Protein-surface Interactions Research Articles

Orientation of Proteorhodopsin in Artificial Membranes is affected by Lipid Bilayer Composition

In artificial membrane systems we want to study the structure and function of proteins as they would behave in their natural environment, so they can be used in applications that require an ex vivo approach. For instance, proteins are increasingly used in FET biosensors, thin-film protein arrays, or bio-fuel cells. These applications require a controlled protein orientation after immobilization onto varying surfaces. When two orientations are present in a system, the average functionality may not always be sufficient to acquire a signal. However, an anisotropic orientation can lead to homogeneity in transport direction and therefore increase signal response. Our objective was to determine whether the surface charge on a liposome plays a key role in determining transmembrane protein orientation and functionality during formation of proteoliposomes. To study this, we reconstitute a model ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and observe the resultant protein orientation. We used four different assays to study the electrostatic protein-surface interactions and light-driven ion transport in proteoliposomes. The surface treatments and proteolysis of proteoliposomes showed physical evidence of preferential orientation, while functional assays provided evidence of vectorial ion transport. We show that the manipulation of lipid composition can control orientation of protein in liposomes. This technique opens up a simple route for controlling protein orientation in many applications ranging from solution vesicle assays to complex bioelectronic devices and sensors that use membrane proteins.

Optofluidic Lab-on-a-Chip Monitoring of Subsurface Bacterial Transport

An optofluidic lab-on-a-chip system and subsequent sampling procedure were developed for detecting bacteria from soil samples utilizing light scattering detection of immunoagglutination assay. This system and protocol detected the presence of Escherichia coli K12 from soil particles in near real-time (10 min) with a detection limit down to 1 CFU mL-1, which is superior to the conventional methods, such as plate counting or polymerase chain reaction (PCR) assays. E. coli solutions were applied to the surface of a mock soil system and incubated overnight. The light scattering immunoagglutination assays using the optofluidic lab-on-a-chip showed two E. coli peaks over the soil depth, one at 1 cm and the other at 4 cm. Comparison with bacterial viability assay and Bradford protein assay revealed that smaller E. coli colonies were found at 1 cm depth and larger colonies at 4 cm, while free antigens adsorbed and desorbed more reversibly at both locations. The two peaks were explained by the two-step process of protein-surface interaction and gravitational force. The target molecules with small sizes (free antigens and single cells) arrived at the soil particle surface faster according to the diffusion model, and the larger E. coli colonies arrived later where the soil surface was already occupied. Because the free antigens adsorbed and desorbed in a more reversible manner, they could be found throughout the depth of the mock-up soil system, whereas the larger E. coli colonies traveled through the void space within soil particles via gravitational force and accumulated at the bottom of where the liquid reached. This work also demonstrates a device and procedure that could be potentially implemented in field studies. With proper soil sample handling protocol and light scattering detection of immunoagglutination assay in an optofluidic lab-on-a-chip, developing more complete bacteria subsurface transport models with actual field results can be achieved.

Acid Denaturation and Refolding of Cytochrome c on Silica Surface

AbstractDenaturation of oxidized cytochrome c (cyt c) adsorbed to a hydrophilic fused silica surface was studied by UV‐VIS attenuated total reflection (ATR) spectroscopy using a multiple optical pass system newly developed by this lab. Cyt c surface adsorption at neutral pH gave an adsorption equilibrium constant of Ka = 2 × 105 M−1 and a surface coverage at 63% of a monolayer saturation. Protein unfolding by acid denaturation was studied by equilibrating surface bound cyt c with acid buffers ranging in pH from 5 to 2. Protein orientation and surface coverage were calculated based on a theoretical model developed in previous work. The average heme tilt angle (44°) was found to be independent of pH, implicating protein‐surface interactions as the dominant factor governing adsorption. A non‐random molecular orientation distribution of cyt c on the surface was observed, providing further support for the dominance of protein‐surface interactions. It was shown that when denaturing acid buffers were removed and replaced with a neutral buffer cyt c refolded, assuming their original conformation. The combination of unique, yet applicable, science and laboratory skills involved in this project had a tremendous impact on the authors‘ undergraduate curriculum, making it ideal for capstone project development.

