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D M Engelman

Publications and source records attributed to D M Engelman.

At least 55 records · Page 3Linked to original sources
PubMed1996

A zinc-binding domain involved in the dimerization of RAG1.

Recombination-activating gene 1 (RAG1), as well as RAG2, are the only lymphoid-specific genes required for V(D)J recombination. RAG1 protein contains a C3HC4 zinc-binding motif (zinc ring finger) that binds two zinc ions. We have found that RAG1 contains additional zinc-binding motifs in the form of two separate C2H2 zinc finger sequences. One of the zinc fingers, in combination with the C3HC4 subdomain, forms a highly specific dimerization domain. A combination of biophysical techniques has been used to determine the energetics of association, the overall shape of the dimerization domain, and the relative orientation of the monomeric subunits within the dimer. These results provide direct evidence that a C3HC4 motif is involved in a protein-protein interaction, in this case via homodimer formation. In addition, the observation that the dimerization domain includes multi-class zinc binding motifs, namely both a zinc finger and a C3HC4 subdomain, has important implications for other C3HC4-containing proteins. The position of this dimerization domain in the N-terminal third of the RAG1 sequence of 1040 amino acid residues may have a significant influence on the activities associated with the C-terminal domains of the protein.

Amino Acid Sequence↗
PubMed1996

Coassembly of synthetic segments of shaker K+ channel within phospholipid membranes.

Increasing evidence suggests that membrane-embedded hydrophobic segments can interact within the phospholipid milieu of the membrane with varying degrees of specificity and thus contribute to the folding and oligomerization of proteins. We have used synthetic peptides corresponding to segments from the hydrophobic core of the Shaker potassium channel as a model system to study interactions between membrane-embedded segments. Three synthetic segments of the Shaker K+ channel, comprising the hydrophobic S2, S3, and S4 sequences, were used, and their secondary structure, their interactions with, and orientation within phospholipid membranes were examined. Secondary structure studies revealed that though S3 and S4 both adopt certain fractions of alpha-helical structures in membrane mimetic environments, the alpha-helical content of S3 is lower. Both S3 and S4 bind strongly to zwitterionic phospholipids, with partition coefficients in the order of 10(4) and 10(5) M-1. ATR-FTIR studies showed that while the S4 peptide is oriented parallel to the membrane surface, S3 tends to a more transmembranal orientation. Enzymatic cleavage experiments demonstrated that the presence of S3 induces some change in the proteolytic accessibility of the S4 segment. Resonance energy transfer measurements, done in high lipid/peptide molar ratios, revealed that S3 and S4 cannot self-associate in zwitterionic phospholipid vesicles but can associate with each other and with the S2 segment of the channel. Furthermore, S3 does not interact with the homologous S4 region from the first repeat of the eel sodium channel, demonstrating specificity in the interactions. These results are in line with data indicating that functionally important interactions indeed exist between the negatively charged S2 and S3 regions and the positively charged S4 region [Papazian, D. M., et al (1995) Neuron 14, 1293-1301; Planells-Cases, R., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 92, 9422-9426]. From a broader point of view, these results provide further support to the notion that interactions (either specific or nonspecific) may exist between transmembrane segments of integral membrane proteins and therefore can contribute to their assembly and organization.

Amino Acid Sequence↗
PubMed1996

Improved prediction for the structure of the dimeric transmembrane domain of glycophorin A obtained through global searching.

A more global search method, using fewer assumptions, has been used to predict the structure of the dimeric transmembrane region of the protein glycophorin A. The resulting model significantly differs from that previously determined. In particular, the arrangement between the two transmembrane helices is now more symmetric resulting in improved interaction energies and an increased buried surface area. An increase in the van der Waals interaction energy due to tighter packing compensates for the loss of the interhelical hydrogen bond observed between Thr-87 of each helix in the previous model.

Computer Simulation↗
PubMed1996

Surface point mutations that significantly alter the structure and stability of a protein's denatured state.

Significantly different m values (1.9-2.7 kcal mol-1 M-1) were observed for point mutations at a single, solvent-exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m-value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m-) m-value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 S-1 for the m+ mutant and 952 S-1 for the m- mutant) and the rates of unfolding (11 S-1 for m+ mutant and 3 S-1 for the m- mutant) were significantly different, as determined by stopped-flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (alpha ++ = 0.3). Small-angle X-ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd approximately 23 A); whereas, the Rgd of the m- mutant increased from approximately 17 A to 23 A with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl-induced denatured state of these two proteins.

Bacterial Proteins↗
PubMed1996

Leucine side-chain rotamers in a glycophorin A transmembrane peptide as revealed by three-bond carbon-carbon couplings and 13C chemical shifts.

