTD. As a consequence, this strand detaches and exposes a hydrophobic surface patch, which enables binding to the p53 client. We propose that the charged linker plays an important regulatory role by coupling the Hsp90 NTD−MD arrangement with the accessibility of a client binding site on the NTD.

Peptide bond planarity constrains hydrogen bond geometry and influences secondary structure conformations.

An extensive database study of hydrogen bonds in different protein environments showed systematic variations in donor-acceptor-acceptor antecedent angle (Ĥ) and donor-acceptor distance. Protein environments were characterized by depth (distance of amino acids from bulk solvent), secondary structure, and whether the donor/acceptor belongs to the main chain (MC) or side chain (SC) of amino acids. The MC-MC hydrogen bonds (whether in secondary structures or not) have Ĥ angles tightly restricted to a value of around 155°, which was distinctly different from other Ĥ angles. Quantum chemical calculations attribute this characteristic MC-MC Ĥ angle to the nature of the electron density distribution around the planar peptide bond. Additional classical simulations suggest a causal link between MC-MC Ĥ angle and the conformation of secondary structures in proteins. We also showed that donor-acceptor distances are environment dependent, which has implications on protein stability. Our results redefine hydrogen bond geometries in proteins and suggest useful refinements to existing molecular mechanics force fields.

Structural and functional properties of perilla protein isolate extracted from oilseed residues and its utilization in Pickering emulsions

The objective of this study was to investigate the effects of defatting methods (hot-pressing, cold-pressing and solvent-extraction) on the functional and structural properties of perilla protein isolate (PPI) extracted from oilseed residues. Cold-pressing residues isolated protein (CRIP) exhibited the highest foaming ability (90.67%), emulsification capacity (3390.09 m2/g) and water holding capacity (2.17 g/g), which were attributed to its high surface hydrophobicity (119.29) and solubility (78.71%) and small particle size (318 nm). However, the oil holding capacity of CRIP (2.31 g/g) was slightly lower than that of hot-pressing residues isolated protein (HRIP). Furthermore, the water/oil holding capacity, foaming capacity and emulsifying stability of CRIP and HRIP were significantly higher than that of solvent-extraction residues isolated protein (SRIP). Interestingly, there were no significant differences in the secondary structure and ternary conformation among CRIP, HRIP and SRIP. At the oil-water ratio of 5:5 and PPI nanoparticles concentration of 2.0%, the creaming index of CRIP, HRIP and SRIP nanoparticle-stabilized Pickering emulsions after 7 d of storage were 26.01%, 29.42% and 31.45%, respectively. The emulsifying stability of CRIP nanoparticle-stabilized emulsions was better than that of HRIP and SRIP nanoparticle-stabilized emulsions, which was ascribed to the small droplet size (27.55 μm) and high ζ-potential (−52.74 mV) of CRIP nanoparticle-stabilized emulsion. These results indicated that PPI extracted from cold-pressing residues demonstrated excellent functional properties and could be used as a potential food-grade emulsifier for Pickering emulsions fabrication.

Effects of pretreatments on physicochemical and structural properties of proteins isolated from canola seeds after oil extraction by supercritical-CO2 process

The present research was to investigate the influences of pretreatments on the structural and physicochemical properties of proteins isolated from canola seeds (CSP) after oil extraction by supercritical CO2 fluid. The results showed that the CSPs pretreated with microwave treatment (MV-CSP) and ultrasound treatment (UL-CSP) significantly differed in terms of molecular weight distribution, amino acid composition, and the primary conformation of CSP. The crystalline properties and secondary structural conformation as well as average particle sizes of CSPs were not significantly influenced by pretreatments, but the morphological characteristics were remarkably changed. The physicochemical properties including solubility, water/oil holding capacity, emulsifying capacity and stability, as well as foaming capacity and stability were substantially altered by the pretreatments. The MV-CSP and UL-CSP possessed better physicochemical characteristics. Therefore, microwave- and ultrasound-pretreatments could serve as potential technologies to improve the physicochemical properties of canola seeds proteins. Moreover, different oil extraction methods greatly influenced the quality and functional properties of isolated CSPs. The supercritical CO2 (SC–CO2) process protected intact functional properties and enhanced the overall quality of the isolated CSPs compared to solvent extraction.

Structural Plasticity of EAK-16 Peptide Inducing Vesicle Membrane Leakage.