Effect of Surface Wettability on Ion-Specific Protein Adsorption

We have systematically investigated the effect of surface wettability on ion-specific adsorption of bovine serum albumin (BSA) by using quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR). The changes in frequency (Δf) and resonance unit (ΔRU) show a nonmonotonous change of the adsorbed amount of BSA as a function of molar fraction of 1-dodecanethiol (x(DDT)) of the self-assembled monolayer at pH 3.8, while the amount of adsorbed protein gradually increases with the x(DDT) at pH 7.4. The small changes of dissipation (ΔD) indicate that BSA molecules form a quite rigid protein layer on the surfaces, which results in only a slight difference in the adsorbed mass between the mass-uptake estimations from the Sauerbrey equation and the Voigt model. The difference in the adsorbed mass between QCM-D and SPR measurements is attributed to the coupled water in the protein layer. On the other hand, specific anion effect is observed in the BSA adsorption at pH 3.8 with the exception of the surface at x(DDT) of 0%, but no obvious cation specificity can be observed at pH 7.4. The ΔD-Δf plots show that the BSA adsorption at pH 3.8 has two distinct kinetic processes. The first one dominated by the protein-surface interactions is an anion-nonspecific process, whereas the second one dominated by the protein structural rearrangements is an anion-specific process. At pH 7.4, the second kinetic process can only be observed at the relatively hydrophobic surfaces, and no cation specificity is observed in the first and second kinetic processes.

Molecular Dynamics Simulation of Lysozyme Adsorption/Desorption on Hydrophobic Surfaces

In this work, we present a series of fully atomistic molecular dynamics (MD) simulations to study lysozyme's orientation-dependent adsorption on polyethylene (PE) surface in explicit water. The simulations show that depending on the orientation of the initial approach to the surface the protein may adsorb or bounce from the surface. The protein may completely leave the surface or reorient and approach the surface resulting in adsorption. The success of the trajectory to adsorb on the surface is the result of different competing interactions, including protein-surface interactions and the hydration of the protein and the hydrophobic PE surface. The difference in the hydration of various protein sites affects the protein's orientation-dependent behavior. Side-on orientation is most likely to result in adsorption as the protein-surface exhibits the strongest attraction. However, adsorption can also happen when lysozyme's longest axis is tilted on the surface if the protein-surface interaction is large enough to overcome the energy barrier that results from dehydrating both the protein and the surface. Our study demonstrates the significant role of dehydration process on hydrophobic surface during protein adsorption.

Single and binary adsorption of proteins on ion‐exchange adsorbent: The effectiveness of isothermal models

Simultaneous and sequential adsorption equilibria of single and binary adsorption of bovine serum albumin and bovine hemoglobin on Q Sepharose FF were investigated in different buffer constituents and initial conditions. The results in simultaneous adsorption showed that both proteins underwent competitive adsorption onto the adsorbent following greatly by protein-surface interaction. Preferentially adsorbed albumin complied with the universal rule of ion-exchange adsorption whereas buffer had no marked influence on hemoglobin adsorption. Moreover, an increase in initial ratios of proteins was benefit to a growth of adsorption density. In sequential adsorption, hemoglobin had the same adsorption densities as single-component adsorption. It was attributed to the displacement of preadsorbed albumin and multiple layer adsorption of hemoglobin. Three isothermal models (i.e. extended Langmuir, steric mass-action, and statistical thermodynamic (ST) models) were introduced to describe the ion-exchange adsorption of albumin and hemoglobin mixtures. The results suggested that extended Langmuir model gave the lowest deviation in describing preferential adsorption of albumin at a given salt concentration while steric mass-action model could very well describe the salt effect in albumin adsorption. For weaker adsorbed hemoglobin, ST model was the preferred choice. In concert with breakthrough data, the research further revealed the complexity in ion-exchange adsorption of proteins.

