Low Ligand Density Research Articles

Lipid rafts are required for efficient signal transduction by CD1d

Plasma membranes of eukaryotic cells are not uniform, possessing distinct cholesterol- and sphingolipid-rich lipid raft microdomains which constitute critical sites for signal transduction through various immune cell receptors and their co-receptors. CD1d is a conserved family of major histocompatibility class I-like molecules, which has been established as an important factor in lipid antigen presentation to natural killer T (NKT) cells. Unlike conventional T cells, recognition of CD1d by the T cell receptor (TCR) of NKT cells does not require CD4 or CD8 co-receptors, which are critical for efficient TCR signaling. We found that murine CD1d (mCD1d) was constitutively present in the plasma membrane lipid rafts on antigen presenting cells, and that this restricted localization was critically important for efficient signal transduction to the target NKT cells, at low ligand densities, even without the involvement of co-receptors. Further our results indicate that there may be additional regulatory molecule(s), co-located in the lipid raft with mCD1d for NKT cell signaling.

Extended Langmuir approach to analyze the telomeric sequence using a biochromatographic concept

Immobilization of single-stranded DNA (ssDNA) on chromatographic support, i.e. copolymerized particles of styrene and glycidyl methacrylate via formation of amino groups with either N-methyl-1,3-propanediamine (stationary phase 1) or hexamethylenediamine (stationary phase 2) is a promise method to separate sequence specific DNA. However, the low ligand density of nonporous particles led to nonspecific interaction of the complementary oligonucleotide DNA in the sample with the stationary phase (1 or 2). In this paper, the binding process of telomere (tel; TTAGGG) to the complementary ssDNA immobilized (AATCCC) on the two chromatographic supports was studied for the first time using extended Langmuir equation. It appeared that the nonspecific adsorption of the tel due to electrostatic interaction between the polynucleotide sample (tel) and the residual amino groups on the particle surface via amination with hexamethylenediamine (stationary phase 2) was significant and could be reduced by using a high salt (NaCl) concentration in the bulk solvent. In contrast, the nonspecific adsorption of tel was neglected in the column using DNA-immobilized particles via amination with N-methyl-1,3-propanediamine (stationary phase 1). Thus, the affinity chromatography prepared via amination by N-methyl-1,3-propanediamine was more effective for analysis of sequence-specific DNA than the one prepared via amination by hexamethylenediamine. Moreover, for the two stationary phases, increasing NaCl concentration in the bulk solvent enhanced the hybridization between tel and the complementary immobilized oligonucleotide.

Cholesterol-independent Interactions with CD47 Enhance αvβ3 Avidity

Expression in OV10 cells of either wild-type CD47 or its extracellular IgV domain linked to a glycosylphosphatidylinositol anchor-(IgV-GPI) enhanced ligand-induced alpha(v)beta(3) activation as detected by the binding of LIBS1 and LIBS6 mAbs. The amplitude of LIBS binding was greater with both CD47 and IgV-GPI expression, indicating an increase in the population of "activable" integrin molecules. Expression of either CD47 species also increased alpha(v)beta(3)-mediated adhesion to vitronectin, and to surfaces coated with the anti-beta(3) antibody AP3, because of enhanced clustering of alpha(v)beta(3) as confirmed by chemical cross-linking. Cholesterol depletion with methyl-beta-cyclodextrin did not prevent the increase in anti-LIBS binding, but reduced cell adhesion to vitronectin and AP3. However, cells expressing CD47 were partially insulated against this disruption, and IgV-GPI was even more effective. Both CD47 and IgV-GPI were found in cholesterol-rich rafts prepared in the absence of detergent, but only CD47 could recruit alpha(v)beta(3) and its associated signaling molecules to these domains. Thus CD47-alpha(v)beta(3) complexes in cholesterol-rich raft domains appear to engage in G(i)-dependent signaling whereas CD47-alpha(v)beta(3) interactions that lead to integrin clustering are also detergent resistant, but are insensitive to cholesterol depletion and do not require the transmembrane region of CD47.

