Surfactant effects on protein structure examined by electrospray ionization mass spectrometry (original) (raw)
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Analysis of Peptide and Protein Samples Containing Surfactants by MALDI-MS
Analytical Chemistry, 1997
Protein samples containing ionic surfactants such as sodium dodecyl sulfate (SDS) can be analyzed by matrixassisted laser desorption/ionization mass spectrometry, contrary to what is currently accepted. SDS at varying concentrations was added to four polypeptide samples. As expected, a decrease in the signal was observed for increasing concentrations of SDS up to 0.1% (w/v), at which no signal was observed for three of the analytes. However, a recovery of the signals of all the analytes was observed at concentrations of SDS above 0.3%, with useful spectra at concentrations as high as 10%. The SDS concentration at which signal recovery starts is always the same, regardless of the type and the concentration of the analyte. Another two surfactants 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and octylphenolpoly(ethylene glycol ether)10 (Triton X-100) were also tested. The trend for signal variation with CHAPS addition is similar to that for SDS addition, but for Triton X-100, the signal always diminishes with increasing surfactant concentration. For protein samples containing surfactants with anionic groups, addition of more surfactant allows the acquisition of useful mass spectrometric data.
Lipid Specificity of Surfactant Protein B Studied by Time-of-Flight Secondary Ion Mass Spectrometry
Biophysical Journal, 2006
One of the key functions of mammalian pulmonary surfactant is the reduction of surface tension to minimal values. To fulfill this function it is expected to become enriched in dipalmitoylphosphatidylcholine either on its way from the alveolar type II pneumocytes to the air/water interface of the lung or within the surface film during compression and expansion of the alveoli during the breathing cycle. One protein that may play a major role in this enrichment process is the surfactant protein B. The aim of this study was to identify the lipidic interaction partner of this protein. Time-of-flight secondary ion mass spectrometry was used to analyze the lateral distribution of the components in two SP-B-containing model systems. Either native or partly isotopically labeled lipids were analyzed. The results of both setups give strong indications that, at least under the specific conditions of the chosen model systems (e.g., concerning pH and lipid composition), the lipid interacting with surfactant protein B is not phosphatidylglycerol as generally accepted, but dipalmitoylphosphatidylcholine instead.
Journal of Biological Chemistry, 2011
D-mannose, D-␣-methylmannose, and glycerol, which represent subdomains of glycans on pathogen surfaces. Comparison of these complexes with the unliganded SP-A neck and carbohydrate recognition domain revealed an unexpected ligand-associated conformational change in the loop region surrounding the lectin site, one not previously reported for the lectin homologs SP-D and mannan-binding lectin. The net result of the conformational change is that the SP-A lectin site and the surrounding loop region become more compact. The Glu-202 side chain of unliganded SP-A extends out into the solvent and away from the calcium ion; however, in the complexes, the Glu-202 side chain translocates 12.8 Å to bind the calcium. The availability of Glu-202, together with positional changes involving water molecules, creates a more favorable hydrogen bonding environment for carbohydrate ligands. The Lys-203 side chain reorients as well, extending outward into the solvent in the complexes, thereby opening up a small cation-friendly cavity occupied by a sodium ion. Binding of this cation brings the large loop, which forms one wall of the lectin site, and the adjacent small loop closer together. The ability to undergo conformational changes may help SP-A adapt to different ligand classes, including microbial glycolipids and surfactant lipids. Surfactant protein A (SP-A) 2 is an abundant protein associated with pulmonary surfactant. Together with the lung pro-* This work was supported, in whole or in part, by National Institutes of Health Grant AI083222 from NIAID (to B. A. S.). This work was also supported by a Department of Veterans Affairs merit award (to F. X. M.). The atomic coordinates and structure factors (codes 3PAK, 3PAQ, 3PAR, and 3PBF) have been deposited in the Protein . 2 The abbreviations used are: SP-A, surfactant protein A; MBP, mannosebinding protein; CRD, carbohydrate recognition domain; NCRD, neck and carbohydrate recognition domain; ␣-MMA, ␣-methyl-D-mannoside; r.m.s.d., root mean square deviation. http://www.jbc.org/ Downloaded from FIGURE 4. Comparison of SP-A complex and native structures. Upper, stereo diagram of the C␣ trace of the SP-A NCRD⅐mannose complex (green) superimposed on the unliganded SP-A NCRD (orange). Middle and lower, stereo diagrams of an electron density map calculated with Fourier coefficients F o Ϫ F c of unliganded SP-A (middle) at 2.3 Å resolution and the SP-A⅐mannose complex (lower), omitting atoms from residues 197-203 from the calculation. The lectin calcium and sodium ions are shown as yellow and gray spheres, respectively.
Molecular Membrane Biology, 2011
Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-β-D-maltoside ('C12-b-M') as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b(6 )f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M.