Tearing the Top Off ‘Top-Down’ Proteomics

Tearing the Top Off ‘Top-Down’ Proteomics

RETRACTED ARTICLE: Plasma Protein Adsorption to Zwitterionic Poly (Carboxybetaine Methacrylate) Modified Surfaces: Chain Chemistry and End-Group Effects on Protein Adsorption Kinetics, Adsorbed Amounts and Immunoblots

Protein-surface interactions are crucial to the overall biocompatability of biomaterials, and are thought to be the impetus towards the adverse host responses such as blood coagulation and complement activation. Only a few studies hint at the ultra-low fouling potential of zwitterionic poly(carboxybetaine methacrylate) (PCBMA) grafted surfaces and, of those, very few systematically investigate their non-fouling behavior. In this work, single protein adsorption studies as well as protein adsorption from complex solutions (i.e. human plasma) were used to evaluate the non-fouling potential of PCBMA grafted silica wafers prepared by nitroxide-mediated free radical polymerization. PCBMAs used for surface grafting varied in charge separating spacer groups that influence the overall surface charges, and chain end-groups that influence the overall hydrophilicity, thereby, allows a better understanding of these effects towards the protein adsorption for these materials. In situ ellipsometry was used to quantify the adsorbed layer thickness and adsorption kinetics for the adsorption of four proteins from single protein buffer solutions, viz, lysozyme, α-lactalbumin, human serum albumin and fibrinogen. Total amount of protein adsorbed on surfaces differed as a function of surface properties and protein characteristics. Finally, immunoblots results showed that human plasma protein adsorption to these surfaces resulted, primarily, in the adsorption of human serum albumin, with total protein adsorbed amounts being the lowest for PCBMA-3 (TEMPO). It was apparent that surface charge and chain hydrophilicity directly influenced protein adsorption behavior of PCBMA systems and are promising materials for biomedical applications.

Apparent Activation Energies Associated with Protein Dynamics on Hydrophobic and Hydrophilic Surfaces

With the use of single-molecule total internal reflection fluorescence microscopy (TIRFM), the dynamics of bovine serum albumin (BSA) and human fibrinogen (Fg) at low concentrations were observed at the solid-aqueous interface as a function of temperature on hydrophobic trimethylsilane (TMS) and hydrophilic fused silica (FS) surfaces. Multiple dynamic modes and populations were observed and characterized by their surface residence times and squared-displacement distributions (surface diffusion). Characteristic desorption and diffusion rates for each population/mode were generally found to increase with temperature, and apparent activation energies were determined from Arrhenius analyses. The apparent activation energies of desorption and diffusion were typically higher on FS than on TMS surfaces, suggesting that protein desorption and mobility were hindered on hydrophilic surfaces due to favorable protein-surface and solvent-surface interactions. The diffusion of BSA on TMS appeared to be activationless for several populations, whereas diffusion on FS always exhibited an apparent activation energy. All activation energies were small in absolute terms (generally only a few kBT), suggesting that most adsorbed protein molecules are weakly bound and move and desorb readily under ambient conditions.

Direct Observation of Phenylalanine Orientations in Statherin Bound to Hydroxyapatite Surfaces

Extracellular biomineralization proteins such as salivary statherin control the growth of hydroxyapatite (HAP), the principal component of teeth and bones. Despite the important role that statherin plays in the regulation of hard tissue formation in humans, the surface recognition mechanisms involved are poorly understood. The protein-surface interaction likely involves very specific contacts between the surface atoms and the key protein side chains. This study demonstrates for the first time the power of combining near-edge X-ray absorption fine structure (NEXAFS) spectroscopy with element labeling to quantify the orientation of individual side chains. In this work, the 15 amino acid N-terminal binding domain of statherin has been adsorbed onto HAP surfaces, and the orientations of phenylalanine rings F7 and F14 have been determined using NEXAFS analysis and fluorine labels at individual phenylalanine sites. The NEXAFS-derived phenylalanine tilt angles have been verified with sum frequency generation spectroscopy.

DnaK Prevents Human Insulin Amyloid Fiber Formation on Hydrophobic Surfaces

We have developed a multiwell-based protein aggregation assay to study the kinetics of insulin adsorption and aggregation on hydrophobic surfaces and to investigate the molecular mechanisms involved. Protein-surface interaction progresses in two phases: (1) a lag phase during which proteins adsorb and prefibrillar aggregates form on the material surface and (2) a growth phase during which amyloid fibers form and then are progressively released into solution. We studied the effect of three bacterial chaperones, DnaK, DnaJ, and ClpB, on insulin aggregation kinetics. In the presence of ATP, the simultaneous presence of DnaK, DnaJ, and ClpB allows good protection of insulin against aggregation. In the absence of ATP, DnaK alone is able to prevent insulin aggregation. Furthermore, DnaK binds to insulin adsorbed on hydrophobic surfaces. This process is slowed in the presence of ATP and can be enhanced by the cochaperone DnaJ. The peptide LVEALYL, derived from the insulin B chain, is known to promote fast aggregation in a concentration- and pH-dependent manner in solution. We show that it also shortens the lag phase for insulin aggregation on hydrophobic surfaces. As this peptide is also a known DnaK substrate, our data indicate that the peptide and the chaperone might compete for a common site during the process of insulin aggregation on hydrophobic surfaces.

