Characterization of the structure of RAMP1 by mutagenesis and molecular modeling

Receptor activity modifying proteins (RAMPs) are a family of single-pass transmembrane proteins that dimerize with G-protein-coupled receptors. They may alter the ligand recognition properties of the receptors (particularly for the calcitonin receptor-like receptor, CLR). Very little structural information is available about RAMPs. Here, an ab initio model has been generated for the extracellular domain of RAMP1. The disulfide bond arrangement (Cys 27-Cys82, Cys40-Cys72, and Cys 57-Cys104) was determined by site-directed mutagenesis. The secondary structure (a-helices from residues 29-51, 60-80, and 87-100) was established from a consensus of predictive routines. Using these constraints, an assemblage of 25,000 structures was constructed and these were ranked using an all-atom statistical potential. The best 1000 conformations were energy minimized. The lowest scoring model was refined by molecular dynamics simulation. To validate our strategy, the same methods were applied to three proteins of known structure; PDB:1HP8, PDB:1V54 chain H (residues 21-85), and PDB:1T0P. When compared to the crystal structures, the models had root mean-square deviations of 3.8 Å, 4.1 Å, and 4.0 Å, respectively. The model of RAMP1 suggested that Phe93, Tyr 100, and Phe101 form a binding interface for CLR, whereas Trp74 and Phe92 may interact with ligands that bind to the CLR/RAMP1 heterodimer. © 2006 by the Biophysical Society.

The pharmacology of adrenomedullin receptors and their relationship to CGRP receptors

Adrenomedullin (AM) has two specific receptors formed by the calcitonin-receptor-like receptor (CL) and receptor activity-modifying protein (RAMP) 2 or 3. These are known as AM1 and AM2 receptors, respectively. In addition, AM has appreciable affinity for the CGRP1 receptor, composed of CL and RAMP1. The AM1 receptor has a high degree of selectivity for AM over CGRP and other peptides, and AM 22-52 is an effective antagonist at this receptor. By contrast, the AM2 receptor shows less specificity for AM, having appreciable affinity for βCGRP. Here, CGRP8-37 is either equipotent or more effective as an antagonist than AM22-52, depending on the species from which the receptor components are derived. Thus, under the appropriate circumstances it seems that βCGRP might be able to activate both CGRP 1 and AM2 receptors and AM could activate both AM 1 and AM2 receptors as well as CGRP1 receptors. Current peptide antagonists are not sufficiently selective to discriminate between these three receptors. The CGRP-selectivity of RAMP1 and RAMP3 may be conferred by a putative disulfide bond from the N-terminus to the middle of the extracellular domain of these molecules. This is not present in RAMP2. Copyright © 2004 Humana Press Inc. All rights of any nature whatsoever reserved.

Protein oxidation:role in signalling and detection by mass spectrometry

Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while reversible modifications are thought to be relevant in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM, such as oxidation, chlorination or nitration. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to disulfide, glutathionylated or sulfenic acid forms that can be reversed by thiol reductants. Proline hydroxylation in HIF signalling is also quite well characterized, and there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. For some proteins regulated by cysteine oxidation, the residues and molecular mechanism involved have been extensively studied and are well understood, such as the protein tyrosine phosphatase PTP1B and MAP3 kinase ASK1, as well as transcription factor complex Keap1-Nrf2. The advances in understanding of the role oxPTMs in signalling have been facilitated by advances in analytical technology, in particular tandem mass spectrometry techniques. Combinations of peptide sequencing by collisionally induced dissociation and precursor ion scanning or neutral loss to select for specific oxPTMs have proved very useful for identifying oxidatively modified proteins and mapping the sites of oxidation. The development of specific labelling and enrichment procedures for S-nitrosylation or disulfide formation has proved invaluable, and there is ongoing work to establish analogous methods for detection of nitrotyrosine and other modifications.