We have used a spin-echo difference NMR pulse sequence to measure three-bond J couplings between lambda- and alpha-carbons of the leucine residues in a micelle-associated helical peptide dimer that corresponds to residues 62-101 of the transmembrane erythrocyte protein glycophorin A. The observed 3J couplings correlate strongly with the 13C chemical shift of the lambda-methyl groups, and within experimental error both the shift distribution of the methyl carbons and the variations in 3J can be accounted for by variations in side-chain rotamer populations. We infer that all leucine side chains in this peptide dimer are in fast exchange among chi 2 rotamers and sample two of the three possible rotameric states, even when the side chain forms part of the dimer interface. The observed correlation of chemical shift with couplings can be traced to a gamma-gauche interaction of methyl and alpha-carbons. This correlation may provide an alternate route to rotamer analysis in some protein systems.

Carbon Isotopes↗
PubMed1996

Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban.

Phospholamban is a 52-amino acid residue membrane protein that regulates Ca(2+)-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca(2+)-selective ion channel. We have used polarized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLB. The frequency and dichroism of the amide I and II bands appearing at 1658 cm-1 and 1544 cm-1, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidycholine bilayer membranes is predominantly alpha-helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating 13C at two positions in the protein backbone. A small band seen near 1614 cm-1 is assigned to the amide I mode of the 13C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are alpha-helical, with an axial orientation that is approximately 30 degrees relative to the membrane normal. Upon exposure to 2H2O (D2O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope-labeled hPLB films, our results strongly support models of PLB that predict the existence of an alpha-helical hydrophobic region spanning the membrane domain.

Amino Acid Sequence↗
PubMed1996

Mapping the lipid-exposed surfaces of membrane proteins.

Phospholamban forms a stable complex of five long transmembrane helices. We show that the relative rotational orientation of the helices in the pentameric complex can be distinguished by S-H to S-D exchange of cysteine sulphydryl groups located in the transmembrane segment of the protein and exposed to the lipid environment. Of the three cysteine residues in phospholamban, two residues (Cys 36 and Cys 46) are oriented towards the helix interface and protected from exchange, while the third cysteine (Cys 41) is oriented towards the lipid interface and undergoes exchange with water diffused into the bilayer. Distinguishing the external and internal faces of a membrane protein by sulphydryl exchange provides a general approach for determining the three-dimensional fold of membrane proteins and enhances model building efforts to generate high-resolution structures.

Adenosine Triphosphatases↗
PubMed1995

Structural model of the phospholamban ion channel complex in phospholipid membranes.

Phospholamban is a 52 amino acid residue membrane protein involved with the regulation of calcium levels across sarcoplasmic reticulum membranes in cardiac muscle cells. The N-terminal 30 amino acid residues of the protein are largely hydrophilic and include two sites whose phosphorylation is thought to dissociate an inhibitory complex between phospholamban and Ca2+ ATPase. The C-terminal 22 amino acid residues are largely hydrophobic, anchor the protein in the membrane and are responsible for Ca2+ selective ion conductance. Specific interactions between the transmembrane domains stabilize a pentameric protein complex. We have obtained circular dichroism (CD), transmission Fourier transform infrared (FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra of the full-length protein and have compared these results to those from a 28 residue peptide that includes the transmembrane domain. Both proteins reconstituted into phospholipid membranes are largely alpha-helical by CD and FTIR. Polarized ATR-FTIR measurements show that both the cytosolic and transmembrane helices are oriented perpendicular to the membrane plane with a tilt of 28 (+/- 6) degrees with respect to the membrane normal. This tilt angle is in close agreement to that calculated from a model for the transmembrane domain of phospholamban suggested by mutagenesis and molecular modeling. Phosphorylation does not significantly change the secondary structure or orientation of the protein. The pentameric complex is modeled as a left-handed coiled-coil of five long helices (40 (+/- 3) residues) that extend across the membrane from the lumenal carboxy terminus to the phosphorylation site in the cytoplasm. The helix bundle forms a perpendicular ion pore that may begin at a distance (17 to 29 A) from the membrane surface. Based on the above, we propose a mechanism by which phospholamban regulates Ca2+ levels across membranes that takes into account both its selective ion conductance and inhibitory association with the Ca2+ pump.

Amino Acid Sequence↗
PubMed1995

Small angle x-ray scattering studies of magnetically oriented lipid bilayers.

