Rob C. A. Keller

Mode of Insertion of the Signal Sequence of a Bacterial Precursor Protein into Phospholipid Bilayers As Revealed by Cysteine-Based Site-Directed Spectroscopy

The interactions between a bacterial precursor protein and phospholipids in bilayer-based model membrane systems is addressed in this study. The precursor-lipid interactions were assessed from the side of the lipid phase by fluorescence and electron spin resonance spectroscopy, using the precursor of the Escherichia coli outer membrane protein PhoE. The role of the signal sequence, as part of the precursor, in this interaction was investigated by using cysteine-based site-directed spectroscopy. For this purpose, purified cysteine-containing mutants of prePhoE, which were made by site-directed mutagenesis of the signal sequence part and of the mature part, and defined lipids were used. The location of the fluorescently labeled cysteine residues was established by resonance energy transfer and quenching experiments and those of the corresponding spin-labeled cysteine residues by paramagnetic relaxation enhancement. It was demonstrated that precursor-phospholipid interactions exist in model membrane systems and also that these interactions were dependent on the presence of anionic phospholipids and resulted in a deep insertion of (parts of) the precursor into the lipid bilayer. Furthermore, the results with the cysteine mutations in the signal sequence of the precursor indicate that both termini of the signal sequence are located near or at the membrane surface, with only the fluorescence of the labeled cysteines in the signal sequence part being protected against aqueous quenchers. The results demonstrate that, when part of the intact precursor, the signal sequence experiences similar lipid-protein interactions as do isolated signal peptides. They also indicate that the signal sequence inserts entirely as a looped structure into the membrane. In addition, the data also indicate that the mature part of the precursor has an affinity for the membrane.

Nucleotide and negatively charged lipid-dependent vesicle aggregation caused by SecA. Evidence that SecA contains two lipid-binding sites.

SecA which is an overall acidic protein was found to induce an increase in the turbidity of a solution of vesicles consisting of negatively charged phospholipids. This increase was found to be due to an aggregation of the vesicles mediated by SecA. The SecA‐mediated vesicle aggregation was not found for zwitterionic 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine and showed a large dependence on both temperature and ionic strength. Furthermore it was shown that ATP and to a lesser extent ADP+Pi were able to reduce the SecA‐mediated vesicle aggregation, while no effect could be seen for a non‐hydrolysable ATP analog AMP‐PNP. Using the steady state fluorescence anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene present in 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoglycerol vesicles we could show that SecA inserts in the bilayer. Monolayer studies confirmed that SecA is able to cause close contact between two membranes and gave a direct insight into the different types of lipid‐protein interactions involved. From our results we propose that the SecA monomer possesses two lipid‐binding sites which in the functional dimer conformation are responsible for the SecA‐mediated vesicle aggregation.

SecA restricts in a nucleotide-dependent manner acyl chain mobility up to the center of a phospholipid bilayer

The effects of SecA—lipid interactions on lipid mobility were studied by electron spin resonance (ESR) spectroscopy in bilayer systems containing phospholipids spin‐labeled at different positions along the acyl chain. The SecA protein, which functions in protein translocation at the cytosolic side of the E. coli inner membrane, was found to decrease the mobility of the lipids upon its interaction with the membrane. The restriction of lipid motion, at all chain positions measured, reflects the ability of SecA to penetrate the membrane. At a 49:1 lipid/protein molar ratio, a second, motionally more restricted component is observed in ESR spectra of phospholipids spin‐labeled close to the methyl ends of the chains (12th and 14th positions). Furthermore, SecA was found to eliminate the order‐to‐disorder phase transition of 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphoglycerol bilayers. A remarkably strong reduction in the ability of SecA to penetrate the membrane was found when the nucleotides ATP and ADP + Pi were present. The presence of the non‐hydrolyzable analogue AMP‐PNP had no effect. These results clearly demonstrate that SecA perturbs, in a nucleotide dependent manner, lipid mobility upon insertion into the bilayer. The implications of these findings for translocation of precursor proteins across the E. coli inner membrane are discussed.

The prediction of novel multiple lipid-binding regions in protein translocation motor proteins: A possible general feature

Protein translocation is an important cellular process. SecA is an essential protein component in the Sec system, as it contains the molecular motor that facilitates protein translocation. In this study, a bioinformatics approach was applied in the search for possible lipid-binding helix regions in protein translocation motor proteins. Novel lipid-binding regions in Escherichia coli SecA were identified. Remarkably, multiple lipid-binding sites were also identified in other motor proteins such as BiP, which is involved in ER protein translocation. The prokaryotic signal recognition particle receptor FtsY, though not a motor protein, is in many ways related to SecA, and was therefore included in this study. The results demonstrate a possible general feature for motor proteins involved in protein translocation.

