Cationic Residues Research Articles

The evolution and functional characterization of CXC chemokines and receptors in lamprey

Chemokines are a large family of soluble peptides guiding cell migration in development and immune defense. They interact with chemokine receptors and are essential for the coordination of cell migration in diverse physiological processes. The CXC subfamily is one of the largest groups in the chemokine family and consists of multiple members. In this study, we identified homologues of three chemokine ligands (CXCL8, CXCL_F5 and CXCL12) and two CXC receptor like molecules (CXCR_L1 and CXCR_L2) in lamprey. Sequence analysis revealed that they share the same genomic organization with their counterparts in jawed vertebrates but synteny was not conserved. Lamprey CXCL8 and CXCL12 have four conserved cysteine residues whilst the CXCL_F5 has two additional cysteine residues. In addition, CXCL_F5 is evolutionarily related to the fish specific CXC chemokine groups previously identified and contains multiple cationic aa residues in the extended C- terminal region. The two CXCRs possess seven transmembrane domains and conserved structural elements for receptor activation and signaling, including the DRYXXI(V)Y motif in TM2, the disulphide bond connecting ECL2 and TM3, the WXP motif in TM6 and NPXXY motif in TM7. The identified CXC chemokines and receptors were constitutively expressed in tissues including the liver, kidney, intestine, heart, gills, supraneural body and primary leukocytes, but exhibited distinct expression patterns. Relatively high expression was detected in the gills for CXCL8, CXCL_F5 and CXCR_L1 and in the supraneural body for CXCL12 and CXCR_L2. All the genes except CXCL12 were upregulated by stimulation with LPS, pokeweed and bacterial infection, and the CXCL8 and CXCL_F5 was induced by poly (I:C). Functional analysis showed that the CXCL8 and CXCL_F5 specifically interacted with CXCR_L1 and CXCR_L2, respectively. Our results demonstrate that the CXC chemokine system had diversified in jawless fish.

Crystal structure and site-directed mutagenesis of circular bacteriocin plantacyclin B21AG reveals cationic and aromatic residues important for antimicrobial activity

Plantacyclin B21AG is a circular bacteriocin produced by Lactiplantibacillus plantarum B21 which displays antimicrobial activity against various Gram-positive bacteria including foodborne pathogens, Listeria monocytogenes and Clostridium perfringens. It is a 58-amino acid cyclised antimicrobial peptide, with the N and C termini covalently linked together. The circular peptide backbone contributes to remarkable stability, conferring partial proteolytic resistance and structural integrity under a wide temperature and pH range. Here, we report the first crystal structure of a circular bacteriocin from a food grade Lactobacillus. The protein was crystallised using the hanging drop vapour diffusion method and the structure solved to a resolution of 1.8 Å. Sequence alignment against 18 previously characterised circular bacteriocins revealed the presence of conserved charged and aromatic residues. Alanine substitution mutagenesis validated the importance of these residues. Minimum inhibitory concentration analysis of these Ala mutants showed that Phe8Ala and Trp45Ala mutants displayed a 48- and 32-fold reduction in activity, compared to wild type. The Lys19Ala mutant displayed the weakest activity, with a 128-fold reduction. These experiments demonstrate the relative importance of aromatic and cationic residues for the antimicrobial activity of plantacyclin B21AG and by extension, other circular bacteriocins sharing these evolutionarily conserved residues.

Targeted Amino Acid Substitutions in a Trichoderma Peptaibol Confer Activity against Fungal Plant Pathogens and Protect Host Tissues from Botrytis cinerea Infection.

Fungal species belonging to the Trichoderma genus are commonly used as biocontrol agents against several crop pathogens. Among their secondary metabolites, peptaibols are helical, antimicrobial peptides, which are structurally stable even under extreme pH and temperature conditions. The promise of peptaibols as agrochemicals is, however, hampered by poor water solubility, which inhibits efficient delivery for practical use in crop protection. Using a versatile synthetic strategy, based on green chemistry procedures, we produced water-soluble analogs of the short-length peptaibol trichogin. Although natural trichogin was inactive against the tested fungal plant pathogens (Botrytis cinerea, Bipolaris sorokiniana, Fusarium graminearum, and Penicillium expansum), three analogs completely inhibited fungal growth at low micromolar concentrations. The most effective peptides significantly reduced disease symptoms by B. cinerea on common bean and grapevine leaves and ripe grape berries without visible phytotoxic effects. An in-depth conformational analysis featuring a 3D-structure–activity relationship study indicated that the relative spatial position of cationic residues is crucial for increasing peptide fungicidal activity.

