RESUMEN
Aromatic amino acids of membrane proteins are enriched at the lipid-water interface. The role of tryptophan on the folding and stability of an integral membrane protein is investigated with ultraviolet resonance Raman and fluorescence spectroscopy. We investigate a model system, the ß-barrel outer membrane protein A (OmpA), and focus on interfacial tryptophan residues oriented toward the lipid bilayer (trp-7, trp-170, or trp-15) or the interior of the ß-barrel pore (trp-102). OmpA mutants with a single tryptophan residue at a nonnative position 170 (Trp-170) or a native position 7 (Trp-7) exhibit the greatest stability, with Gibbs free energies of unfolding in the absence of denaturant of 9.4 and 6.7 kcal/mol, respectively. These mutants are more stable than the tryptophan-free OmpA mutant, which exhibits a free energy of unfolding of 2.6 kcal/mol. Ultraviolet resonance Raman spectra of Trp-170 and Trp-7 reveal evolution of a hydrogen bond in a nonpolar environment during the folding reaction, evidenced by systematic shifts in hydrophobicity and hydrogen bond markers. These observations suggest that the hydrogen bond acceptor is the lipid acyl carbonyl group, and this interaction contributes significantly to membrane protein stabilization. Other spectral changes are observed for a tryptophan residue at position 15, and these modifications are attributed to development of a tryptophan-lipid cation-π interaction that is more stabilizing than an intraprotein hydrogen bond by â¼2 kcal/mol. As expected, there is no evidence for lipid-protein interactions for the tryptophan residue oriented toward the interior of the ß-barrel pore. These results highlight the significance of lipid-protein interactions, and indicate that the bilayer provides more than a hydrophobic environment for membrane protein folding. Instead, a paradigm of lipid-assisted membrane protein folding and stabilization must be adopted.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Dobles de Lípidos/metabolismo , Pliegue de Proteína , Espectrometría Raman , Triptófano/metabolismo , Rayos Ultravioleta , Adsorción , Membrana Dobles de Lípidos/química , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
Antimicrobial peptides play an important role in host defense against pathogens. Recently, phenol-soluble modulins (PSMs) from Staphylococcus epidermidis (S. epidermidis) were shown to interact with lipid membranes, form complexes, and exert antimicrobial activity. Based on the abundance and innocuity of the cutaneous resident S. epidermidis, we hypothesized that their PSMs contribute to host defense. Here we show that S. epidermidis delta-toxin (PSMgamma) is normally present in the epidermis and sparsely in the dermis of human skin using immunohistochemistry. Synthetic delta-toxin interacted with neutrophil extracellular traps (NETs) and colocalized with cathelicidin while also inducing NET formation in human neutrophils. In antimicrobial assays against Group A Streptococcus (GAS), delta-toxin cooperated with CRAMP, hBD2, and hBD3. In whole blood, addition of delta-toxin exerted a bacteriostatic effect on GAS, and in NETs, delta-toxin increased their killing capacity against this pathogen. Coimmunoprecipitation and tryptophan spectroscopy demonstrated direct binding of delta-toxin to host antimicrobial peptides LL-37, CRAMP, hBD2, and hBD3. Finally, in a mouse wound model, GAS survival was reduced (along with Mip-2 cytokine levels) when the wounds were pretreated with delta-toxin. Thus, these data suggest that S. epidermidis-derived delta-toxin cooperates with the host-derived antimicrobial peptides in the innate immune system to reduce survival of an important human bacterial pathogen.
Asunto(s)
Antiinfecciosos/farmacología , Toxinas Bacterianas/farmacología , Péptidos/farmacología , Staphylococcus epidermidis/metabolismo , Streptococcus pyogenes/efectos de los fármacos , Animales , Toxinas Bacterianas/metabolismo , Humanos , Inmunohistoquímica , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Neutrófilos/citología , Unión Proteica , Piel/efectos de los fármacos , Piel/metabolismo , Análisis Espectral/métodosRESUMEN
Antimicrobial peptides serve as a first line of innate immune defense against invading organisms such as bacteria and viruses. In this study, we hypothesized that peptides produced by a normal microbial resident of human skin, Staphylococcus epidermidis, might also act as an antimicrobial shield and contribute to normal defense at the epidermal interface. We show by circular dichroism and tryptophan spectroscopy that phenol-soluble modulins (PSMs) gamma and delta produced by S. epidermidis have an alpha-helical character and a strong lipid membrane interaction similar to mammalian AMPs such as LL-37. Both PSMs directly induced lipid vesicle leakage and exerted selective antimicrobial action against skin pathogens such as Staphylococcus aureus. PSMs functionally cooperated with each other and LL-37 to enhance antimicrobial action. Moreover, PSMs reduced Group A Streptococcus (GAS) but not the survival of S. epidermidis on mouse skin. Thus, these data suggest that the production of PSMgamma and PSMdelta by S. epidermidis can benefit cutaneous immune defense by selectively inhibiting the survival of skin pathogens while maintaining the normal skin microbiome.