Magnetically oriented lipid/detergent bilayers are potentially useful for studies of membrane-associated molecules and complexes using x-ray scattering and nuclear magnetic resonance (NMR). To establish whether the system is a reasonable model of a phospholipid bilayer, we have studied the system using x-ray solution scattering to determine the bilayer thickness, interparticle spacing, and orientational parameters for magnetically oriented lipid bilayers. The magnetically orientable samples contain the phospholipid L-alpha-dilauroylphosphatidylcholine (DLPC) and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) in a 3:1 molar ratio in 70% water (w/v) and are similar to magnetically orientable samples used as NMR media for structural studies of membrane-associated molecules. A bilayer thickness of 30 A was determined for the DLPC/CHAPSO particles, which is the same as the bilayer thickness of pure DLPC vesicles, suggesting that the CHAPSO is not greatly perturbing the lipid bilayer. These data, as well as NMR data on molecules incorporated in the oriented lipid particles, are consistent with the sample consisting of reasonably homogeneous and well dispersed lipid particles. Finally, the orientational energy of the sample suggests that the size of the cooperatively orienting unit in the samples is 2 x 10(7) phospholipid molecules.

Biophysical Phenomena↗
PubMed1995

Computational searching and mutagenesis suggest a structure for the pentameric transmembrane domain of phospholamban.

Structural and environmental constraints greatly simplify the folding problem for membrane proteins. Computational methods can be used in a global search to find a small number of chemically reasonable models within these constraints, such that a modest set of experimental data can distinguish among them. We show that, for phospholamban, the global search can be further simplified by reducing the problem to two-body, rather than many-body, interactions. This method of a constrained global search combined with experimental mutagenesis data yields a three-dimensional structure for this pentameric ion channel. The model is a left-handed symmetric homopentamer of alpha-helices with a well-defined channel, lined solely by hydrophobic residues.

Amino Acid Sequence↗
PubMed1994

Structural organization of the pentameric transmembrane alpha-helices of phospholamban, a cardiac ion channel.

Phospholamban is a 52 amino acid calcium regulatory protein found as pentamers in cardiac SR membranes. The pentamers form through interactions between its transmembrane domains, and are stable in SDS. We have employed a saturation mutagenesis approach to study the detailed interactions between the transmembrane segments, using a chimeric protein construct in which staphylococcal nuclease (a monomeric soluble protein) is fused to the N-terminus of phospholamban. The chimera forms pentamers observable in SDS-PAGE, allowing the effects of mutations upon the oligomeric association to be determined by electrophoresis. The disruptive effects of amino acid substitutions in the transmembrane domain were classified as sensitive, moderately sensitive or insensitive. Residues of the same class lined up on faces of a 3.5 amino acids/turn helical projection, allowing the construction of a model of the interacting surfaces in which the helices are associated in a left-handed pentameric coiled-coil configuration. Molecular modeling simulations (to be described elsewhere in detail) confirm that the helices readily form a left-handed coiled-coil helical bundle and have yielded molecular models for the interacting surfaces, the best of which is identical to that predicted by the mutagenesis. Residues lining the pore show considerable structural sensitivity to mutation, indicating that care must be taken in interpreting the results of mutagenesis studies of channels. The cylindrical ion pore (minimal diameter of 2 A) appears to be defined largely by hydrophobic residues (I40, L43 and I47) with only two mildly polar elements contributed by sulfurs in residues C36 and M50.

Amino Acid Sequence↗
PubMed1994

Specificity and promiscuity in membrane helix interactions.

Transmembrane alpha-helices can associate with one another in lipid bilayers. This association is important in the folding and oligomerization of many integral membrane proteins, and may also play a role in their function. The interactions between helices may be highly specific or relatively non-specific, and their roles may differ accordingly. These two cases are discussed.

Amino Acid Sequence↗
PubMed1994

Glycophorin A helical transmembrane domains dimerize in phospholipid bilayers: a resonance energy transfer study.

Glycophorin A and its isolated transmembrane region (GpATM) are each known to form sequence-specific dimers in SDS micelles. Whether this behavior accurately reflects behavior in red cell membranes or lipid bilayers, however, has remained unclear. Resonance energy transfer between labeled GpATM peptides has been used to observe dimerization of GpATM in bilayers. Separate populations of GpATM peptides were labeled with 2,6-dansyl chloride as the donor chromophore and dabsyl chloride as the acceptor. Quenching of the 2,6-dansyl chloride by the dabsyl group demonstrated an association of the labeled peptides. The quenching was not affected by increases in the amount of lipid present or by unlabeled heterologous peptides but was decreased by the addition of unlabeled GpATM. GpATM was determined to form dimers by fitting the observed energy transfer for a number of donor to acceptor ratios and fitting to the expected number of donor labeled peptides in an oligomer with an acceptor as a function of oligomer number. The finding that the GpATM peptide forms helical dimers in lipid bilayers supports the idea that GpA is a dimer in the erythrocyte membrane. The resonance energy transfer approach may extend to the study of other oligomeric complexes.

