Jaume Torres

Site-specific examination of secondary structure and orientation determination in membrane proteins: The peptidic 13C18O group as a novel infrared probe

Detailed site‐specific information can be exceptionally useful in structural studies of macromolecules in general and proteins in particular. Such information is usually obtained from spectroscopic studies using a label/probe that can reflect on particular properties of the protein. A suitable probe must not modify the native properties of the protein, and should yield interpretable structural information, as is the case with isotopic labels used by Fourier transform infrared (FTIR) spectroscopy. In particular, 1‐13CO labels have been shown to relay site‐specific secondary structure and orientational information, although limited to small peptides. The reason for this limitation is the high natural abundance of 13C and the lack of baseline resolution between the main amide I band and the isotope‐edited peak. Herein, we dramatically extend the utility of isotope edited FTIR spectroscopy to proteins of virtually any size through the use of a new 1‐13C18O label. The double‐isotope label virtually eliminates any contribution from natural abundance 13C. More importantly, the isotope‐edited peak is further red‐shifted (in accordance with ab initio Hartree–Fock calculations) and is now completely baseline resolved from the main amide I band. Taken together, this new label enables determination of site specific secondary structure and orientation in proteins of virtually any size. Even in small peptides 1‐13C18O is far preferable as a label in comparison to 1‐13C16O since it enables analysis without the need for any deconvolution or peak fitting procedures. Finally, the results obtained herein represent the first stage in the application of site‐directed dichroism to the structural elucidation of polytopic membrane proteins.

A new method to model membrane protein structure based on silent amino acid substitutions.

The importance of accurately modeling membrane proteins cannot be overstated, in lieu of the difficulties in solving their structures experimentally. Often, however, modeling procedures (e.g., global searching molecular dynamics) generate several possible candidates rather then pointing to a single model. Herein we present a new approach to select among candidate models based on the general hypothesis that silent amino acid substitutions, present in variants identified from evolutionary conservation data or mutagenesis analysis, do not affect the stability of a native structure but may destabilize the non‐native structures also found. The proof of this hypothesis has been tested on the α‐helical transmembrane domains of two homodimers, human glycophorin A and human CD3‐ζ, a component of the T‐cell receptor. For both proteins, only one structure was identified using all the variants. For glycophorin A, this structure is virtually identical to the structure determined experimentally by NMR. We present a model for the transmembrane domain of CD3‐ζ that is consistent with predictions based on mutagenesis, homology modeling, and the presence of a disulfide bond. Our experiments suggest that this method allows the prediction of transmembrane domain structure based only on widely available evolutionary conservation data. Proteins 2001;44:370–375.

Topology of the Salmonella invasion protein SipB in a model bilayer

A critical early event in Salmonella infection is entry into intestinal epithelial cells. The Salmonellainvasion protein SipB is required for the delivery of bacterial effector proteins into target eukaryotic cells, which subvert signal transduction pathways and cytoskeletal dynamics. SipB inserts into the host plasma membrane during infection, and the purified protein has membrane affinity and heterotypic membrane fusion activity in vitro. We used complementary biochemical and biophysical techniques to inves‐tigate the topology of purified SipB in a model membrane. We show that the 593 residue SipB is predominantly α‐helical in aqueous solution, and that no significant change in secondary structural content accompanies lipid interaction. SipB contains two α‐helical transmembrane domains (residues 320–353 and 409–427), which insert deeply into the bilayer. Their integration allowed the hydrophilic region between the hydrophobic domains (354–408) to cross the bilayer. SipB membrane integration required both the hydrophobic domains and an additional helical C‐terminal region (428–593). Further spectroscopic analysis of these domains in isolation showed that the hydrophobic regions insert obliquely into the bilayer, whereas the C‐terminal domain associates with the bilayer surface, tilted parallel to the membrane. The combined data suggest a topological model for membrane‐inserted SipB.

The orientation of the antibiotic peptide maculatin 1.1 in DMPG and DMPC lipid bilayers. Support for a pore-forming mechanism

Maculatin 1.1 is an antimicrobial peptide isolated from the Australian tree frog Litoria genimaculata that adopts an amphipathic, α‐helical structure in solution. Its orientation and conformation when incorporated to pre‐formed DMPG (1,2‐dimyristoyl‐sn‐glycero‐3‐phosphoglycerol) and DMPC (1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine) vesicles was determined using polarised Fourier transform infrared–attenuated total reflection infrared and deuterium exchange experiments. For DMPG membranes, our results show insertion of ∼70% of the maculatin 1.1 molecules, with an angle of insertion of approximately 35° to the membrane normal and with a predominant α‐helical structure. These results suggest that maculatin 1.1 acts through a pore‐forming mechanism to lyse bacterial membranes. A similar degree of insertion in DMPG (65%) and α‐helical structure was observed for a biologically inactive, less amphipathic maculatin 1.1 analogue, P15A, although the helix tilt was found to be greater (46°) than for maculatin 1.1. Similar experiments performed using DMPC liposomes showed poor insertion, less than 5%, for both maculatin 1.1 and its analogue. In addition, the shape of the amide I band in these samples is consistent with α‐helix, β‐structure and disordered structures being present in similar proportion. These results clearly show that maculatin 1.1 inserts preferentially in negatively charged membranes (DMPG) which mimic the negatively charged membrane of Gram‐positive bacteria. We attribute the high percentage of insertion of the biologically inactive analogue in DMPG to the fact that its concentration on the membrane surface in our experiments is likely to be much higher than that found in physiological conditions.