Alternating Cationic-Hydrophobic Peptide/Peptoid Hybrids: Influence of Hydrophobicity on Antibacterial Activity and Cell Selectivity.

The influence of hydrophobicity on antibacterial activity versus the effect on the viability of mammalian cells for peptide/peptoid hybrids was examined for oligomers based on the cationic Lys-like peptoid residue combined with each of 28 hydrophobic amino acids in an alternating sequence. Their relative hydrophobicity was correlated to activity against both Gram-negative and Gram-positive species, human red blood cells, and HepG2 cells. This identified hydrophobic side chains that confer potent antibacterial activity (e. g., MICs of 2-8 μg/mL against E. coli) and low toxicity toward mammalian cells (<10 % hemolysis at 400 μg/mL and IC50 >800 μg/mL for HepG2 viability). Most peptidomimetics retained activity against drug-resistant strains. These findings corroborate the hypothesis that for related peptidomimetics two hydrophobicity thresholds may be identified: i) it should exceed a certain level in order to confer antibacterial activity, and ii) there is an upper limit, beyond which cell selectivity is lost. It is envisioned that once identified for a given subclass of peptide-like antibacterials such thresholds can guide further optimisation.

Insights into how innocuous foods or proteins deserving of immune ignorance can become allergens.

Allergic disorders include food allergy, allergic rhinitis, and certain forms of asthma resulting from the inappropriate development of immune responses to otherwise innocuous aeroallergens and foods. In this issue of the JCI, Thouvenot and Roitel et al. explore transcription infidelity as a mechanism that underlies the ability of these benign proteins to become allergens. Some foods and bioaerosols that produce allergies have RNA polymerase with a propensity to generate RNA gaps, thereby causing translational frameshifts. These frameshifts often create cationic carboxy-terminus residues that replace hydrophobic amino acids and have enhanced MHC binding, resulting in the tendency to provoke immune responses. IgE antibody responses initiated by these variant transcripts can later lead to IgE against the native molecule and also explain how anaphylaxis may occur in individuals who lacked specific IgE when tested using native protein reagents. This study has the potential to transform the diagnosis and treatment of allergic disorders.

Autoinhibition of TRPV6 Channel and Regulation by PIP2.

SummaryTransient receptor potential vanilloid 6 (TRPV6), a calcium-selective channel possessing six transmembrane domains (S1-S6) and intracellular N and C termini, plays crucial roles in calcium absorption in epithelia and bone and is involved in human diseases including vitamin-D deficiency, osteoporosis, and cancer. The TRPV6 function and regulation remain poorly understood. Here we show that the TRPV6 intramolecular S4-S5 linker to C-terminal TRP helix (L/C) and N-terminal pre-S1 helix to TRP helix (N/C) interactions, mediated by Arg470:Trp593 and Trp321:Ile597 bonding, respectively, are autoinhibitory and are required for maintaining TRPV6 at basal states. Disruption of either interaction by mutations or blocking peptides activates TRPV6. The N/C interaction depends on the L/C interaction but not reversely. Three cationic residues in S5 or C terminus are involved in binding PIP2 to suppress both interactions thereby activating TRPV6. This study reveals “PIP2 - intramolecular interactions” regulatory mechanism of TRPV6 activation-autoinhibition, which will help elucidating the corresponding mechanisms in other TRP channels.

Conformational preferences of cationic β-peptide in water studied by CCSD(T), MP2, and DFT methods

The conformational preferences of the cationic nylon-3 βNM [(3R,4)-diaminobutanoic acid, dAba] dipeptide in water were explored as the first step to understand the mode of action of polymers of βNM against phylogenetically diverse and intrinsically drug-resistant pathogenic fungi. The CCSD(T), MP2, M06-2X, ωB97X-D, B2PLYP-D3BJ, and DSD-PBEP86-D3BJ levels of theory with various basis sets were assessed for relative energies of the 45 local minima of the cationic Ac-dAba-NHMe located at the SMD M06-2X/6-31+G(d) level of theory in water against the benchmark CCSD(T)/CBS-limit energies in water. The best performance was obtained at the double-hybrid DSD-PBEP86-D3BJ/def2-QZVP level of theory with RMSD = 0.12 kcal/mol in water. The M06-2X/def2-QZVP level of theory predicted reasonably the conformational preference with RMSD = 0.38 kcal/mol in water and may be an alternative level of theory with marginal deviations for the calculation of conformational energies of relatively longer cationic peptides in water. In particular, the H14–helical structures appeared to be the most feasible conformations for the cationic Ac-dAba-NHMe populated at 48–64% by relative free energies in water. The hexamer built from the H14–structure of the cationic Ac-dAba-NHMe adopted a left-handed 314-helix, which has a slightly narrower radius and a longer rise than the regular 314-helix of β-peptides. Hence, the 314-helices of oligomers or polymers of the cationic dAba residues are expected to be the active conformation to exhibit the ability to bridge between charged lipid head groups that might cause a local depression or invagination of the membrane of fungi.