New user-friendly approach to obtain an Eisenberg plot and its use as a practical tool in protein sequence analysis.

The Eisenberg plot or hydrophobic moment plot methodology is one of the most frequently used methods of bioinformatics. Bioinformatics is more and more recognized as a helpful tool in Life Sciences in general, and recent developments in approaches recognizing lipid binding regions in proteins are promising in this respect. In this study a bioinformatics approach specialized in identifying lipid binding helical regions in proteins was used to obtain an Eisenberg plot. The validity of the Heliquest generated hydrophobic moment plot was checked and exemplified. This study indicates that the Eisenberg plot methodology can be transferred to another hydrophobicity scale and renders a user-friendly approach which can be utilized in routine checks in protein–lipid interaction and in protein and peptide lipid binding characterization studies. A combined approach seems to be advantageous and results in a powerful tool in the search of helical lipid-binding regions in proteins and peptides. The strength and limitations of the Eisenberg plot approach itself are discussed as well. The presented approach not only leads to a better understanding of the nature of the protein–lipid interactions but also provides a user-friendly tool for the search of lipid-binding regions in proteins and peptides.

Identification and in silico analysis of helical lipid binding regions in proteins belonging to the amphitropic protein family

The role of protein–lipid interactions is increasingly recognized to be of importance in numerous biological processes. Bioinformatics is being increasingly used as a helpful tool in studying protein–lipid interactions. Especially recently developed approaches recognizing lipid binding regions in proteins can be implemented. In this study one of those bioinformatics approaches specialized in identifying lipid binding helical regions in proteins is expanded. The approach is explored further by features which can be easily obtained manually. Some interesting examples of members of the amphitropic protein family have been investigated in order to demonstrate the additional features of this bioinformatics approach. The results in this study seem to indicate interesting characteristics of amphitropic proteins and provide insight into the mechanistic functioning and overall understanding of this intriguing class of proteins. Additionally, the results demonstrate that the presented bioinformatics approach might be either an interesting starting point in protein–lipid interactions studies or a good tool for selecting new focus points for more detailed experimental research of proteins with known overall protein–lipid binding abilities.

Prediction of Lipid-Binding Regions in Cytoplasmic and Extracellular Loops of Membrane Proteins as Exemplified by Protein Translocation Membrane Proteins

The presence of possible lipid-binding regions in the cytoplasmic or extracellular loops of membrane proteins with an emphasis on protein translocation membrane proteins was investigated in this study using bioinformatics. Recent developments in approaches recognizing lipid-binding regions in proteins were found to be promising. In this study a total bioinformatics approach specialized in identifying lipid-binding helical regions in proteins was explored. Two features of the protein translocation membrane proteins, the position of the transmembrane regions and the identification of additional lipid-binding regions, were analyzed. A number of well-studied protein translocation membrane protein structures were checked in order to demonstrate the predictive value of the bioinformatics approach. Furthermore, the results demonstrated that lipid-binding regions in the cytoplasmic and extracellular loops in protein translocation membrane proteins can be predicted, and it is proposed that the interaction of these regions with phospholipids is important for proper functioning during protein translocation.

Chapter 8 Lipid involvement in protein translocation

Publisher Summary In membrane biogenesis and protein targeting, polypeptide chains very often insert into and move across biological membranes. Correct membrane assembly and functioning critically depends on the spatial and temporal interactions between the two main membrane components, i.e. proteins and lipids. It can therefore a priori be reasoned that protein-lipid interactions must be involved in membrane insertion and translocation of newly synthesized proteins. Such an involvement will be of a general nature such as to correctly assemble a putative proteinaceous insertion/translocation device in a membrane and to provide and maintain the essential barrier function of the membrane during the translocation process. In addition, membrane lipids could play more direct roles to provide alone or in combination with proteins an insertion and translocation pathway. The specific, dynamical, and complex lipid composition of biomembranes together with the unique structural and motional properties of individual membrane lipid classes offer fascinating possibilities for protein-lipid interactions to be involved in binding, insertion, translocation and release of proteins in transit across membranes. The approach presented in the chapter to analyze these possibilities is to study selected protein transport pathways in the main cellular protein trafficking routes via a combination of biochemical and biophysical techniques using both model and biological membranes. This chapter summarizes the current insights into the involvement of lipids in protein translocation, in protein secretion of prokaryotes, and in protein import in mitochondria and chloroplasts.