Insertion of X-ray structures of proteins in membranes

Basyn, Frédéric; Spies, Benoit; Bouffioux, Olivier; Thomas, A. G.; Brasseur, Robert

doi:10.1016/s1093-3263(03)00122-0

Cited by 26 publications

(33 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Interestingly, NMR studies on Table 1 The parameters of amide hydrogen exchange of the N-terminal helix and the internal helices of human pancreatic PLA 2 free in the buffer and bound to POPC/POPG (4:1) membranes. In both cases, the buffer was 100 mM NaCl, 1 mM NaN 3 a porcine pancreatic PLA 2 , which shares 88% sequence identity with its human counterpart, identified that the N-terminal helix of PLA 2 acquires a rigid structure in the complex with phospholipid micelles and a substrate mimic [20], which is consistent with our findings. In addition to characterization of the dynamic structural properties of the protein, polarized ATR-FTIR spectra of the segmentally 13 C-labeled PLA 2 were used to determine the precise membrane binding mode of the protein, which has been described elsewhere [19].…”

Section: Resultssupporting

confidence: 86%

“…Determination of the structures of these complexes is key to understanding the molecular mechanisms underlying protein function. While the most valuable structural information on macromolecular complexes has been provided by atomic-resolution techniques such as X-ray crystallography and NMR, structural characterization of protein-membrane complexes is a particularly challenging problem for both methods [3,6,8,10,11,15]. Therefore, there is an urgent need to develop new biophysical or other technologies to characterize the structure and dynamics of proteins in their membrane-bound state and to resolve structural changes occurring in specific domains or segments of the protein.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Structural analysis of proteins by isotope-edited FTIR spectroscopy

Tatulian

2010

Spectroscopy

View full text Add to dashboard Cite

Abstract. Structure determination of multidomain proteins or protein-membrane complexes is one of the most challenging tasks in modern structural biology. High-resolution techniques, like NMR or X-ray crystallography, are limited to molecules of moderate size or those that can be crystallized easily. Both methods encounter serious technical obstacles in structural analysis of protein-membrane systems. This work describes an emerging biophysical technique that combines segmental isotope labeling of proteins with Fourier transform infrared (FTIR) spectroscopy, which provides site-specific structural information on proteins and allows structural characterization of protein-membrane complexes. Labeling of a segment of the protein with 13 C results in infrared spectral resolution of the labeled and unlabeled parts and thus allows identification of structural changes in specific domains/segments of the protein that accompany functional transitions. Segmental isotope labeling also allows determination of the precise configuration of protein-membrane complexes by polarized attenuated total reflection FTIR (ATR-FTIR) spectroscopy. These new developments offer solutions to functionally important site-specific structural changes in proteins and protein-membrane complexes that are hard to approach using conventional methods.

show abstract

Section: Resultssupporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Structural analysis of proteins by isotope-edited FTIR spectroscopy

Tatulian

2010

Spectroscopy

View full text Add to dashboard Cite

show abstract

“…The interactions between the peptide and the interface are approached by two energy restraint terms that vary with z. As previously described (20,21), one restraint term describes the hydrophobicity effect that pushes hydrophilic atoms in water and draws hydrophobic atoms in the lipid phase, whereas the other one, the lipid perturbation, pushes any molecule out of the bilayer. In our assay, the peptide structure remains helical throughout the test.…”