Structure and mechanism of the Mrp complex, an ancient cation/proton antiporter.

Multiple resistance and pH adaptation (Mrp) antiporters are multi-subunit Na+ (or K+)/H+ exchangers representing an ancestor of many essential redox-driven proton pumps, such as respiratory complex I. The mechanism of coupling between ion or electron transfer and proton translocation in this large protein family is unknown. Here, we present the structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. It is a dimer of seven-subunit protomers with 50 trans-membrane helices each. Surface charge distribution within each monomer is remarkably asymmetric, revealing probable proton and sodium translocation pathways. On the basis of the structure we propose a mechanism where the coupling between sodium and proton translocation is facilitated by a series of electrostatic interactions between a cation and key charged residues. This mechanism is likely to be applicable to the entire family of redox proton pumps, where electron transfer to substrates replaces cation movements.

A Noncytotoxic Temporin L Analogue with In Vivo Antibacterial and Antiendotoxin Activities and a Nonmembrane-Lytic Mode of Action.

Cytotoxic frog antimicrobial peptide Temporin L (TempL) is an attractive molecule for the design of lead antimicrobial agents due to its short size and versatile biological activities. However, noncytotoxic TempL variants with desirable biological activities have rarely been reported. TempL analogue Q3K,TempL is water-soluble and possesses a significant antiendotoxin property along with comparable cytotoxicity to TempL. A phenylalanine residue, located at the hydrophobic face of Q3K,TempL and the "d" position of its phenylalanine zipper sequence, was replaced with a cationic lysine residue. This analogue, Q3K,F8K,TempL, showed reduced hydrophobic moment and was noncytotoxic with lower antimicrobial activity. Interestingly, swapping between tryptophan at the fourth and serine at the sixth positions turned Q3K,F8K,TempL totally amphipathic as reflected by its helical wheel projection with clusters of hydrophobic and hydrophilic residues and the highest hydrophobic moment among these peptides. Surprisingly, this analogue, SW,Q3K,F8K,TempL, was as noncytotoxic as Q3K,F8K,TempL but showed augmented antimicrobial and antiendotoxin properties, comparable to that of TempL and Q3K,TempL. SW,Q3K,F8K,TempL exhibited appreciable survival of mice against P.aeruginosa infection and a lipopolysaccharide (LPS) challenge. Unlike TempL and Q3K,TempL, SW,Q3K,F8K,TempL adopted an unordered secondary structure in bacterial membrane mimetic lipid vesicles and did not permeabilize them or depolarize the bacterial membrane. Overall, the results demonstrate the design of a nontoxic TempL analogue that possesses clusters of hydrophobic and hydrophilic residues with impaired secondary structure and shows a nonmembrane-lytic mechanism and in vivo antiendotoxin and antimicrobial activities. This paradigm of design of antimicrobial peptide with clusters of hydrophobic and hydrophilic residues and high hydrophobic moment but low secondary structure could be attempted further.

Anticancer peptide: Physicochemical property, functional aspect and trend in clinical application (Review).

Cancer is currently ineffectively treated using therapeutic drugs, and is also able to resist drug action, resulting in increased side effects following drug treatment. A novel therapeutic strategy against cancer cells is the use of anticancer peptides (ACPs). The physicochemical properties, amino acid composition and the addition of chemical groups on the ACP sequence influences their conformation, net charge and orientation of the secondary structure, leading to an effect on targeting specificity and ACP-cell interaction, as well as peptide penetrating capability, stability and efficacy. ACPs have been developed from both naturally occurring and modified peptides by substituting neutral or anionic amino acid residues with cationic amino acid residues, or by adding a chemical group. The modified peptides lead to an increase in the effectiveness of cancer therapy. Due to this effectiveness, ACPs have recently been improved to form drugs and vaccines, which have sequentially been evaluated in various phases of clinical trials. The development of the ACPs remains focused on generating newly modified ACPs for clinical application in order to decrease the incidence of new cancer cases and decrease the mortality rate. The present review could further facilitate the design of ACPs and increase efficacious ACP therapy in the near future.