L-selectin Dimerization Enhances Tether Formation to Properly Spaced Ligand

Selectin counterreceptors are glycoprotein scaffolds bearing multiple carbohydrate ligands with exceptional ability to tether flowing cells under disruptive shear forces. Bond clusters may facilitate formation and stabilization of selectin tethers. L-selectin ligation has been shown to enhance L-selectin rolling on endothelial surfaces. We now report that monoclonal antibodies-induced L-selectin dimerization enhances L-selectin leukocyte tethering to purified physiological L-selectin ligands and glycopeptides. Microkinetic analysis of individual tethers suggests that leukocyte rolling is enhanced through the dimerization-induced increase in tether formation, rather than by tether stabilization. Notably, L-selectin dimerization failed to augment L-selectin-mediated adhesion below a threshold ligand density, suggesting that L-selectin dimerization enhanced adhesiveness only to properly clustered ligand. In contrast, an epidermal growth factor domain substitution of L-selectin enhanced tether formation to L-selectin ligands irrespective of ligand density, suggesting that this domain controls intrinsic ligand binding properties of L-selectin without inducing L-selectin dimerization. Strikingly, at low ligand densities, where L-selectin tethering was not responsive to dimerization, elevated shear stress restored sensitivity of tethering to selectin dimerization. This is the first indication that shear stress augments effective selectin ligand density at local contact sites by promoting L-selectin encounter of immobilized ligand.

A Response Calculus for Immobilized T Cell Receptor Ligands

To address the molecular mechanism of T cell receptor (TCR) signaling, we have formulated a model for T cell activation, termed the 2D-affinity model, in which the density of TCR on the T cell surface, the density of ligand on the presenting surface, and their corresponding two-dimensional affinity determine the level of T cell activation. When fitted to T cell responses against purified ligands immobilized on plastic surfaces, the 2D-affinity model adequately simulated changes in cellular activation as a result of varying ligand affinity and ligand density. These observations further demonstrated the importance of receptor cross-linking density in determining TCR signaling. Moreover, it was found that the functional two-dimensional affinity of TCR ligands was affected by the chemical composition of the ligand-presenting surface. This makes it possible that cell-bound TCR ligands, despite their low affinity in solution, are of optimal two-dimensional affinity thereby allowing effective TCR binding under physiological conditions, i.e. at low ligand densities in cellular interfaces.

Concentration of MHC class II molecules in lipid rafts facilitates antigen presentation.

The plasma membranes of eukaryotic cells are not uniform and possess distinct cholesterol- and sphingolipid-rich raft microdomains that are enriched in proteins known to be essential for cellular function. Lipid raft microdomains are important for T cell receptor (TCR)-mediated activation of T cells. However, the importance of lipid rafts on antigen presenting cells (APCs) and their role in major histocompatibility (MHC) class II-restricted antigen presentation has not been examined. MHC class II molecules were found to be constitutively present in plasma membrane lipid rafts in B cells. Disruption of these microdomains dramatically inhibited antigen presentation at limiting concentrations of antigen. The inhibitory effect of raft disruption on antigen presentation could be overcome by loading the APCs with exceptionally high doses of antigen, showing that raft association concentrates MHC class II molecules into microdomains that allow efficient antigen presentation at low ligand densities.

Substrate microtopography can enhance cell adhesive and migratory responsiveness to matrix ligand density.

