An official website of the United States government
Members of the saposin-fold protein family and related proteins sharing a similar fold (saposin-like proteins; SAPLIP) are peripheral-membrane binding proteins that perform essential cellular functions. Saposins and SAPLIPs are abundant in both plant and animal kingdoms, and peripherally bind to lipid membranes to play important roles in lipid transfer and hydrolysis, defense mechanisms, surfactant stabilization, and cell proliferation. However, quantitative studies on the interaction between proteins and membranes are challenging due to the different nature of the two components in relation to size, structure, chemical composition, and polarity. Using liposomes and the saposin-fold member saposin C (sapC) as model systems, we describe here a method to apply solution NMR and dynamic light scattering to study the interaction between SAPLIPs and synthetic membranes at the quantitative level. Specifically, we prove with NMR that sapC binds reversibly to the synthetic membrane in a pH-controlled manner and show the dynamic nature of its fusogenic properties with dynamic light scattering. The method can be used to infer the optimal pH for membrane binding and to determine an apparent dissociation constant (KDapp) for protein-liposome interaction. We propose that these experiments can be applied to other proteins sharing the saposin fold.
more »
« less
ssPINE: Probabilistic Algorithm for Automated Chemical Shift Assignment of Solid-State NMR Data from Complex Protein Systems
The heightened dipolar interactions in solids render solid-state NMR (ssNMR) spectra more difficult to interpret than solution NMR spectra. On the other hand, ssNMR does not suffer from severe molecular weight limitations like solution NMR. In recent years, ssNMR has undergone rapid technological developments that have enabled structure–function studies of increasingly larger biomolecules, including membrane proteins. Current methodology includes stable isotope labeling schemes, non-uniform sampling with spectral reconstruction, faster magic angle spinning, and innovative pulse sequences that capture different types of interactions among spins. However, computational tools for the analysis of complex ssNMR data from membrane proteins and other challenging protein systems have lagged behind those for solution NMR. Before a structure can be determined, thousands of signals from individual types of multidimensional ssNMR spectra of samples, which may have differing isotopic composition, must be recognized, correlated, categorized, and eventually assigned to atoms in the chemical structure. To address these tedious steps, we have developed an automated algorithm for ssNMR spectra called “ssPINE”. The ssPINE software accepts the sequence of the protein plus peak lists from a variety of ssNMR experiments as inputs and offers automated backbone and side-chain assignments. The alpha version of ssPINE, which we describe here, is freely available through a web submission form.
more »
« less
- Award ID(s):
- 2051595
- PAR ID:
- 10425026
- Date Published:
- Journal Name:
- Membranes
- Volume:
- 12
- Issue:
- 9
- ISSN:
- 2077-0375
- Page Range / eLocation ID:
- 834
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Protein’s magic function stems from its structure and various analytical techniques have been developed for it. Among proteins, membrane proteins are encoded 20–30% of genomes, whereas cause challenges for many analytical techniques. For example, lots of membrane proteins cannot form single crystal structure required by X-ray crystallography. As for NMR, the measurements were hindered by the low tumbling rates of membrane (i.e., phospholipid bilayers) where membrane proteins exist. In addition, membrane proteins usually lay parallel to the surface of phospholipid bilayers or form transmembrane structure. No matter parallel or perpendicular to phospholipid bilayers surface, membrane proteins form monolayer structure which is also difficult for X-ray and NMR to provide high-resolution results. Because NMR and X-ray crystallography are the two major analytical techniques to address protein’s structure, membrane proteins only contribute 2.4% to the solved protein databank. Surface FT-IR techniques can evaluate the conformation and orientation of membrane proteins by amide I band. Specifically for α-helical peptides/proteins, the orientation of the axis is critical to decide whether proteins form transmembrane structure. Notice that the traditional FT-IR can only provide “low-resolution” results.Here,13C isotope was introduced into the nonamyloid component (NAC), which spans residues 61–95 of α-synuclein (α-syn). Then, p-polarized multiple-angle incidence resolution spectrometry (pMAIRS) was used to determine the orientation of a specific residue of α-helical NAC in monolayer. In general, pMAIRS is a novel technique to work complementary with X-ray and NMR to address membrane peptides/proteins structure with high resolution even in monolayer. Graphical abstractmore » « less
-
Membrane proteins work in large complexes to perceive and transduce external signals and to trigger a cellular response leading to the adaptation of the cells to their environment. Biochemical assays have been extensively used to reveal the interaction between membrane proteins. However, such analyses do not reveal the unique and complex composition of the membrane proteins of the different plant cell types. Here, we conducted a comprehensive analysis of the expression of Arabidopsis membrane proteins in the different cell types composing the root. Specifically, we analyzed the expression of genes encoding membrane proteins interacting in large complexes. We found that the transcriptional profiles of membrane protein-encoding genes differ between Arabidopsis root cell types. This result suggests that different cell types are characterized by specific sets of plasma membrane proteins, which are likely a reflection of their unique biological functions and interactions. To further explore the complexity of the Arabidopsis root cell membrane proteomes, we conducted a co-expression analysis of genes encoding interacting membrane proteins. This study confirmed previously reported interactions between membrane proteins, suggesting that the co-expression of genes at the single cell-type level can be used to support protein network predictions.more » « less
-
Accurate signal assignments can be challenging for congested solid-state NMR (ssNMR) spectra. We describe an automatic sequential assignment program (ASAP) to partially overcome this challenge. ASAP takes three input files: the residue type assignments (RTAs) determined from the better-resolved NCACX spectrum, the full peak list of the NCOCX spectrum, and the protein sequence. It integrates our auto-residue type assignment strategy (ARTIST) with the Monte Carlo simulated annealing (MCSA) algorithm to overcome the hurdle for accurate signal assignments caused by incomplete side-chain resonances and spectral congestion. Combined, ASAP demonstrates robust performance and accelerates signal assignments of large proteins (>200 residues) that lack crystalline order.more » « less
-
Atomistic molecular dynamics simulations of concentrated protein solutions in the presence of a phospholipid bilayer are presented to gain insights into the dynamics and interactions at the cytosol–membrane interface. The main finding is that proteins that are not known to specifically interact with membranes are preferentially excluded from the membrane, leaving a depletion zone near the membrane surface. As a consequence, effective protein concentrations increase, leading to increased protein contacts and clustering, whereas protein diffusion becomes faster near the membrane for proteins that do occasionally enter the depletion zone. Since protein–membrane contacts are infrequent and short-lived in this study, the structure of the lipid bilayer remains largely unaffected by the crowded protein solution, but when proteins do contact lipid head groups, small but statistically significant local membrane curvature is induced, on average.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/membranes12090834