Redox signalling and detection of protein oxidation by mass spectrometry

It is now recognised that redox control of proteins plays an important role in many signalling pathways both in health and disease. Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while the reversible modifications are thought to be most important in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM (chlorination, nitration, etc.), and the susceptibilities of residues vary depending on their structural location. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to the disulfide, glutathionylated or sulfenic acid forms, but there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. Well understood examples of oxidative regulation include protein tyrosine phosphatases, e.g. PTP1B/C, and members of the MAPK pathways such as MEKK1 and ASK1. Transcription factors such as NFkB and Nrf-2 are also regulated by redox-active cysteines. Improved methods for analysing specific oxPTMs in biological samples are critical for understanding the physiological and pathological roles of these changes, and tandem or MS3 mass spectrometry techniques interfaced with nano-LC separation are being now used. MS3 fragmentation markers for a variety of oxidized residues including tyrosine, tryptophan and proline have been identified, and a precursor ion scanning method that allows the selective identification of these oxPTMs in complex samples has been developed. Such advances in technology offer potential for biomarker development, disease diagnosis and understanding pathology.

Molecular basis for reduced estrone sulfate transport and altered modulator sensitivity of transmembrane helix (TM) 6 and TM17 mutants of multidrug resistance protein 1 (ABCC1)

Multidrug resistance protein 1 (MRP1) confers drug resistance and also mediates cellular efflux of many organic anions. MRP1 also transports glutathione (GSH); furthermore, this tripeptide stimulates transport of several substrates, including estrone 3-sulfate. We have previously shown that mutations of Lys(332) in transmembrane helix (TM) 6 and Trp(1246) in TM17 cause different substrate-selective losses in MRP1 transport activity. Here we have extended our characterization of mutants K332L and W1246C to further define the different roles these two residues play in determining the substrate and inhibitor specificity of MRP1. Thus, we have shown that TM17-Trp(1246) is crucial for conferring drug resistance and for binding and transport of methotrexate, estradiol glucuronide, and estrone 3-sulfate, as well as for binding of the tricyclic isoxazole inhibitor N-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo-[4,3-c]quinolin-5-yl)-cyclohexylmethyl]-benzamide (LY465803). In contrast, TM6-Lys(332) is important for enabling GSH and GSH-containing compounds to serve as substrates (e.g., leukotriene C(4)) or modulators (e.g., S-decyl-GSH, GSH disulfide) of MRP1 and, further, for enabling GSH (or S-methyl-GSH) to enhance the transport of estrone 3-sulfate and increase the inhibitory potency of LY465803. On the other hand, both mutants are as sensitive as wild-type MRP1 to the non-GSH-containing inhibitors (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571), 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-yl)butoxy]phenyl]-ethanone (LY171883), and highly potent 6-[4'-carboxyphenylthio]-5[S]-hydroxy-7[E], 11[Z]14[Z]-eicosatetrenoic acid (BAY u9773). Finally, the differing abilities of the cysteinyl leukotriene derivatives leukotriene C(4), D(4), and F(4) to inhibit estradiol glucuronide transport by wild-type and K332L mutant MRP1 provide further evidence that TM6-Lys(332) is involved in the recognition of the gamma-Glu portion of substrates and modulators containing GSH or GSH-like moieties.

Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23-60 of the calcitonin receptor-like receptor

The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs.

The interaction of sodium chlorite with phospholipids and glutathione:a comparison of effects in vitro, in mammalian and in microbial cells

In this study the interaction of the preservative sodium chlorite with unsaturated lipids and glutathione was investigated, in comparison with peroxides, sodium hypochlorite, and benzalkonium chloride. The aim was to determine whether the action of sodium chlorite could involve membrane lipid damage or antioxidant depletion, and how this related to toxicity in both mammalian and microbial cells. The treatment of phospholipids with chlorite yielded low levels of hydroperoxides, but sodium chlorite oxidized the thiol-containing antioxidant glutathione to its disulfide form very readily in vitro, with a 1:4 oxidant:GSH stoichiometry. In cultured cells, sodium chlorite also caused a substantial depletion of intracellular glutathione, whereas lipid oxidation was not very prominent. Sodium chlorite had a lower toxicity to ocular mammalian cells than benzalkonium chloride, which could be responsible for the different effects of long-term application in the eye. The fungal cells, which were most resistant to sodium chlorite, maintained higher percentage levels of intracellular glutathione during treatment than the mammalian cells. The results show that sodium chlorite can cause oxidative stress in cells, and suggest that cell damage is more likely to be due to interaction with thiol compounds than with cell membrane lipids. The study also provides important information about the differential resistance of ocular cells and microbes to various preservatives and oxidants.