The regulation of cell motility by ligand density on substrates with variable microtopography is not well understood. In this report, we studied the adhesion and motility behavior of HepG2 cells on microtextured poly(glycolic-co-lactic)acid (PGLA) copolymer substrates, whose surface bioactivity was differentially modified through the adsorption of 0-5.5 ng/cm(2) collagen. Microtextured PGLA substrates were fabricated as thin films with a uniform surface distribution of micropores of median size of 3.1 +/- 1.5 microm and three-dimensional root mean squared roughness of 0.253 microm. Even in the absence of collagen, cells on microtextured substrates responded to substrate topography by exhibiting a 200% increase in adhesion strength compared with untextured controls and ventral localization of the intracellular adhesion protein vinculin. Further enhancement in adhesion strength (420% over untextured, untreated substrates) was demonstrated with bioactivated, microtextured surfaces, indicating that cell adhesion responses to topography and surface ligand density were cooperative. Our motility studies of cells on untextured substrates adsorbed with different levels of collagen demonstrated that a classical biphasic relationship between the cell population averaged migration rate, mu, and the collagen ligand density was preserved. However, comparison of cell motility responses between untextured and microtextured substrates indicates that the motility versus ligand density curve shifted, such that equivalent levels of cell motility were achieved at lower ligand density on microtextured surfaces. Furthermore, the maximum mu values achieved on the microtextured substrates exceeded those on untextured substrates by twofold. Taken together, we show that the magnitude of subcellular scale microtexture of a polymer substrate can sensitize the cell motility responsiveness to substrate ligand concentration; we suggest that the underlying mechanisms involve alteration in the degree of cell-substrate adhesivity as well as changes in the nature of ligand-induced cell activation processes.

Analysis of the Binding of the Fab Fragment of Monoclonal Antibody NC10 to Influenza Virus N9 Neuraminidase from Tern and Whale Using the BIAcore Biosensor: Effect of Immobilization Level and Flow Rate on Kinetic Analysis

The binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase, isolated from tern and whale, was measured using an optical biosensor. Both neuraminidases, homotetramers of 190 kDa, were immobilized to avoid multivalent binding, and the binding of the monovalent NC10 Fab to immobilized neuraminidase was analyzed using the 1:1 Langmuir binding model. A contribution of mass transport to the kinetic constants was demonstrated at higher surface densities and low flow rates, and was minimized at low ligand densities and relatively high flow rates (up to 100 μl/min). Application of a global fitting algorithm to a 1:1 binding model incorporating a correction term for mass transport indicated that mass transport was minimized under appropriate experimental conditions; analysis of binding data with a mass transport component, using this model, yielded kinetic constants similar to those obtained with the 1:1 Langmuir binding model applied to binding data where mass transport had been minimized experimentally. The binding constant for binding of NC10 Fab to N9 neuraminidase from tern influenza virus (KA = 6.3 ± 1.3 × 107 M−1) was about 15-fold higher than that for the NC10 Fab binding to N9 neuraminidase from whale influenza virus (KA = 4.3 ± 0.7 × 106 M−1). This difference in binding affinity was mainly attributable to a 12-fold faster dissociation rate constant of the whale neuraminidase-NC10 Fab complex and may be due to either (i) the long-range structural effects caused by mutation of two residues distant from the binding epitope or (ii) differences in carbohydrate residues, attached to Asn200, which form part of the binding epitope on both neuraminidases to which NC10 Fab binds.

Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports

A poly-His tag was fused in the glutaryl acylase (GA) from Acinetobacter sp. strain YS114 cloned in E. coli yielding a fully active enzyme. Biochemical analyses showed that the tag did not alter the maturation of the chimeric GA (poly-His GA) that undergoes a complex post-translational processing from an inactive monomeric precursor to the active heterodimeric enzyme. This enzyme has been used as a model to develop a novel and very simple procedure for one-step purification of poly-His proteins via immobilized metal-ion affinity chromatography on tailor-made supports. It was intended to improve the selectivity of adsorption of the target protein on tailor-made chelate supports instead of performing a selective desorption. The rate and extent of the adsorption of proteins from a crude extract from E. coli and of pure poly-His tagged GA on different metal chelate supports was studied. Up to 90% of proteins from E. coli were adsorbed on commercial chelate supports having a high density of ligands attached to the support through long spacer arms, while this adsorption becomes almost negligible when using low ligand densities, short spacer arms and Zn 2+ or Co 2+ as cations. On the contrary, poly-His GA adsorbs strongly enough on all supports. A strong affinity interaction between the poly-His tail and a single chelate moiety seems to be the responsible for the adsorption of poly-His GA. By contrast, multipoint weak interactions involving a number of chelate moieties seem to be mainly responsible for adsorption of natural proteins. By using tailor-made affinity supports, a very simple procedure for one-step purification of GA with minimal adsorption of host proteins could be performed. Up to 20 mg of GA were adsorbed on each ml of chelate support while most of accompanying proteins were hardly adsorbed on such supports. Following few washing steps, the target enzyme was finally recovered (80% yield) by elution with 50 m M imidazole with a very high increment of specific activity (up to a 120 purification factor).