Aggregation-induced conformational transitions in bovine β-lactoglobulin adsorbed onto open chitosan structures

Chitosan is a natural polysaccharide which strongly interacts with whey proteins in solution. Certain protein–polyelectrolyte complexes and electrostatically assembled thin films have been shown to be good platforms for the preservation of globular and small proteins in their native state mainly due to the hydrophilic nature of these polyelectrolytes and to the lack of physical space for embedded proteins to relax. As a consequence, the use of natural polyelectrolytes in new nanocomposite materials for medicine, chemical analysis, catalysis, etc. has exploded in the last few years. Nevertheless, in many of these applications proteins are immobilized in more open structures without physical restrictions to undergo conformational changes. In this work, we investigate the nature of these transitions for a model whey protein, β-LG, on a chitosan-decorated Au surface in a scenario for which protein–surface interactions compete with boosted protein–protein non-coulombic forces. The adsorption kinetics, protein mass uptake (plus associated water), and flexibility of the adsorbed layers have been followed in situ by the quartz crystal microbalance with dissipation monitoring (QCM-D). Further ex situ characterization has been performed by atomic force microscopy (AFM) and non-invasive scanning electron microscopy (SEM). The balance between both types of interactions yielded surfaces heavily loaded with protein and water in which orientational transitions seemed restricted. The kinetics of the process was registered in a wide range of concentrations and successfully fitted to a double exponential equation derived from the RSA theory accounting for the establishment of slow post-adsorption conformational transitions.

Conformational Dynamics of Lipid-Reconstituted LeuT Studied at the Single Vesicle and Single Molecule Level

Conformational changes are an essentially prerequisite for the function of transmembrane(TM) proteins. Ensemble studies typically average dynamics of the conformational changes that consequently become obscure. Therefore an approach for revealing the dynamics of conformational changes is to study each protein at the single molecule (SM) level. At present only two reports have addressed the conformational dynamics of any TM protein at SM level1,2 reflecting the difficulties and importance of these experiments. Proteins in these experiments were solubilized in detergent micelles. Here we investigate conformational dynamics of the Leucine transporter(LeuT) reconstituted in lipid vesicles, to understand the influence of the membrane on the transporter. We are focusing on single vesicle and SM microscopy measurements of allosteric transitions and oscillations between different states of the sixth TM helix (TM6) of LeuT.We have developed a unique strategy3,4,5,6 for immobilization of TM proteins under conditions that minimize non-specific interactions with the surface and thus minimize denaturation. We reconstitute membrane proteins into vesicles, which are anchored on a Neutravidin coated surface with biotinilated lipids. In this manner vesicle thus serves as a 3D scaffold that minimizes protein-surface interactions. By employing this method we have successfully reconstituted LeuT. The protein is labeled with the tetramethylrhodamine (TMR) dye on TM6 (at position 192C), which is quenched by Histidine (position 7H) when LeuT is in an inactive conformation. Variation in distance or orientation between the quencher and TMR, which are induced by the conformational changes of the protein during binding of substrate affect fluorescence signal intensity of the TMR. The dynamics of conformational changes are monitored by Total Internal Reflection microscopy at the single vesicle and the SM level.