Section: Methodsmentioning

confidence: 99%

Role of the Lid Hydrophobicity Pattern in Pancreatic Lipase Activity

Thomas

Allouche

Basyn

et al. 2005

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Pancreatic lipase is a soluble globular protein that must undergo structural modifications before it can hydrolyze oil droplets coated with bile salts. The binding of colipase and movement of the lipase lid open access to the active site. Mechanisms triggering lid mobility are unclear. The *KNILSQIVDIDGI* fragment of the lid of the human pancreatic lipase is predicted by molecular modeling to be a tilted peptide. Tilted peptides are hydrophobicity motifs involved in membrane fusion and more globally in perturbations of hydrophobic/hydrophilic interfaces. Analysis of this lid fragment predicts no clear consensus of secondary structure that suggests that its structure is not strongly sequence determined and could vary with environment. Point mutations were designed to modify the hydrophobicity profile of the [240 -252] fragment and their consequences on the lipase-mediated catalysis were tested. Two mutants, in which the tilted peptide motif was lost, also have poor activity on bile saltcoated oil droplets and cannot be reactivated by colipase. Conversely, one mutant in which a different tilted peptide is created retains colipase dependence. These results suggest that the tilted hydrophobicity pattern of the [240 -252] fragment is neither important for colipase binding to lipase, nor for interfacial binding but is important to trigger the maximal catalytic efficiency of lipase in the presence of bile salt.The pancreatic lipase-colipase complex plays a key role in dietary fat absorption in the intestine by converting triglycerides into more polar products able to cross the brush-border membrane of enterocytes. The lipase is fully active on water-insoluble substrates that form lipid/water interfaces, a phenomenon called interfacial activation. In vivo, oil droplets consist of a bulk substrate phase surrounded by a monolayer of amphiphilic compounds, mainly biliary lipids (phosphatidylcholine, cholesterol and bile salts) that prevent lipase adsorption. To counteract the inhibitory effect of biliary lipids, the pancreas secretes a small protein, colipase, which anchors lipase to the biliary lipid-coated water/ lipid interface. The water-soluble lipase must therefore partition between aqueous and lipid phases before lipolysis can occur.Structural studies (1-3) have shown that lipases possess a two-domain organization, the N-terminal domain bearing the active site and the C-terminal domain bearing the colipase binding site. One specific feature of lipase is the shielding of its catalytic site by a surface loop (lid) controlling the access of the substrate. Therefore, movement of the lid domain is an absolute requirement for the lipase to adopt an active conformation.The role of the lid in lipolysis has been investigated by site-directed mutagenesis, lid exchange, or lid deletion (4 -6). It was first thought to account for the interfacial activation of pancreatic lipase but the presence of a full-length lid in most pancreatic lipase-related proteins that display no interfacial activation rules out this explanation. Actua...

show abstract

“…It should be noted that several other methods exist for this purpose. Including Garlik, 39 IMPALA, 40 or PPM. 13 In a comparison of the different methods, Lomize et al 13 showed that different estimates of bilayer thickness may be obtained.…”

Section: Discussionmentioning

confidence: 99%

Dissecting membrane protein architecture: An annotation of structural complexity

2009

View full text Add to dashboard Cite

alpha-Helical membrane proteins exist in an anisotropic environment which strongly influences their folding, stability, and architecture, which is far more complex than a simple bundle of transmembrane helices, notably due to helix deformations, prosthetic groups and extramembrane structures. However, the role and the distribution of such heterogeneity in the supra molecular organization of membrane proteins remains poorly investigated. Using a nonredundant subset of alpha-helical membrane proteins, we have annotated and analyze the statistics of several types of new elements such as incomplete helices, intramembrane loops, helical extensions of helical transmembrane domains, extracellular loops, and helices lying parallel to the membrane surface. The relevance of the annotation scheme was studied using residue composition, statistics, physical chemistry, and symmetry of their distribution in relation to the immediate membrane environment. Calculation of hydrophobicity using different scales show that different structural elements appear to have affinities coherent with their position in the membrane. Examination of the annotation scheme suggests that there is considerable information content in the amino acid compositions of the different elements suggesting that it might be useful for structural prediction. More importantly, the proposed annotation will help to decipher the complex hierarchy of interactions involved in membrane protein architecture.

show abstract

Insertion of X-ray structures of proteins in membranes

Cited by 26 publications

References 47 publications

Structural analysis of proteins by isotope-edited FTIR spectroscopy

Structural analysis of proteins by isotope-edited FTIR spectroscopy

Role of the Lid Hydrophobicity Pattern in Pancreatic Lipase Activity

Dissecting membrane protein architecture: An annotation of structural complexity

Contact Info

Product

Resources

About