Ionic complementary peptide EAK-16 has been studies for anticancer drug delivery application. This is a 16 residues, short sequence peptide has ability to trosnform into micro/nanoparticle via self-assembly. However, it is still not clear that how this can bind with cell membrane to induce membrane leakage or delivering their cargo inside cell membrane. The main objective of this work was to understand behaviour of secondary structure conformation of peptide in solution and at lipid membrane interfaces and membrane permeability of synthetic ionic complementary peptide EAK-16. The corresponding secondary structure conformation was evaluated. We performed biophysical investigation to probe the interaction of synthesised ionic complementary peptide (EAK-16) with dimyristoylphospholcholine (DMPC) and dimyristoylphosphoserine (DMPS) membrane interfaces. The folding behaviours of EAK-16 were studied with Circular Dichroism (CD) spectroscopy. Membrane leakage with peptide was confirmed with calcein leakage assay. Our finding of this study showed that in aqueous phase EAK-16 was predominantly folded into β-sheets. The temperature could alter the β-sheets. However, in DMPC and DMPS membrane interfaces, EAK-16 adopted helical conformation. EAK-16 has preference in perturbing anionic compared Zwitterionic lipid vesicles. This study proposed that hydrophobic grooves of EAK-16 might be a key in the association with lipid bilayers. Secondly, a charge distribution of ionic residues would also support the orientation at lipid bilayers. This peptide membrane association would facilitate the membrane destabilisation. This study demonstrated the supporting evidence that EAK-16 could interact with lipid membranes and conforming to helical structure, while the helical conformation induced the lipid membrane leakage. Overall, this study provides a physical rationale that ionic complementary peptide can be a useful tool for designing and development of novel antibiotics and anticancer agents along its previous drug delivery applications.

Enzyme mimics based membrane reactor for di(2-ethylhexyl) phthalate degradation

Di(2-ethylhexyl) phthalate (DEHP), the most abundantly used plasticizer, was considered to be a hazardous chemical that was difficult to be degraded naturally. In this study, inspired by the “catalytic triad’’ in serine proteases, an enzyme mimic material was developed by combining the proteases’s active sites of serine, histidine and aspartate (S-H-D) with the self-assembling sequence of LKLKLKL and the aromatic group of fluorenylmethyloxycarbonyl (Fmoc). By mixing the monomer of peptides containing separate S, H and D residues with a ratio of 2:1:1, the enzyme mimics were found to co- assemble into nanofibers (Co-HSD) and showed the highest activity towards DEHP degradation because of the synergistic effects of active sites, orderly secondary structure and stable molecular conformation. To further improve ability and applicability, the high active mimetic enzyme was immobilized onto regenerated cellulose (RC) membranes for DEHP degradation in a continuous recycling mode. The RC membranes were first functionalized by the NaIO4 oxidation method to form aldehyde groups and then conjugated with the enzyme mimics via Schiff-base reaction. As a biocatalytic membrane, this membrane could not only effectively degrade DEHP, but also showed good stability, thus establishing a promising biomaterial for large scale biodegradation of DEHP in water decontamination and liquid food depollution.

CD-NuSS: A Web Server for the Automated Secondary Structural Characterization of the Nucleic Acids from Circular Dichroism Spectra Using Extreme Gradient Boosting Decision-Tree, Neural Network and Kohonen Algorithms

Nucleic acids exhibit a repertoire of conformational preference depending on the sequence and environment. Circular dichroism (CD) is an essential and valuable tool for monitoring such secondary structural conformations of nucleic acids. Nonetheless, the CD spectral diversity associated with these structures poses a challenge in obtaining the quantitative information about the secondary structural content of a given CD spectrum. To this end, the competence of the extreme gradient boosting decision-tree (XGBoost), Kohonen and neural network (nnet) algorithms have been exploited here to predict the diverse secondary structures of nucleic acids. A curated library of 450 CD spectra corresponding to 16 different secondary structures of nucleic acids has been created and used as a training dataset. The hyper-parameters corresponding to the aforementioned algorithms have been optimized using holdout and k-fold (here, k = 5) cross-validation methods. For a test dataset of 150 CD spectra, both the nnet and XGBoost algorithms have exhibited nearly similar prediction accuracy in the range of 85% and 87% (the latter exhibited a slightly higher prediction accuracy). Thus, the nnet and XGBoost algorithms tested here can be employed for predicting the hybrid nucleic acid topologies in future. For the sake of accessibility, the entire process has been automated and implemented as a webserver, called CD-NuSS (CD to nucleic acids secondary structure) and is freely accessible at https://project.iith.ac.in/cdnuss/.

Optimization of Molecular Dynamics Simulations of c-MYC1-88-An Intrinsically Disordered System.