Selective in vitro glycosylation of recombinant proteins:semi-synthesis of novel homogeneous glycoforms of human erythropoietin

Background: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure–function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity. Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His10-WThEPO, His10-Asn24Cys, His10-Asn38Cys, His10-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l−1 from Escherichia coli. Chemical glycosylation with glycosyl-β-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn→Cys mutations at natural glycosylation sites can be glycosylated using β-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins. Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His10-WThEPO, His10-Asn24Cys, His10-Asn38Cys, His10-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l−1 from Escherichia coli. Chemical glycosylation with glycosyl-β-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn→Cys mutations at natural glycosylation sites can be glycosylated using β-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins

Conformational dynamics associated with ligand binding to vertebrate hexa-coordinate hemoglobins

Neuroglobin (Ngb) and cytoglobin (Cygb) are two new additions to the globin family, exhibiting heme iron hexa-coordination, a disulfide bond and large internal cavities. These proteins are implicated in cytoprotection under hypoxic-ischemic conditions, but the molecular basis of their cytoprotective function is unclear. Herein, a photothermal and spectroscopic study of the interactions of diatomic ligands with Ngb, Cygb, myoglobin and hemoglobin is presented. The impact of the disulfide bond in Ngb and Cygb and role of conserved residues in Ngb His64, Val68, Cys55, Cys120 and Tyr44 on conformational dynamics associated with ligand binding/dissociation were investigated. Transient absorption and photoacoustic calorimetry studies indicate that CO photo-dissociation from Ngb leads to a volume expansion (13.4±0.9 mL mol-1), whereas a smaller volume change was determined for Ngb with reduced Cys (ΔV=4.6±0.3 mL mol-1). Furthermore, Val68 side chain regulates ligand migration between the distal pocket and internal hydrophobic cavities since Val68Phe geminate quantum yield is ∼2.7 times larger than that of WT Ngb. His64Gln and Tyr44Phe mutations alter the thermodynamic parameters associated with CO photo-release indicating that electrostatic/hydrogen binding network that includes heme propionate groups, Lys 67, His64, and Tyr 44 in Ngb modulates the energetics of CO photo-dissociation. In Cygb, CO escape from the protein matrix is fast (< 40 ns) with a ΔH of 18±2 kcal mol-1 in Cygbred, whereas disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4 kcal mol-1. Therefore, the disulfide bond modulates conformational dynamics in Ngb and Cygb. I propose that in Cygb with reduced Cys the photo-dissociated ligand escapes through the hydrophobic tunnel as occurs in Ngb, whereas the CO preferentially migrates through the His64 gate in Cygbox. To characterize Cygb surface 1,8-ANS interactions with Cygb were investigated employing fluorescence spectroscopy, ITC and docking simulations. Two 1,8-ANS binding sites were identified. One binding site is located close to the extended N-terminus of Cygb and was also identified as a binding site for oleate. Furthermore, guanidinium hydrochloride-induced unfolding studies of Cygb reveal that the disulfide bond does not impact Cygb stability, whereas binding of cyanide slightly increases the protein stability.

A Comparison Between Graphene and WS2 as Solid Lubricant Additives to Aluminum for Automobile Applications

The purpose of this thesis was to compare graphene nanoplatelets (GNP) and WS2 as solid lubricant additives to aluminum in order to reduce friction and wear. The central hypothesis of this work relied on lubricating properties of 2D materials, which consist layers that slip under a shear force. Two aluminum composites were made (Al-2 vol.% GNP and Al-2 vol.% WS2) by spark plasma sintering. Tribological properties were evaluated by ball-on-disk wear tests at room temperature (RT) and 200°C. WS2 not only presented the lowest COF (0.66) but also improved the wear resistance of aluminum by 54% at RT. Al-2 vol.% GNP composite displayed poor densification (91%) and low hardness resulting in poor wear resistance. The wear rate of Al-2 vol.% GNP composite increased by 233% at RT and 48% at 200°C as compared to pure aluminum. GNP addition also resulted in lower COF (0.79) as compared to pure aluminum (0.87).