De Novo Design of Cell-Penetrating Foldamers.

Cell-penetrating peptides (CPPs) have gained much attention as carriers of hydrophilic molecules, such as drugs, peptides, and nucleic acids, into cells. CPPs are mainly composed of cationic amino acid residues, which play an important role in their intracellular uptake via interactions with acidic groups on cell surfaces. In addition, the secondary structures of CPPs also affect their cell-membrane permeability. Based on this knowledge, a variety of cell-penetrating foldamers (oligomers that form organized secondary structures) have been developed to date. In this account, we describe recent attempts to develop cell-penetrating foldamers containing various building blocks, and their application as DDS carriers.

Chain-End Modifications and Sequence Arrangements of Antimicrobial Peptoids for Mediating Activity and Nano-Assembly.

Poly(N-substituted glycine) “peptoids” are an interesting class of peptidomimics that can resist proteolysis and mimic naturally found antimicrobial peptides (AMPs), which exhibit wide spectrum activity against bacteria. This work investigates the possibility of modifying peptoid AMP mimics (AMPMs) with aliphatic lipid “tails” to generate “lipopeptoids” that can assemble into micellar nanostructures, and evaluates their antimicrobial activities. Two families of AMPMs with different distributions of hydrophobic and cationic residues were employed—one with a uniform repeating amphiphilicity, the other with a surfactant-like head-to-tail amphiphilicity. To further evaluate the interplay between self-assembly and activity, the lipopeptoids were variously modified at the AMPM chain ends with a diethylene glycol (EG2) and/or a cationic group (Nlys-Nlys dipeptoid) to adjust amphiphilicity and chain flexibility. Self-assembly was investigated by critical aggregation concentration (CAC) fluorescence assays and dynamic light scattering (DLS). The structure of a key species was also verified by small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM). To screen for antibacterial properties, we measured the minimum inhibitory concentrations (MIC) against S. aureus, E. coli, and P. aeruginosa. We found that certain combinations of lipid tail and AMPM sequences exhibit increased antibacterial activity (i.e., decreased MICs). Perhaps counter-intuitively, we were particularly interested in increased MICs in combination with low CACs. Concealing antimicrobial interactions due to packing of AMPMs in nano-assemblies could pave the way to AMPMs that may be “inert” even if unintentionally released and prevent microbes from gaining resistance to the lipopeptoids. Overall, incorporation of EG2 significantly improved lipopeptoids packing while the hydrophobic tail length was found to have a major influence over the MIC. One particular sequence, which we named C15-EG2-(kss)4, exhibited a very low CAC of 34 μM (0.0075 wt.%) and a significantly increased MIC above values for the unmodified AMPM. With the sequence design trends uncovered from this study, future work will focus on discovering more species such as C15-EG2-(kss)4 and on investigating release mechanisms and the potency of the released lipopeptoids.

Synthesis of Fmoc-Triazine Amino Acids and Its Application in the Synthesis of Short Antibacterial Peptidomimetics.

To combat the escalating rise of antibacterial resistance, the development of antimicrobial peptides (AMPs) with a unique mode of action is considered an attractive strategy. However, proteolytic degradation of AMPs remains the greatest challenge in their transformation into therapeutics. Herein, we synthesized Fmoc-triazine amino acids that differ from each other by anchoring either cationic or hydrophobic residues. These unnatural amino acids were adopted for solid-phase peptide synthesis (SPPS) to synthesize a series of amphipathic antimicrobial peptidomimetics. From the antimicrobial screening, we found that the trimer, BJK-4 is the most potent short antimicrobial peptidomimetic without showing hemolytic activity and it displayed enhanced proteolytic stability. Moreover, the mechanism of action to kill bacteria was found to be an intracellular targeting.

Cellular Uptake and Cytosolic Delivery of a Cyclic Cystine Knot Scaffold.