Many of the proteins involved in key cellular regulatory events contain extensive intrinsically disordered regions that are not readily amenable to conventional structure/function dissection. The oncoprotein c-MYC plays a key role in controlling cell proliferation and apoptosis and more than 70% of the primary sequence is disordered. Computational approaches that shed light on the range of secondary and tertiary structural conformations therefore provide the only realistic chance to study such proteins. Here, we describe the results of several tests of force fields and water models employed in molecular dynamics simulations for the N-terminal 88 amino acids of c-MYC. Comparisons of the simulation data with experimental secondary structure assignments obtained by NMR establish a particular implicit solvation approach as highly congruent. The results provide insights into the structural dynamics of c-MYC1-88, which will be useful for guiding future experimental approaches. The protocols for trajectory analysis described here will be applicable for the analysis of a variety of computational simulations of intrinsically disordered proteins.

Effect of nano titanium dioxide on mechanical properties of regenerated silk

Silk and spider silk have excellent mechanical properties, and their superior physical properties are closely related to their complex structure. Here, we reported the wet spinning behavior of nanomaterials mixed with silk proteins and study the effect of the different sizes of TiO2 nanoparticles (NPs) on the mechanical properties of regenerated silk. Our study retained silk protein nanofibers in CaCl2-FA solution, and optimized the wet spinning process, and produced high-quality regenerated silk fiber (RSF) with excellent mechanical properties by using TiO2 NPs. Among them, when the TiO2 NPs of 5-10 nm was 4 mg/g(TiO2 NPs/Silk Fibroin(SF)), the tensile stress of the RSF reached at 312.44 ± 20.31 MPa, which is 113.11% higher than the control group; the tensile strain reached 46.55 ± 2.76%, which is 152.85% higher than the control group. And the secondary structure conformation of RSF tended to transform from silk I to silk II, and its β-sheet content was 47.27 ± 0.25%. We found that the mechanical properties of RSF decreased with the size of TiO2 NPs increased. This may be due to the smaller the size of TiO2 NPs and the larger the specific surface area, which can provide more binding sites between TiO2 NPs and silk proteins, and also transformed the RSF conformation from silk I to silk II. The characteristics of this spinning method are simple operation, environmentally friendly, low cost, and large-scale industrialization potential. The method of producing RSF has great potential in the production of textiles and multifunctional materials.

In vitro inhibitory effects of Chinese bayberry (Myrica rubra Sieb. et Zucc.) leaves proanthocyanidins on pancreatic α-amylase and their interaction

Chinese bayberry leaves proanthocyanidins (BLPs) belongs to the prodelphinidin category with potent EGCG unit, whose inhibition effect on α-amylase and their interaction were investigated by in vitro digestion and enzyme kinetic analysis, multi fluorescence spectroscopies (fluorescence quenching, synchronous fluorescence, and three-dimensional fluorescence), circular dichroism spectra, Fourier transform infrared spectroscopy and in silico modelling. The results revealed that BLPs was a mixed inhibitor to α-amylase with the IC50 value of 3.075 ± 0.073 μg/mL. BLPs could lead to a static fluorescence quenching of α-amylase, mainly by means of interacting with amino acids (mainly Try and Tyr residues) in one site on α-amylase molecule under the action of hydrogen bonding and/or Van der Waals force. This interaction further induced the change of secondary conformational structure, functional group structure and hydrophobicity of α-amylase, thus resulting in lowering activity. Molecular docking simulated that this binding occurred in a cavity on the surface of the α-amylase molecule, and BLPs trimer showed a relatively high binding energy. The present study provided a new insight of BLPs as an α-amylase inhibitor, which could be considered in anti-diabetic therapy.

Biophysical Stress Responses of the Yeast Lachancea thermotolerans During Dehydration Using Synchrotron-FTIR Microspectroscopy.

During industrial yeast production, cells are often subjected to deleterious hydric variations during dehydration, which reduces their viability and cellular activity. This study is focused on the yeast Lachancea thermotolerans, particularly sensitive to dehydration. The aim was to understand the modifications of single-cells biophysical profiles during different dehydration conditions. Infrared spectra of individual cells were acquired before and after dehydration kinetics using synchrotron radiation-based Fourier-transform infrared (S-FTIR) microspectroscopy. The cells were previously stained with fluorescent probes in order to measure only viable and active cells prior to dehydration. In parallel, cell viability was determined using flow cytometry under identical conditions. The S-FTIR analysis indicated that cells with the lowest viability showed signs of membrane rigidification and modifications in the amide I (α-helix and β-sheet) and amide II, which are indicators of secondary protein structure conformation and degradation or disorder. Shift of symmetric C–H stretching vibration of the CH2 group upon a higher wavenumber correlated with better cell viability, suggesting a role of plasma membrane fluidity. This was the first time that the biophysical responses of L. thermotolerans single-cells to dehydration were explored with S-FTIR. These findings are important for clarifying the mechanisms of microbial resistance to stress in order to improve the viability of sensitive yeasts during dehydration.