Thermodynamic properties for the solute transfer from the mobile to the stationary phase in reversed phase liquid chromatography obtained by squalane-impregnated C 18 bonded phase

We have devised a reversed phase chromatographic system which secured a simple retention mechanism and showed reproducible solute retention over a long period of time. We used a squalane impregnated C 18 phase in a thermostated system with extreme care, and obtained retention data of some selected solutes (benzene, toluene, ethylbenzene, phenol, and acetophenone) at 25, 30, 35, 40, 45, and 50°C in aqueous methanol eluents. The van’t Hoff plots were nicely linear, thus we calculated dependable enthalpies and entropies of solute transfer from the mobile to the stationary phase based on three independent retention measurements on different days (or weeks). We observed that the solute transfer from the mobile to the stationary phase was enthalpically favorable and entropically unfavorable in general except for highly aqueous mobile phases. The enthalpic contribution to the overall solute transfer free energy was found generally more important than the entropic contribution. The cavity formation effect was found the major factor that governs the solute distribution between the mobile and stationary phases for methanol-rich mobile phases while the hydrophobic effect became significant in highly aqueous mobile phases. Comparison of our data with the literature data leaded to the conclusion that C 18 stationary phases with a high ligand density generally follow a partition mechanism while a C 18 phase with a low ligand density follows an adsorption-like mechanism.

Serial TCR Engagement and Down-Modulation by Peptide:MHC Molecule Ligands: Relationship to the Quality of Individual TCR Signaling Events

In the present study, we examined the relationships among quantitative aspects of TCR engagement as measured by receptor down-modulation, functional responses, and biochemical signaling events using both mouse and human T cell clones. For T cells from both species, ligands that are more potent in inducing functional responses promote TCR down-modulation more efficiently than weaker ligands. At low ligand density, the number of down-modulated TCR exceeds the number of available ligands by as much as 80-100:1 in the optimal human case, confirming the previous description of serial ligand engagement of TCR (Valitutti, et al. 1995. Nature 375:148-151). A previously unappreciated relationship involving TCR down-modulation, the pattern of proximal TCR signaling, and the extent of serial engagement was revealed by analyzing different ligands for the same TCR. Functionally, more potent ligands induce a higher proportion of fully tyrosine phosphorylated zeta-chains and a greater amount of phosphorylated ZAP-70 than less potent ligands, and the number of TCR down-modulated per available ligand is higher with ligands showing this full agonist-like pattern. The large number of receptors showing partial zeta phosphorylation following exposure to weak ligands indicates that the true extent of TCR engagement and signaling, and thus the amount of sequential engagement, is underestimated by measurement of TCR down-modulation alone, which depends on full receptor activation. These data provide new insight into T cell activation by revealing a clear relationship among intrinsic ligand quality, signal amplification by serial engagement, functional T cell responses, and observable TCR clearance from the cell surface.

An automatic braking system that stabilizes leukocyte rolling by an increase in selectin bond number with shear.