Cyclotides are macrocyclic peptides with exceptionally stable structures and have been reported to penetrate cells, making them promising scaffolds for the delivery of inhibitory peptides to target intracellular proteins. However, their cellular uptake and cytosolic localization have been poorly understood until now, which has limited their therapeutic potential. In this study, the recently developed chloroalkane penetration assay was combined with established assays to characterize the cellular uptake and cytosolic delivery of the prototypic cyclotide, kalata B1. We show that kalata B1 enters the cytosol at low efficiency. A structure-activity study of residues in loop 6 showed that some modifications, such as increasing cationic residue content, did not affect delivery efficiency, whereas others, including introducing a single hydrophobic amino acid, did significantly improve cytosolic delivery. Our results provide a foundation for the further development of a structurally unique class of scaffolds for the delivery of therapeutic cargoes into cells.

Improved performance of 3D hierarchical NiAl-LDHs micro-flowers via a surface anchored ZIF-8 for rapid multiple-pollutants simultaneous removal and residues monitoring

In this paper, we report a new type of 3D ZIF-8@NiAl-LDHs micro-flowers material consisting of sandwich-like structured 2D nanopetals (highly compact ZIF-8 film anchored on both sides of petals). ZIF-8 was successfully incorporated into NiAl-LDHs (ZIF-8@NiAl-LDHs) via seeding strategy directed growth of ZIFs on the surface of LDHs nanopetals. The coating of ZIF-8 significantly increased the adsorption ability to organic pollutants and inorganic cation. 3D ZIF-8@NiAl-LDHs with excellent enrichment and filtration properties has been exploited for the application in water purification, and exhibit superior high adsorption rate and adsorption efficiency of organic (nonsteroidal anti-inflammatory drugs: ketoprofen, flurbiprofen, indometacin and ibuprofe; anionic dyes: congo red, orange g; cationic dyes: methylene blue, rhodamine b) and inorganic cation (Cu2+, Pb2+) residues due to their novel hierarchical and submicroscopic structures. Further, 3D ZIF-8@NiAl-LDHs as filter membrane to extraction four kind of trace anti-inflammatory drugs followed by direct quantification detection of targets with HPLC was demonstrated. The validated method was successfully applied for analysis of four anti-inflammatory drugs in environmental water and human urine samples. This work provided a feasible way to design and construct purification materials for wastewater treatment and contaminant detection.

PIP2 Binds Multiple Cationic Sites of ENaC

Phosphatidylinositol 4,5‐bisphosphate (PIP2) regulates the activity and gating of many different ion channels to include the epithelial Na+ channel (ENaC). The negatively charged phosphoryl groups of the PIP2 headgroup are expected to interact with cationic residues of the cytoplasmic tails of ENaC. To date, understanding of the biophysical interaction between ENaC and PIP2 has been limited to indirect functional assays. Consequently, the precise binding sites and their affinities have not been defined. We began this study using secondary structure prediction tools to show that helical conformation that may cluster the cationic residues of ENaC cytoplasmic tails. Such clustering would form an electropositive region attractive to the PIP2 headgroup. We used a CIBN/CRY2‐5‐ptase optogenetic dimerization system in HEK cells to show that the C5 phosphoryl group of PIP2 plays a role in regulating ENaC activity. To more precisely define the PIP2 binding sites of ENaC, we designed short peptides corresponding to the cationic clusters of ENaC. Steady state intrinsic fluorescence ligand binding data showed that PIP2 interacted with the extreme amino terminus of the β‐ENaC subunit (βN1 peptide) and the amino and carboxy termini of γ‐ENaC nearest the transmembrane domains (γN2 and γC peptides, respectively), but no significant changes were observed with the other peptides or mutant controls. Microscale thermophoresis data revealed that the βN1 and γN2 sites has the strongest interaction with the PIP2 headgroup with Kd ~5.2 and 15 μM, respectively, whereas the γC site interacts with much weaker affinity. Again, no interaction was observed between PIP2 and the other peptides and mutant controls. These results are consistent with PIP2 binding ENaC to stabilize the open conformation of channel via moderate and weak electrostatic interactions within the cytoplasmic termini.Support or Funding InformationThis work is supported by NIH/HHLBI T32 HL07446 to CRA and JDS, and NIH/NIDDK R01 DK987460 and DK117909 to JDS.

Investigation of the structure-activity relationship in ponericin L1 from Neoponera goeldii.