Structural and physicochemical properties of soya bean protein isolate/maltodextrin mixture and glycosylation conjugates

AbstractThe effect of mixture and conjugation with maltodextrin (MD) in aqueous solution on the structural and physicochemical properties of soya bean protein isolate (SPI) was investigated. Although the mixing of MD would not change the distribution of secondary structure and tertiary conformation of SPI, the protein aggregation was promoted through hydrophobic interaction, which resulted in the decrease in solubility and the improvement of gel strength and water holding capacity (WHC) of SPI/MD mixture. However, glycosylation decreased α‐helix and increased β‐structure. The bathochromic shift of the fluorescence emission maximum wavelength for SPI/MD conjugates was promoted, resulting in less compact tertiary conformation. These structural modifications might be the reason for the changes in the physicochemical properties of conjugates. After glycosylation, the surface hydrophobicity and solubility were increased, the average particle size was decreased, and the gelation properties were weakened including gel strength and WHC.

Investigation of the antifreeze mechanism and effect on quality characteristics of largemouth bass (Micropterus salmoides) during F-T cycles by hAFP

The interaction between herring antifreeze protein (hAFP) and ice crystals was studied by molecular dynamics simulation in this paper. On this basis, the effect of hAFP on the quality attributes of largemouth bass after three freezing-thawing (F-T) cycles was studied. Scanning electron microscope was conducted to analyze the microstructure changes of muscle fibers. The content of dityrosine/total sulfhydryl/carbonyl and the Ca2+-ATPase activity were measured to explore the degree of protein oxidation. Raman and intrinsic fluorescence spectra were used to measure the protein secondary structure and tertiary conformation. Results showed that hAFP protected the organisms from freezing by binding to the ice crystals, decreasing the freezing point and inhibiting the recrystallization. Furthermore, hAFP combined with chitosan magnetic (CS@Fe3O4) nanoparticles or vacuum impregnation hAFP was shown to be an effective method to reduce the mechanical damage of ice crystals to samples, and decrease the oxidation degree of samples during F-T cycles.

The effect of salt and temperature on the conformational changes of P1LEA-22, a repeat unit of plant Late Embryogenesis Abundant proteins.

The effect of choline chloride on the conformational dynamics of the 11-mer repeat unit P1LEA-22 of group 3 Late Embryogenesis Abundant (G3LEA) proteins was studied. Circular dichroism data of aqueous solutions of P1LEA-22 revealed that the peptide favors a polyproline II (PPII) helix structure at low temperature, with increasing temperature promoting a gain of unstructured conformations. Furthermore, increases in sample FeCl3 or choline chloride concentrations causes a gain in PPII helical structure at low temperature. The potential role of PPII structure in intrinsically disordered and G3LEA proteins is discussed, including its ability to easily access other secondary structural conformations such as α-helix and β-sheet, which have been observed for dehydrated G3LEA proteins. The observed effect of FeCl3 and choline chloride salts on P1LEA-22 suggests favorable cation interactions with the PPII helix, supporting ion sequestration as a G3LEA protein function. As choline chloride is suggested to improve salt tolerance and protect cell membrane in plants at low temperature, our results support adoption of the PPII structure as a possible damage-preventing measure of Late Embryogenesis Abundant proteins.

Fe(II) catalyzing sodium percarbonate facilitates the dewaterability of waste activated sludge: Performance, mechanism, and implication

In this work, Fe(II) catalyzing sodium percarbonate (Fe(II)/SPC) was managed to facilitate waste activated sludge (WAS) dewatering for the first time. The results showed that after WAS was treated by 20 mg/g total suspended solids (TSS) Fe(II) and 50 mg/g TSS SPC, the water content of sludge cake (WCSC) by press filtration and capillary suction time (CST) dropped from 90.8% ± 1.6% and 96.1 ± 4.0 s (the control) to 55.6% ± 1.4% and 30.1 ± 2.5 s, respectively. The mechanism investigations indicated that four intermediates or products (i.e., •OH, H2O2, Fe(II), and Fe(III)) generated in the Fe(II)/SPC process were responsible for the improved WAS dewaterability, and •OH and Fe(III) were the two major contributors. It was found that •OH collapsed and fragmented extracellular polymeric substances, damaged cell wall and permeabilized cytoplasmic membrane, and transformed conformation of the extracellular proteins secondary structure via both affecting the hydrogen bond maintaining α-helix and cracking disulfide bond in cysteine residues while Fe(III), the oxidization product of Fe(II), decreased the surface electronegativity and water-affinity surface areas of WAS flocs. As a result, the bound water release, flocculability, surface hydrophobicity, drain capability, and flowability of WAS flocs were strengthened whereas the compact surface structure, colloidal forces, network strength, gel-like structure, and apparent viscosity of WAS flocs were weakened. In addition, Fe(II)/SPC process also reduced the recalcitrant organics and fecal coliforms in sludge, which facilitated land application of dewatered sludge. The findings acquired in this work not only deepens our understanding of Fe(II)/SPC-involved WAS treatment process but also may guide engineers to develop both effective and promising strategies to better condition WAS for dewatering in the future.