Wall shear stress in postcapillary venules varies widely within and between tissues and in response to inflammation and exercise. However, the speed at which leukocytes roll in vivo has been shown to be almost constant within a wide range of wall shear stress, i.e., force on the cell. Similarly, rolling velocities on purified selectins and their ligands in vitro tend to plateau. This may be important to enable rolling leukocytes to be exposed uniformly to activating stimuli on endothelium, independent of local hemodynamic conditions. Wall shear stress increases the rate of dissociation of individual selectin-ligand tether bonds exponentially (, ) thereby destabilizing rolling. We find that this is compensated by a shear-dependent increase in the number of bonds per rolling step. We also find an increase in the number of microvillous tethers to the substrate. This explains (a) the lack of firm adhesion through selectins at low shear stress or high ligand density, and (b) the stability of rolling on selectins to wide variation in wall shear stress and ligand density, in contrast to rolling on antibodies (). Furthermore, our data successfully predict the threshold wall shear stress below which rolling does not occur. This is a special case of the more general regulation by shear of the number of bonds, in which the number of bonds falls below one.

Liquid chromatographic selectivity and retention behavior of rare-earth elements on a chelating resin having a propylenediaminetetraacetate type functional group

A new chelating resin that has a functional group such as propylenediaminetetraacetate separated from the polymer matrix by a spacer arm (PDTA resin) was synthesized. The amount of ligand in the PDTA resin was 127 µmol g–1 as the amount of copper adsorbed on the resin. The chromatographic selectivity and retention behavior of rare-earth elements (REEs) on the PDTA resin were evaluated. A good correlation between the capacity factor for a series of REEs on the PDTA resin and the stability constant of the corresponding PDTA complexes was obtained (R2 = 0.963). The increasing flexibility of ligand caused by the spacer arm led to a good correlation between the selectivity of PDTA resin and free PDTA with REEs. The PDTA resin was applied to the column stationary phase for the ion chromatographic separation of REEs. A favorable separation of a series of REEs was achieved within 70 min using a nitric acid gradient. It was demonstrated that the chelating resin that has low ligand density and a spacer arm between ligand and base matrix was suitable as a stationary phase for liquid chromatography and ion chromatography.

Hydrophobic charge induction chromatography: salt independent protein adsorption and facile elution with aqueous buffers

A new form of protein chromatography, hydrophobic charge induction, is described. Matrices prepared by attachment of weak acid and base ligands were uncharged at adsorption pH. At low ligand densities, protein adsorption was typically promoted with lyotropic salts. At higher ligand densities, chymosin, chymotrypsinogen and lysozyme were adsorbed independently of ionic strength. A pH change released the electrostatic potential of the matrix and weakened hydrophobic interactions, inducing elution. Matrix hydrophobicity and titration range could be matched to protein requirements by ligand choice and density. Both adsorption and elution could be carried out within the pH 5–9 range.

Negative Regulation of T Cell Homing by CD43

We report that the cell surface mucin CD43 acts as an anti-adhesin on T lymphocytes. CD43-deficient murine lymphocytes homed significantly more frequently to secondary lymphoid organs than wild-type cells. Intravital microscopy of peripheral lymph node venules revealed that CD43-deficient lymphocytes were twice as likely to tether, roll, and stick than wild-type cells. This effect was due to CD43 interference with the homing receptor, L-selectin, and was most pronounced in venules with low L-selectin ligand density. In vitro, CD43-deficient cells tethered to L-selectin ligands more efficiently and rolled more slowly than wild-type lymphocytes. Thus, CD43 exerts a negative regulatory effect on T cell trafficking by counterbalancing L-selectin–mediated adhesion.