Amino Acid Sequence↗
PubMed1994

Rotational orientation of transmembrane alpha-helices in bacteriorhodopsin. A neutron diffraction study.

The rotational orientation of the seven transmembrane alpha-helices (A-G) in bacteriorhodopsin has been investigated by neutron diffraction. The current model of bacteriorhodopsin is based on an electron density map obtained by high-resolution electron microscopy (EM). Assigning helix rotational positions in the EM model depended on fitting large side-chains, mainly aromatic residues, into bulges in the electron density map. For helix D, which contains no aromatic residues, the EM map is more difficult to interpret. For helices A and B, whose position and orientation had been determined previously by neutron diffraction, the positions defined by EM agree within experimental error with these earlier conclusions. The orientation of all seven helices has been examined by using neutron diffraction on bacteriorhodopsin samples with specifically deuterated valine, leucine and tryptophan residues. Experimental peak intensities were compared to those predicted for an extensive set of structural models. The models were generated by (1) rotating all helices around their axis; (2) moving deuterated residues in the extramembrane loops about their probable positions and changing the weight of their contribution to the neutron diffraction pattern; (3) allowing deuterated side-chains to change their conformation. The analysis confirmed exactly the positions previously determined for helices A and B. For an optimal fit to the data to be obtained, the other five helices, including helix D, must lie either at or within 20 degrees of their position in the current EM model. The complementarity of medium-resolution EM, neutron diffraction and model building for the structural study of integral membrane proteins is discussed.

Bacteriorhodopsins↗
PubMed1994

A dimerization motif for transmembrane alpha-helices.

Specific helix-helix interactions inside lipid bilayers guide the folding and assembly of many integral membrane proteins and their complexes. We report here a pattern of 7 amino acids (LIxxGVxxGVxxT) which when introduced into several hydrophobic transmembrane alpha-helices promotes their specific dimerization. Dimerization is driven by interactions that are specific, dominated by the helix-helix interface, and involve no potentially ionizable groups. The motif may provide a useful tool for the functional analysis of such interactions in a variety of systems. Further, since this particular motif is rare, whilst specific helix association is not, many other such motifs may exist, which could permit sorting within complex membranes as well as guiding folding and oligomerization.

Amino Acid Sequence↗
PubMed1993

Mutations can cause large changes in the conformation of a denatured protein.

Deletion of 13 amino acids from the carboxyl terminus of staphylococcal nuclease (WTSNase delta) results in a denatured, partially unfolded molecule that lacks significant persistent secondary structure but is relatively compact and monomeric under physiological conditions [Shortle & Meeker (1989) Biochemistry 28, 936-944; Flanagan et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 748-752]. Because of these and other properties of the SNase delta polypeptide, it is a useful model system for investigating the conformation of the denatured state of a protein without using extreme temperature or solvent conditions. Moreover, since the modification is a carboxyl-terminal deletion, SNase delta may also resemble a transient state of the polypeptide chain as it emerges from a ribosome prior to its folding. In the present study, we have examined the sizes and conformations of mutated forms of SNase delta, using small-angle X-ray scattering and circular dichroism spectroscopy. Seven mutated forms were studied: four with single substitutions, two with double substitutions, and one triple substitution. When present in the full-length SNase, each of these mutated forms exhibited unusual behavior upon solvent or thermal denaturation. In the case of the truncated form (SNase delta), the small-angle scattering curves of the mutated forms fall into two classes: one resembling the scattering curve of compact native nuclease and the other having features consistent with those expected for an expanded coil-like polymer. In contrast, the scattering curve of WT SNase delta exhibits features intermediate between those observed for globular proteins and random polymers. The amino acid substitutions that gave rise to compact, native-like versions of SNase delta were all of the m--type (m-substitutions are predicted to decrease the size of the denatured state). Those which gave rise to versions of SNase delta that were more extended and coil-like than WT SNase delta were of the m+ type (m+ substitutions are predicted to increase the size of the denatured state). Estimates of the residual secondary structure present in WT SNase delta, as well as both the m+ and m-substituted versions of SNase delta, as determined by CD, suggest that the formation of secondary structure and compaction of the polypeptide chain occur concurrently. Our results show that single amino acid substitutions can radically alter the conformational distribution of a partially condensed polypeptide chain.(ABSTRACT TRUNCATED AT 400 WORDS)

Algorithms↗