Asunto(s)
Toxinas Bacterianas/farmacología , Queratinocitos/microbiología , Staphylococcus epidermidis/metabolismo , Infecciones Estreptocócicas/tratamiento farmacológico , Streptococcus pyogenes , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Toxinas Bacterianas/síntesis química , Permeabilidad de la Membrana Celular , Células Cultivadas , Dicroismo Circular , Células Epidérmicas , Epidermis/inmunología , Epidermis/microbiología , Humanos , Queratinocitos/citología , Queratinocitos/inmunología , Membrana Dobles de Lípidos/metabolismo , Membranas Artificiales , Ratones , Ratones Endogámicos C57BL , Estructura Secundaria de Proteína , Infecciones Cutáneas Estafilocócicas/tratamiento farmacológico , Infecciones Cutáneas Estafilocócicas/inmunología , Infecciones Cutáneas Estafilocócicas/microbiología , Staphylococcus aureus , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , SimbiosisRESUMEN
Refolding curves of the integral membrane protein outer membrane protein A (OmpA) were measured to determine the conformational stabilities of this model system for membrane protein folding. Wild-type OmpA exhibits a free energy of unfolding (DeltaG degrees H2O) of 10.5 kcal/mol. Mutants, containing a single tryptophan residue at the native positions 7, 15, 57, 102, or 143, are less stable than wild-type OmpA, with DeltaG degrees H2O values of 6.7, 4.8, 2.4, 4.7, and 2.8 kcal/mol, respectively. The trend observed here is discussed in terms of noncovalent interactions, including aromatic interactions and hydrogen bonding. The effect of the soluble tail on the conformational stability of the transmembrane domain of OmpA was also investigated via truncated single-Trp mutants; DeltaG degrees H2O values for four of the five truncated mutants are greater by >2.7 kcal/mol relative to the full-length versions, suggesting that the absence of the soluble domain may destabilize the unfolded transmembrane domain. Finally, dynamic light scattering experiments were performed to measure the effects of urea and protein on vesicle size and stability. Urea concentrations greater than 1 M cause an increase in vesicle size, and these diameters are unaltered in the presence of protein. These dynamic light scattering results complement the fluorescence studies and illustrate the important effects of vesicle size on protein conformational stability.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Pliegue de Proteína , Triptófano/metabolismo , Liposomas Unilamelares/química , Proteínas de la Membrana Bacteriana Externa/genética , Modelos Moleculares , Mutación , Conformación Proteica , Renaturación de Proteína , Estabilidad Proteica , Estructura Terciaria de Proteína , Dispersión de Radiación , Solubilidad , Espectrometría de Fluorescencia , Termodinámica , Liposomas Unilamelares/metabolismo , Urea/farmacologíaRESUMEN
The vibrational structure of native anchoring tryptophan (Trp) and tyrosine residues in an integral membrane protein, bacterial outer membrane protein A (OmpA), have been investigated using UV resonance Raman (UVRR) spectroscopy for the first time. Spectra of native OmpA, a single-Trp mutant, and a Trp-less mutant were recorded in folded and unfolded states, and reveal significant changes in tryptophan structure and local environment. Salient alterations upon folding include loss of hydrogen-bonding character of indole N1H, evidenced by a shift in W17 frequency from 874 and 878 cm(-1), and growth in hydrophobicity of the local tryptophan environment, supported by increase in the ratio I1361/I1340. In addition to these site-specific changes in a single tryptophan residue, modification of the vibrational structure of the remaining native tryptophan and tyrosine amino acids is also evident. Finally, the UVRR data presented here indicate that the structures of OmpA folded in vesicle and folded in detergent may differ, and provide important foundations for ongoing studies of membrane protein folding.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Pliegue de Proteína , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana/genética , Modelos Moleculares , Mutación/genética , Estructura Terciaria de Proteína , Espectrometría RamanRESUMEN
Protein folding is an exploding area of research in biophysics and physical chemistry. Here, we describe the integration of several techniques, including absorption spectroscopy, fluorescence spectroscopy, and Förster resonance energy transfer (FRET) measurements, to probe important topics in protein folding. Cytochrome c is used as a model protein; comparison of conformational stabilities ( ΔGH2O∘) measured via two chemical denaturants, urea and guanidinium hydrochloride, illustrate important concepts in protein folding and intermolecular interactions. In addition, the determination of intraprotein distances based upon the FRET pair Trp-59 and the heme group for unfolded states of cytochrome c highlights the evolution of the protein structure under unfolding conditions. Analysis and discussion of these results provide opportunities to gain in-depth understanding of models for protein folding while enhancing students' skills with optical techniques. Collectively, the combination of optical spectroscopy, rigorous quantitative analysis, and a focus on biophysics illustrates the significance of fundamental research at the growing intersection of chemistry, biology, and physics.