Ligand structure of the divinylsulfone-based T-gel

The requirements for divinylsulfone (DVS)-based gels to act as thiophilic adsorbents, binding immunoglobulins in a salt-dependent manner have been examined. No differences in protein binding were observed for a DVS-activated gel reacted with mercaptoethanol (the T-gel), or for the same gel treated at high pH to hydrolyse the active groups and/or allow the formation of cross-links within matrix, indicating that an O atom may be substituted for the thioether without affecting the thiophilic interactions. Extending the time of the activation reaction between DVS and the matrix results in increased amounts of sulfone attached to the gel, but decreased levels of active vinyl groups. When coupled to mercaptoethanol, these adsorbents bound more IgG than gels activated for shorter periods. This provides a convenient method to prepare thiophilic adsorbents of high capacity while minimising the amount of DVS used. The immobilised vinylsulfone must be linked to an electron donating atom for IgG to bind. When the vinyl was instead reduced with sodium borohydride, protein binding was decreased. No IgG bound to amine-coupled DVS-activated adsorbents, perhaps due to an overall positive charge on these gels at pH 7.4. The binding of human IgG to the adsorbents is dependent on ligand density, with little protein binding to gels having less than 16 μmol sulfone per ml. The binding increased with the ligand density above this level, with more than 25 mg IgG binding per ml to an adsorbent having 114 μmol sulfone per ml. The lack of binding at low ligand densities would be expected if the IgG must interact with two or more sulfone ligands to be retained on the adsorbent.

Preparation of continuous beds for electrochromatography and reversed-phase liquid chromatography of low-molecular-mass compounds

High-performance capillary columns for electrochromatography and reversed-phase liquid chromatography are often prepared from silica beads. However, the synthesis of the beads involves many expensive and complicated steps and the packing of reproducible, stable and uniform beds requires great experience. In this paper we propose a simpler and more cost-effective approach, not based on preformed beads but on continuous polymer beds synthesized in situ in the chromatographic tube. The continuous bed columns were prepared by polymerizing two different monomer solutions in two steps directly in the capillary, followed by effective derivatization with hydrophobic ligands (C 18). Electroendosmosis was created by embedding a long, charged polymer (dextran sulfate). The continuous beds, as synthesized previously for the separation of proteins by reversed-phase chromatography, showed low resolution for the separation of low-molecular-mass compounds owing to a relatively low ligand density. This disadvantage was overcome by a new method designed to increase the ligand density and, in addition, to achieve a more rigid gel matrix. For rapid removal of the Joule-heat generated in electrochromatography, it is mandatory to employ very narrow columns. Continuous polymer bed columns with an inner diameter of 25 μm or less are easy to prepare. The potential of this type of capillary column is demonstrated by the separation of non-charged polycyclic aromatic hydrocarbons at an efficiency of 120 000 plates/m for a retained solute using electro-driven buffer flow. The performance is thus comparable to that of electrochromatography columns of 40–50 μm I.D. packed with 3–5 μm silica beads. In accordance with theoretical considerations only a somewhat higher plate height was obtained when the same continuous bed column was eluted with a pressure-driven flow. The columns have the distinct advantage over conventional capillary columns packed with beads that air bubbles seldom form and spoil a run, partly due to the absence of a supporting frit.

MHC genotype controls the capacity of ligand density to switch T helper (Th)-1/Th-2 priming in vivo.

A quantitative mechanism for the differentiation of CD4 T cells into recognized subsets of Th1 and Th2 effectors is controversial. Here, we define the Ag dose more precisely to the density of a minimal immunogenic peptide presented on the surface of a specific APC type. Th1 and Th2 responder MHC genotypes differ by as much as an order of magnitude in the density of this peptide displayed on B7-2+ B cells. We asked whether such B cells presenting a low ligand density primed Th2 effectors in an MHC genotype with predisposed high-density presentation and Th1-type immunity, and whether high ligand density B cells primed Th1 effectors in an MHC genotype that normally presents a low density and the Th2 phenotype. While low ligand density had the capacity to switch phenotype in the Th1 responder, high-density presentation did not alter genetically determined Th2 responder status.