Surface Characterization and Protein Interactions of Segmented Polyisobutylene-Based Thermoplastic Polyurethanes

The surface properties and biocompatibility of a class of thermoplastic polyurethanes (TPUs) with applications in blood-contacting medical devices have been studied. Thin films of commercial TPUs and novel polyisobutylene (PIB)-poly(tetramethylene oxide) (PTMO) TPUs were characterized by contact angle measurements, X-ray photoelectron spectroscopy, and atomic force microscopy (AFM) imaging. PIB-PTMO TPU surfaces have significantly higher C/N ratios and lower amounts of oxygen than the theoretical bulk composition, which is attributed to surface enrichment of PIB. Greater differences in the C/N ratios were observed with the softer compositions due to their higher relative amounts of PIB. The contact angles were higher on PIB-PTMO TPUs than on commercial polyether TPUs, indicating lower surface energy. AFM imaging showed phase separation and increasing domain sizes with increasing hard segment content. The biocompatibility was investigated by quantifying the adsorption of fouling and passivating proteins, fibrinogen (Fg) and human serum albumin (HSA) respectively, onto thin TPU films spin coated onto the electrode of a quartz crystal microbalance with dissipation monitoring (QCM-D). Competitive adsorption experiments were performed with a mixture of Fg and albumin in physiological ratio followed by binding of GPIIb-IIIa, the platelet receptor ligand that selectively binds to Fg. The QCM-D results indicate similar adsorbed amounts of both Fg and HSA on PIB-PTMO TPUs and commercial TPUs. The strength of the protein interactions with the various TPU surfaces measured with AFM (colloidal probe) was similar among the various TPUs. These results suggest excellent biocompatibility of these novel PIB-PTMO TPUs, similar to that of polyether TPUs.

Adsorption and protein-induced metal release from chromium metal and stainless steel

A research effort is undertaken to understand the mechanism of metal release from, e.g., inhaled metal particles or metal implants in the presence of proteins. The effect of protein adsorption on the metal release process from oxidized chromium metal surfaces and stainless steel surfaces was therefore examined by quartz crystal microbalance with energy dissipation monitoring (QCM-D) and graphite furnace atomic absorption spectroscopy (GFAAS). Differently charged and sized proteins, relevant for the inhalation and dermal exposure route were chosen including human and bovine serum albumin (HSA, BSA), mucin (BSM), and lysozyme (LYS). The results show that all proteins have high affinities for chromium and stainless steel (AISI 316) when deposited from solutions at pH 4 and at pH 7.4 where the protein adsorbed amount was very similar. Adsorption of albumin and mucin was substantially higher at pH 4 compared to pH 7.4 with approximately monolayer coverage at pH 7.4, whereas lysozyme adsorbed in multilayers at both investigated pH. The protein–surface interaction was strong since proteins were irreversibly adsorbed with respect to rinsing. Due to the passive nature of chromium and stainless steel (AISI 316) surfaces, very low metal release concentrations from the QCM metal surfaces in the presence of proteins were obtained on the time scale of the adsorption experiment. Therefore, metal release studies from massive metal sheets in contact with protein solutions were carried out in parallel. The presence of proteins increased the extent of metals released for chromium metal and stainless steel grades of different microstructure and alloy content, all with passive chromium(III)-rich surface oxides, such as QCM (AISI 316), ferritic (AISI 430), austentic (AISI 304, 316L), and duplex (LDX 2205).

Lysozyme Adsorption on Polyethylene Surfaces: Why Are Long Simulations Needed?

The adsorption of lysozyme onto a polyethylene (PE) surface in an aqueous environment was investigated via molecular dynamics (MD) simulation. The adsorption can be divided into three processes: diffusion to the surface, dehydration induced by hydrophobic surface-protein interactions, and denaturation. The dehydration process is very long, around 70 ns. Structural deformations start soon after the protein reaches the surface and continue during the whole trajectory. The hydrophobic residues are slowly driven toward the surface, inducing changes in the protein's secondary structure. The protein's secondary structural components near the surface are more disturbed than those farther away from the surface. The lysozyme is adsorbed with its long axis parallel to the surface and displays an anisotropic mobility on the surface that is probably due to the intrinsic structure of the PE surface. Our study demonstrates the need for long-time atomistic simulation in order to gain a complete understanding of the adsorption process.