Use of a single glycine residue to determine the tilt and orientation of a transmembrane helix. A new structural label for infrared spectroscopy.

Site-directed dichroism is an emerging technique for the determination of membrane protein structure. However, due to a number of factors, among which is the high natural abundance of (13)C, the use of this technique has been restricted to the study of small peptides. We have overcome these problems through the use of a double C-deuterated glycine as a label. The modification of a single residue (Gly) in the transmembrane segment of M2, a protein from the Influenza A virus that forms H(+)-selective ion channels, has allowed us to determine its helix tilt and rotational orientation. Double C-deuteration shifts the antisymmetric and symmetric stretching vibrations of the CD(2) group in glycine to a transparent region of the infrared spectrum where the dichroic ratio of these bands can be measured. The two dichroisms, along with the helix amide I dichroic ratio, have been used to determine the helix tilt and rotational orientation of M2. The results are entirely consistent with previous site-directed dichroism and solid-state NMR experiments, validating C-deuterated glycine (GlyCD(2)) as a structural probe that can now be used in the study of polytopic membrane proteins.

Contribution of energy values to the analysis of global searching molecular dynamics simulations of transmembrane helical bundles.

Molecular interactions between transmembrane alpha-helices can be explored using global searching molecular dynamics simulations (GSMDS), a method that produces a group of probable low energy structures. We have shown previously that the correct model in various homooligomers is always located at the bottom of one of various possible energy basins. Unfortunately, the correct model is not necessarily the one with the lowest energy according to the computational protocol, which has resulted in overlooking of this parameter in favor of experimental data. In an attempt to use energetic considerations in the aforementioned analysis, we used global searching molecular dynamics simulations on three homooligomers of different sizes, the structures of which are known. As expected, our results show that even when the conformational space searched includes the correct structure, taking together simulations using both left and right handedness, the correct model does not necessarily have the lowest energy. However, for the models derived from the simulation that uses the correct handedness, the lowest energy model is always at, or very close to, the correct orientation. We hypothesize that this should also be true when simulations are performed using homologous sequences, and consequently lowest energy models with the right handedness should produce a cluster around a certain orientation. In contrast, using the wrong handedness the lowest energy structures for each sequence should appear at many different orientations. The rationale behind this is that, although more than one energy basin may exist, basins that do not contain the correct model will shift or disappear because they will be destabilized by at least one conservative (i.e. silent) mutation, whereas the basin containing the correct model will remain. This not only allows one to point to the possible handedness of the bundle, but can be used to overcome ambiguities arising from the use of homologous sequences in the analysis of global searching molecular dynamics simulations. In addition, because clustering of lowest energy models arising from homologous sequences only happens when the estimation of the helix tilt is correct, it may provide a validation for the helix tilt estimate.

Mapping the energy surface of transmembrane helix-helix interactions.

Transmembrane helices are no longer believed to be just hydrophobic segments that exist solely to anchor proteins to a lipid bilayer, but rather they appear to have the capacity to specify function and structure. Specific interactions take place between hydrophobic segments within the lipid bilayer whereby subtle mutations that normally would be considered innocuous can result in dramatic structural differences. That such specificity takes place within the lipid bilayer implies that it may be possible to identify the most favorable interaction surface of transmembrane alpha-helices based on computational methods alone, as shown in this study. Herein, an attempt is made to map the energy surface of several transmembrane helix-helix interactions for several homo-oligomerizing proteins, where experimental data regarding their structure exist (glycophorin A, phospholamban, Influenza virus A M2, Influenza virus C CM2, and HIV vpu). It is shown that due to symmetry constraints in homo-oligomers the computational problem can be simplified. The results obtained are mostly consistent with known structural data and may additionally provide a view of possible alternate and intermediate configurations.

C-Deuterated Alanine: A New Label to Study Membrane Protein Structure Using Site-Specific Infrared Dichroism

The helix tilt and rotational orientation of the transmembrane segment of M2, a 97-residue protein from the Influenza A virus that forms H(+)-selective ion channels, have been determined by attenuated total reflection site-specific infrared dichroism using a novel labeling approach. Triple C-deuteration of the methyl group of alanine in the transmembrane domain of M2 was used, as such modification shifts the asymmetric and symmetric stretching vibrations of the methyl group to a transparent region of the infrared spectrum. Structural information can then be obtained from the dichroic ratios corresponding to these two vibrations. Two consecutive alanine residues were labeled to enhance signal intensity. The results obtained herein are entirely consistent with previous site-specific infrared dichroism and solid-state nuclear magnetic resonance experiments, validating C-deuterated alanine as an infrared structural probe that can be used in membrane proteins. This new label adds to the previously reported (13)C [double bond] (18)O and C-deuterated glycine as a tool to analyze the structure of simple transmembrane segments and will also increase the feasibility of the study of polytopic membrane proteins with site-specific infrared dichroism.