Response to Comment on “Influence of the Chain Length and Surface Density on the Conformation and Mobility of n-Alkyl Ligands Chemically Immobilized onto a Silica Surface”

Molecular dynamics procedures have been used to investigate the conformation and mobility of n-alkyl ligands immobilized onto an amorphous silica surface, analogous to those utilized with silica-based RP-HPLC sorbents. Molecular models were constructed for n-octyl and n-octadecyl bonded phases with ligand densities of 1.64, 2.67, and 3.69 μmol/m 2 for each ligand length. The hydrocarbon layer thickness, gauche-trans statistics, end-to-end distance distribution of the n-alkyl chains, and diffusion coefficients of individual atoms in the n-alkyl ligands were determined for each system. The results have been compared with data previously obtained with an n-butyl bonded phase. The conformation of the chains, their positions with respect to the surface, and the chain mobilities were characterized as a function of ligand length and surface density. The results demonstrated that the most ordered part of the chain is always the segment that is anchored closest to the silica surface. With decreasing density, the ligands become more bent, with more gauche defects occurring in the middle and at the free end of the chains. This behavior leads to a dramatic decrease in the thickness of the hydrocarbon layer at minimum surface loading. Potentially, this behavior could result in stronger surface interactions with a prospective solute. These simulation studies also indicate that both short and long n-alkyl ligands can layer over the surface at lower ligand density, thus creating additional protection of unreacted surface silanols, with this effect being more pronounced for the longern-alkyl chains. In terms of ligand dynamics, the simulated structures indicated that the n-octyl chain exhibits the highest overall mobility at all studied densities, while the n-octadecyl ligands possess the lowest motional freedom. The degree of mobility for any length ligand decreased with increasing surface density. The mobility of individual carbon atoms within the chains also increased with distance from the silica surface for all ligand lengths and densities. These simulated properties of n-alkyl ligands immobilized onto a silica surface correlated well with other results obtained by FTIR or 13 C-cross polarization magic angle spinning NMR experimental methods and statistical predictions of the behavior of immobilized chains.

Comment on “Influence of the Chain Length and Surface Density on the Conformation and Mobility of n-Alkyl Ligands Chemically Immobilized onto a Silica Surface”

Molecular dynamics procedures have been used to investigate the conformation and mobility of n-alkyl ligands immobilized onto an amorphous silica surface, analogous to those utilized with silica-based RP-HPLC sorbents. Molecular models were constructed for n-octyl and n-octadecyl bonded phases with ligand densities of 1.64, 2.67, and 3.69 μmol/m 2 for each ligand length. The hydrocarbon layer thickness, gauche-trans statistics, end-to-end distance distribution of the n-alkyl chains, and diffusion coefficients of individual atoms in the n-alkyl ligands were determined for each system. The results have been compared with data previously obtained with an n-butyl bonded phase. The conformation of the chains, their positions with respect to the surface, and the chain mobilities were characterized as a function of ligand length and surface density. The results demonstrated that the most ordered part of the chain is always the segment that is anchored closest to the silica surface. With decreasing density, the ligands become more bent, with more gauche defects occurring in the middle and at the free end of the chains. This behavior leads to a dramatic decrease in the thickness of the hydrocarbon layer at minimum surface loading. Potentially, this behavior could result in stronger surface interactions with a prospective solute. These simulation studies also indicate that both short and long n-alkyl ligands can layer over the surface at lower ligand density, thus creating additional protection of unreacted surface silanols, with this effect being more pronounced for the longern-alkyl chains. In terms of ligand dynamics, the simulated structures indicated that the n-octyl chain exhibits the highest overall mobility at all studied densities, while the n-octadecyl ligands possess the lowest motional freedom. The degree of mobility for any length ligand decreased with increasing surface density. The mobility of individual carbon atoms within the chains also increased with distance from the silica surface for all ligand lengths and densities. These simulated properties of n-alkyl ligands immobilized onto a silica surface correlated well with other results obtained by FTIR or 13 C-cross polarization magic angle spinning NMR experimental methods and statistical predictions of the behavior of immobilized chains.