Naturally derived antimicrobial peptides have been an area of great interest because of high selectivity against bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. There are also a significant number of venom-derived peptides that exhibit antimicrobial activity in addition to activity against mammals or other organisms. Many venom peptides share the same net cationic, amphiphilic nature as host-defense peptides, making them an attractive target for development as potential antibacterial agents. The peptide ponericin L1 derived from Neoponera goeldii was used as a model to investigate the role of cationic residues and net charge on peptide activity. Using a combination of spectroscopic and microbiological approaches, the role of cationic residues and net charge on antibacterial activity, lipid bilayer interactions, and bilayer and membrane permeabilization were investigated. The L1 peptide and derivatives all showed enhanced binding to lipid vesicles containing anionic lipids, but still bound to zwitterionic vesicles. None of the derivatives were especially effective at permeabilizing lipid bilayers in model vesicles, in-tact Escherichia coli, or human red blood cells. Taken together the results indicate that the lack of facial amphiphilicity regarding charge segregation may impact the ability of the L1 peptides to effectively permeabilize bilayers despite effective binding. Additionally, increasing the net charge of the peptide by replacing the lone anionic residue with either Gln or Lys dramatically improved efficacy against several bacterial strains without increasing hemolytic activity.

Strong Cationic Radical Initiator‐Based Design of a Thermoresponsive Hydrogel Showing Drastic Volume Transition

Front Cover: In article number 1900507 by Tsukuru Masuda, Madoka Takai, and co-workers, a millimeter-sized thermoresponsive gel is prepared by using a novel strong cationic radical initiator, ADIP. The cationic residue of ADIP is introduced in the gel network. In response to temperature change, the gel shows a drastic volume transition.

Strong Cationic Radical Initiator‐Based Design of a Thermoresponsive Hydrogel Showing Drastic Volume Transition

AbstractIn this study, a millimeter‐sized thermoresponsive poly(N‐isopropylacrylamide) (PNIPAAm) gel is prepared by free radical polymerization using 2,2′‐azobis‐[2‐(1,3‐dimethyl‐4,5‐dihydro‐1H‐imidazol‐3‐ium‐2‐yl)]propane triflate (ADIP), a radical initiator possessing a strong cationic group that is newly developed. By comparing this gel with a PNIPAAm gel prepared using a conventional radical initiator, 2,2′‐azobisisobutyronitrile, the following properties are found: 1) the cationic residue of ADIP is introduced in the gel, and 2) the PNIPAAm gel prepared using ADIP exhibits a discontinuous volume transition as a function of temperature and rapid shrinking in response to temperature jump. This thermoresponsive behavior is attributed to the low polymer fraction in the gel prepared using ADIP. The ADIP‐based design of the thermoresponsive hydrogel investigated herein may contribute to the design of functional soft materials.

Membrane Association of the Intrinsically Disordered N-Terminal Region of ChiZ

Multiple proteins including ChiZ that comprise the cell division machinery (divisome) of Mycobacterium tuberculosis (Mtb; causative agent of tuberculosis) contain disordered regions, often enriched in cationic residues. Given the highly anionic lipid composition, membrane association of Mtb divisome proteins may be expected to play important roles in cell division, a process with implications for both pathogenesis and drug resistance. Here we combined NMR spectroscopy and molecular dynamics (MD) simulations to characterize the membrane association of ChiZ. The 165-residue ChiZ consists of a disordered cytoplasmic N-terminal region (NT; residues 1-64), a transmembrane helix (TM; residues 64-86), and a periplasmic LysM domain (residues 114-165). 1H-15N HSQC spectra showed that NT associated only with highly anionic membranes (7:3 POPG/POPE liposomes), with terminal residues remaining dynamic. 13C-13C INEPT and CP experiments further revealed that most residues, but not some arginines, were dynamic when NT was membrane-bound. PRE experiments indicated that arginines strongly interacted with lipids, both in NT and in full-length ChiZ. Extensive MD simulations found that not all arginines interacted with membranes to the same extent. NT was anchored to membranes in the midsection by Arg37, forming on average 0.93 hydrogen bonds (HBs); arginines forming a least 0.5 HBs extended upstream to Arg33 and downstream to Arg49. When tethered to membranes by TM, the above pattern was largely preserved, except now the TM-proximal Arg62 formed 1.47 HBs. Overall, the second half of NT is more adaptive to membrane association, becoming more compact, whereas the first half is more autonomous. This difference is largely attributable to the exclusive occurrence of anionic residues in the first half of the NT sequence. These insights into sequence-conformation-interaction relations have broad significance, as numerous membrane proteins may involve membrane association of disordered regions in their functional processes.