Multimode Analysis of Nanoscale Biomolecular Interactions

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical calculations have been developed for the analysis of biomolecular interactions. Human neuroglobin (hNgb) and cytochrome c from equine heart (Cyt c) proteins have been used to optimize the customized SPR instrument. The obtained Kd value (~13 µM), from SPR results, for Cyt c-hNgb molecular interactions is in general agreement with a previously published result. The SPR results also confirmed no significant impact of the internal disulfide bridge between Cys 46 and Cys 55 on hNgb binding to Cyt c. Using SPR, E. coli topoisomerase I enzyme turnover during plasmid DNA relaxation was found to be enhanced in the presence of Mg2+. In addition, a new theoretical approach of analyzing biphasic SPR data has been introduced based on analytical solutions of the biphasic rate equations. In order to develop a new label free method to quantitatively study protein-protein interactions, quartz nanopipettes were chemically modified. The derived Kd (~20 µM) value for the Cyt c-hNgb complex formations matched very well with SPR measurements (Kd ~16 µM). The finite element numerical simulation results were similar to the nanopipette experimental results. These results demonstrate that nanopipettes can potentially be used as a new class of a label-free analytical method to quantitatively characterize protein-protein interactions in attoliter sensing volumes, based on a charge sensing mechanism. Moreover, the molecule-based selective nature of hydrophobic and nanometer sized carbon nanotube (CNT) pores was observed. This result might be helpful to understand the selective nature of cellular transport through transmembrane protein pores.

Construction and screening of A cDNA library for the C3 gene(s) of the Nurse Shark (Ginglymostoma Cirratum)

Mammalian C3 is a complement protein which consists of an α chain (125kDa) and β chain (75kDa) held together by a disulfide bond. The a chain contains a conserved thiolester site which provides the molecule with opsonic properties. The protein is synthesized as a single pro-C3 molecule which is post-translationally modified. C3 genes have been identified in organisms from different phyla, however, the shark C3 gene remains to be cloned. Sequence data from the shark will contribute to understanding further the evolution of this key protein. To obtain additional sequence data for shark C3 genes a cDNA library was constructed and screened with a DIG-labeled C3 probe. Fifty clones were isolated and sequenced. Analysis identified four sequences that yielded positive alignments with C3 of a variety of organisms including human C3. Deduced amino acid sequence analysis confirmed a β/α cut site (RRRR), the CR3 and properdin binding sites, the catalytic histidine, and the reactive thiolester sequence. In the shark there are at least two C3-like genes as the gene sequence obtained is distinct from that previously described.

Design, Synthesis, and Biological Characterization of Largazole Analogues

Histone deacetylases (HDACs) have been shown to play key roles in tumorigenesis, and

have been validated as effective enzyme target for cancer treatment. Largazole, a marine natural

product isolated from the cyanobacterium Symploca, is an extremely potent HDAC inhibitor that

has been shown to possess high differential cytotoxicity towards cancer cells along with excellent

HDAC class-selectivity. However, improvements can be made in the isoform-selectivity and

pharmacokinetic properties of largazole.

In attempts to make these improvements and furnish a more efficient biochemical probe

as well as a potential therapeutic, several largazole analogues have been designed, synthesized,

and tested for their biological activity. Three different types of analogues were prepared. First,

different chemical functionalities were introduced at the C2 position to probe the class Iselectivity profile of largazole. Additionally, docking studies led to the design of a potential

HDAC8-selective analogue. Secondly, the thiol moiety in largazole was replaced with a wide

variety of othe zinc-binding group in order to probe the effect of Zn2+ affinity on HDAC

inhibition. Lastly, three disulfide analogues of largazole were prepared in order to utilize a

different prodrug strategy to modulate the pharmacokinetic properties of largazole.