Biochemistry, 2004
Although the membrane-associated surfactant protein B (SP-B) is an essential component of lung surfactant, which is itself essential for life, the molecular basis for its activity is not understood. SP-B's biophysical functions can be partially mimicked by subfragments of the protein, including the C-terminus. We have used NMR to determine the structure of a C-terminal fragment of human SP-B that includes residues 63-78. Structure determination was performed both in the fluorinated alcohol hexafluoro-2-propanol (HFIP) and in sodium dodecyl sulfate (SDS) micelles. In both solvents, residues 68-78 take on an amphipathic helical structure, in agreement with predictions made by comparison to homologous saposin family proteins. In HFIP, the five N-terminal residues of the peptide are largely unstructured, while in SDS micelles, these residues take on a well-defined compact conformation. Differences in helical residue side chain positioning between the two solvents were also found, with better agreement between the structures for the hydrophobic face than the hydrophilic face. A paramagnetic probe was used to investigate the position of the peptide within the SDS micelles and indicated that the peptide is located at the water interface with the hydrophobic face of the helix oriented inward, the hydrophilic face of the helix oriented outward, and the N-terminal residues even farther from the micelle center than those on the hydrophilic face of the R-helix. Interactions of basic residues of SP-B with anionic lipid headgroups are known to have an impact on function, and these studies demonstrate structural ramifications of such interactions via the differences observed between the peptide structures determined in HFIP and SDS.
Protein …, 1996
Although the effects of surfactant protein B (SP-B) on lipid surface activity in vitro and in vivo are well known, the relationship between molecular structure and function is still not fully understood. To further characterize protein structure-activity correlations, we have used physical techniques to study conformation, orientation, and molecular topography of N-terminal SP-B peptides in lipids and structure-promoting environments. Fourier transform infrared (FTIR) and CD measurements of SP-B1-25 (residues 1-25) in methanol, SDS micelles, egg yolk lecithin (EYL) liposomes, and surfactant lipids indicate the peptide has a dominant helical content, with minor turn and disordered components. Polarized FTIR studies of SP-B1-25 indicate the long molecular axis lies at an oblique angle to the surface of lipid films. Truncated peptides were similarly examined to assign more accurately the discrete conformations within the SP-B1-25 sequence. Residues Cys-8-Gly-25 are largely alpha-helix in methanol, whereas the N-terminal segment Phe-1-Cys-8 had turn and helical propensities. Addition of SP-B1-25 spin-labeled at the N-terminal Phe (i.e., SP-B1-25) to SDS, EYL, or surfactant lipids yielded electron spin resonance spectra that reflect peptide bound to lipids, but retaining considerable mobility. The absence of characteristic radical broadening indicates that SP-B1-25 is minimally aggregated when it interacts with these lipids. Further, the high polarity of SP-B1-25 argues that the reporter on Phe-1 resides in the headgroup of the lipid dispersions. The blue-shift in the endogenous fluorescence of Trp-9 near the N-terminus of SP-B1-25 suggests that this residue also lies near the lipid headgroup. A summary model based on the above physical experiments is presented for SP-B1-25 interacting with lipids.
Self-association behaviour of protein:surfactant systems in alcohol∕water mixtures
Iee Proceedings - Nanobiotechnology, 2005
The effect of the addition of short-chain monohydric alcohols (ethanol and propan-2-ol) to the protein:surfactant system lysozyme:sodium dodecyl sulfate (Lz:SDS) in aqueous solution was investigated using a conductometric technique. A second protein:surfactant system, bovine serum albumin:SDS (BSA:SDS) was also investigated so that the effect of a different protein conformation and composition could be compared. The critical aggregation concentration (CAC) of the protein forming the complex and the critical micelle concentration (CMC*) of SDS in the presence of protein, at different alcohol concentrations, were determined. It was found in both cases that the addition of alcohol does not produce a significant change in the CAC, whereas the CMC* displays variation with alcohol concentration that shows an inversion in the ranges 0.05-0.06 ethanol mole fraction and 0.02-0.03 propan-2-ol mole fraction. This suggests that, in contrast with the CAC behaviour, the major factor that drives SDS micellisation in the presence of protein is the variation in water structure. Results also suggest that it occurs in the same way for both proteins, where electrostatic interactions are the main force in the formation of the complex. Conversely, hydrophobic interactions play the dominant role at the micellisation stage, and only the extent of the interaction between protein:surfactant aggregates and surfactant species seems to depend on protein nature.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2001
. Surfactant protein A SP-A is an abundant protein found in pulmonary surfactant which has been reported to have multiple functions. In this review, we focus on the structural importance of each domain of SP-A in the functions of protein oligomerization, the structural organization of lipids and the surface-active properties of surfactant, with an emphasis on ultrastructural analyses. The N-terminal domain of SP-A is required for disulfide-dependent protein oligomerization, and for binding and aggregation of phospholipids, but there is no evidence that this domain directly interacts with lipid membranes. The collagen-like domain is important for the stability and oligomerization of SP-A. It also contributes shape and dimension to the molecule, and appears to determine membrane spacing in lipid aggregates such as common myelin and tubular myelin. The neck domain of SP-A is primarily involved in protein trimerization, which is critical for many protein functions, but it does not appear to be directly involved in lipid interactions. The globular C-terminal domain of SP-A clearly plays a central role in lipid binding, and in more complex functions such as the formation andror stabilization of curved membranes. In recent work, we have determined that the maintenance of low surface tension of surfactant in the presence of serum protein inhibitors requires cooperative interactions between the C-terminal and N-terminal domains of the molecule. This effect of SP-A requires a high degree of oligomeric assembly of the protein, and may be mediated by the activity of the protein to alter the form or physical state of surfactant lipid aggregates. ᮊ