Nonlinear spectroscopy of bio-interfaces

In the past five years the Max Planck Research Group for Nonlinear spectroscopy of bio-interfaces has worked at the intersection of surface science, nonlinear optics and soft and biological matter. For hard matter there is molecular level understanding of the surface region. This has enabled researchers to develop the highly complex structures that are nowadays found in (e. g.) computers, and mobile phones. Although biological and soft systems (such as liquid droplets, nanoparticles, liposomes, and viruses) are ubiquitous in our daily lives, our understanding is often limited to macroscopic theories. Consequently, the level of control of soft and biological matter is largely empirical and far behind that of hard matter. To initiate a change, we have in the past period worked on three main themes: (i) The investigation of structure and properties of biologically and medically relevant interfaces (supported lipid bilayers, biopolymer interfaces, water, protein-surface interactions). (ii) Development of nonlinear light scattering techniques as probes for molecular structure at the interfaces of small particles. (iii) The study of molecular interfacial structure, kinetics and dynamics of water, surfactants, phospholipids, sugars and peptides on small objects (nano-droplets, vesicles) in suspensions. The result of our work on these themes is summarized for this special issue article.

Benefits and Limitations of Porous Substrates as Biosensors for Protein Adsorption

Porous substrates have gained widespread interest for biosensor applications based on molecular recognition. Thus, there is a great demand to systematically investigate the parameters that limit the transport of molecules toward and within the porous matrix as a function of pore geometry. Finite element simulations (FES) and time-resolved optical waveguide spectroscopy (OWS) experiments were used to systematically study the transport of molecules and their binding on the inner surface of a porous material. OWS allowed us to measure the kinetics of protein adsorption within porous anodic aluminum oxide membranes composed of parallel-aligned, cylindrical pores with pore radii of 10-40 nm and pore depths of 0.8-9.6 μm. FES showed that protein adsorption on the inner surface of a porous matrix is almost exclusively governed by the flux into the pores. The pore-interior surface nearly acts as a perfect sink for the macromolecules. Neither diffusion within the pores nor adsorption on the surface are rate limiting steps, except for very low rate constants of adsorption. While adsorption on the pore walls is mainly governed by the stationary flux into the pores, desorption from the inner pore walls involves the rate constants of desorption and adsorption, essentially representing the protein-surface interaction potential. FES captured the essential features of the OWS experiments such as the initial linear slopes of the adsorption kinetics, which are inversely proportional to the pore depth and linearly proportional to protein concentration. We show that protein adsorption kinetics allows for an accurate determination of protein concentration, while desorption kinetics could be used to capture the interaction potential of the macromolecules with the pore walls.

Improving Protein Transfer Efficiency and Selectivity in Affinity Contact Printing by Using UV-Modified Surfaces

Affinity contact printing (αCP) is a technique that allows the selective capture of a target protein from solutions to a polymeric stamp decorated with an antibody, and then the target protein is printed onto a solid surface. The success of αCP critically relies on the precise control of protein-surface interactions. Here, we report a study on the effect of UV on the protein-surface interactions between protein and polydimethylsiloxane stamps and between protein and glass slides decorated with N,N-dimethyl-n-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP). Our results show that UV-modified surfaces can be used to improve the transfer efficiency and selectivity of proteins during αCP. For example, the protein transfer efficiency of human IgG onto a DMOAP-coated slide increases from 7.2% to 45.1% after the UV treatment. On the basis of these results, UV-modified surfaces were employed to develop a αCP system for protein detection. The detection limit of anti-IgG in this system is around 10 ng/mL, and the dynamic range is 4 orders of magnitude.

Control of Secondary Granule Release in Neutrophils by Ral GTPase

Neutrophil (polymorphonuclear leukocyte; PMN) inflammatory functions, including cell adhesion, diapedesis, and phagocytosis, are dependent on the mobilization and release of various intracellular granules/vesicles. In this study, we found that treating PMN with damnacanthal, a Ras family GTPase inhibitor, resulted in a specific release of secondary granules but not primary or tertiary granules and caused dysregulation of PMN chemotactic transmigration and cell surface protein interactions. Analysis of the activities of Ras members identified Ral GTPase as a key regulator during PMN activation and degranulation. In particular, Ral was active in freshly isolated PMN, whereas chemoattractant stimulation induced a quick deactivation of Ral that correlated with PMN degranulation. Overexpression of a constitutively active Ral (Ral23V) in PMN inhibited chemoattractant-induced secondary granule release. By subcellular fractionation, we found that Ral, which was associated with the plasma membrane under the resting condition, was redistributed to secondary granules after chemoattractant stimulation. Blockage of cell endocytosis appeared to inhibit Ral translocation intracellularly. In conclusion, these results demonstrate that Ral is a critical regulator in PMN that specifically controls secondary granule release during PMN response to chemoattractant stimulation.