Inactivation and structural changes of polyphenol oxidase in quince (Cydonia oblonga Miller) juice subjected to ultrasonic treatment.

Polyphenol oxidase (PPO) is considered a problem in the food industry because it starts browning reactions during fruit and vegetable processing. Ultrasonic treatment is a technology used to inactivate the enzyme; however, the mechanism behind PPO inactivation is still unclear. For this reason, the inactivation, aggregation, and structural changes in PPO from quince juice subjected to ultrasonic treatments were investigated. Different intensities and times of ultrasonic treatment were used. Changes in the activity, aggregation, conformation, and structure of PPO were investigated through different structural analyses. Compared to untreated juice, the PPO activity in treated juice was reduced to 35% at a high ultrasonic intensity of 400 W for 20 min. The structure of PPO determined from particle size distribution (PSD) analysis showed that ultrasound treatment caused initial dissociation and subsequent aggregation leading to structural modification. The spectra of circular dichroism (CD) analysis of ultrasonic treated PPO protein showed a significant loss of α-helix, and reorganization of secondary structure. Fluorescence analysis showed a significant increase in fluorescence intensity of PPO after ultrasound treatment with evident blue shift, revealing disruption in the tertiary structure. In summary, ultrasonic treatment triggered protein aggregation, distortion of tertiary structure, and loss of α-helix conformation of secondary structure causing inactivation of the PPO enzyme. Hence, ultrasound processing at high intensity and duration could cause the inactivation of the PPO enzyme by inducing aggregation and structural modifications. © 2019 Society of Chemical Industry.

Unravelling the distinctive mode of cooperative and independent interaction mechanism of 1-butyl-2,3-dimethylimidazolium tetrafluoroborate ionic liquid with model transport proteins by comprehensive spectroscopic and computational studies

In the present investigation, a detailed spectroscopic analysis on the interaction mechanism of 1-Butyl-2,3-dimethylimidazolium tetrafluoroborate [Bdmim][BF4] with human serum albumin (HSA) and bovine serum albumin (BSA) has been carried out by multispectroscopic studies. The results obtained from the fluorescence titration experiments indicated the existence of a weak interaction between HSA/BSA and [Bdmim][BF4]. The binding parameters revealed that [Bdmim][BF4] binds to HSA by cooperative process, while in the case of BSA the binding of [Bdmim][BF4] is found to be independent to the binding sites having the similar affinity. The outcome of the CD and absorbance spectral experiments of HSA/BSA-[Bdmim][BF4] systems revealed the occurrence of very little modifications in the micro-environmental and secondary structural conformations of HSA/BSA. Autodocking and molecular dynamics simulation studies indicated that the positively charged imidazolium moieties of ionic liquid (IL) is located in the vicinity of subdomain IIA and subdomain IB in HSA and BSA, respectively. The enzyme-like activity of HSA/BSA is inhibited slightly upon complexation with [Bdmim][BF4].

Surface chemistry and spectroscopic studies of the native phenylalanine dehydrogenase Langmuir monolayer at the air/aqueous NaCl interface

This study investigates the main aspects of the surface behavior of the native phenylalanine dehydrogenase (PheDH) enzyme at the air/aqueous interface employing a saline subphase to induce the enzyme surface activity. Surface chemistry experiments were performed in order to determine the surface packing and stability of the formed layer, while spectroscopic experiments provided information regarding its secondary structure conformation. It was found that the PheDH enzyme forms a fluid film, which is quite homogeneous throughout its entire compression, being stable for long periods of time with no significant evidence of aggregates or irreversible domains during interfacial compression/decompression processes. The main secondary structures of the interfacial PheDH film were accessed via in situ reflectance-absorbance infrared spectroscopy, indicating a majority presence of α-helices, which were maintained after the film transfer to solid muscovite supports. The immobilized films presented a homogeneous and regular deposition, with controlled roughness and a mean thickness in the range of 8–10 nm.