Through these analogues it was shown that C2 position can be modified significantly

without a major loss in activity while also eliciting minimal changes in isoform-selectivity. While

the Zn2+-binding group plays a major role in HDAC inhibition, it was also shown that the thiol

can be replaced by other functionalities while still retaining inhibitory activity. Lastly, the use of

a disulfide prodrug strategy was shown to affect pharmacokinetic properties resulting in varying

functional responses in vitro and in vivo.

v

Largazole is already an impressive HDAC inhibitor that shows incredible promise.

However, in order to further develop this natural product into an anti-cancer therapeutic as well as

a chemical probe, improvements in the areas of pharmacokinetics as well as isoform-selectivity

are required. Through these studies we plan on building upon existing structure–activity

relationships to further our understanding of largazole’s mechanism of inhibition so that we may

improve these properties and ultimately develop largazole into an efficient HDAC inhibitor that

may be used as an anti-cancer therapeutic as well as a chemical probe for the studying of

biochemical systems.

Sulphur mineralogy and geochemistry of serpentinites and gabbros from the Atlantis Massif

In-situ uplifted portions of oceanic crust at the central dome of the Atlantis Massif (Mid-Atlantic Ridge, 30°N) were drilled during Expeditions 304 and 305 of the Integrated Ocean Drilling Program (IODP) and a 1.4 km section of predominantly gabbroic rocks with minor intercalated ultramafic rocks were recovered. Here we characterize variations in sulfur mineralogy and geochemistry of selected samples of serpentinized peridotites, olivine-rich troctolites and diverse gabbroic rocks recovered from Hole 1309D. These data are used to constrain alteration processes and redox conditions and are compared with the basement rocks of the southern wall of the Atlantis Massif, which hosts the Lost City Hydrothermal Field, 5 km to the south. The oceanic crust at the central dome is characterized by Ni-rich sulfides reflecting reducing conditions and limited seawater circulation. During uplift and exhumation, seawater interaction in gabbroic-dominated domains was limited, as indicated by homogeneous mantle-like sulfur contents and isotope compositions of gabbroic rocks and olivine-rich troctolites. Local variations from mantle compositions are related to magmatic variability or to interaction with seawater-derived fluids channeled along fault zones. The concomitant occurrence of mackinawite in olivine-rich troctolites and an anhydrite vein in a gabbro provide temperature constraints of 150-200 °C for late circulating fluids along local brittle faults below 700 m depth. In contrast, the ultramafic lithologies at the central dome represent domains with higher seawater fluxes and higher degrees of alteration and show distinct changes in sulfur geochemistry. The serpentinites in the upper part of the hole are characterized by high total sulfide contents, high d34Ssulfide values and low d34Ssulfate values, which reflect a multistage history primarily controlled by seawater-gabbro interaction and subsequent serpentinization. The basement rocks at the central dome record lower oxygen fugacities and more limited fluid fluxes compared with the serpentinites and gabbros of the Lost City hydrothermal system. Our studies are consistent with previous results and indicate that sulfur speciation and sulfur isotope compositions of altered oceanic mantle sequences commonly evolve over time. Heterogeneities in sulfur geochemistry reflect the fact that serpentinites are highly sensitive to local variations in fluid fluxes, temperature, oxygen and sulfur fugacities, and microbial activity.

Dielectric screening in atomically thin boron nitride nanosheets

Two-dimensional (2D) hexagonal boron nitride (BN) nanosheets are excellent dielectric substrate for graphene, molybdenum disulfide, and many other 2D nanomaterial-based electronic and photonic devices. To optimize the performance of these 2D devices, it is essential to understand the dielectric screening properties of BN nanosheets as a function of the thickness. Here, electric force microscopy along with theoretical calculations based on both state-of-the-art first-principles calculations with van der Waals interactions under consideration, and nonlinear Thomas-Fermi theory models are used to investigate the dielectric screening in high-quality BN nanosheets of different thicknesses. It is found that atomically thin BN nanosheets are less effective in electric field screening, but the screening capability of BN shows a relatively weak dependence on the layer thickness.