Biomedical subjects

C M Deber

Publications and source records attributed to C M Deber.

At least 19 recordsLinked to original sources

PubMed1995

Accessibility and dynamics of Cys residues in Bacteriophage IKe and M13 major coat protein mutants.

The filamentous bacteriophage major coat protein occurs as a membrane-spanning assembly intermediate prior to incorporation into the lipid-free virion. To gain insight into how this small, multifunctional protein is able to be stably incorporated into both of these distinct environments, the reactive sulfhydryl group of IKe and M13 coat protein Cys mutants was exploited to probe the mobility and environment of this residue at several loci within the hydrophobic domain of these proteins. IKe mutants P30C, G39C, and G39C-V36A and M13 mutant Y24C-V31A, each previously obtained from randomized mutagenesis, were characterized in the intact virion, the intermediate spheroidal S-form, and in membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The accessibility of the Cys sulfhydryl in the virion was examined by reaction with [14C]iodoacetamide (14C-IAN) and other alkylating agents. The IKe mutants G39C and G39C-V36A were found to be the most reactive with 14C-IAN and thus the most accessible, although this accessibility was subject to strict steric constraints since only the smallest sulfhydryl-specific alkylating agents were able to modify the Cys39 locus. The spin probe proxyliodoacetamide (PIAN) was used to characterize Cys side chain mobility by electron paramagnetic resonance (EPR) spectroscopy. The M13 mutant Y24C-V31A Cys side chain in the phage was observed to be the most mobile, with slightly less mobility for IKe mutant P30C and considerably less for G39C mutants. The SDS micelle-bound forms of the Cys mutants all exhibited enhanced side chain mobility compared to the virion form, with the extent of mobility dependent upon the specific location of the Cys residue. EPR and fluorescence quenching results show that the Cys side chain in the Y24C-V31A S-form is largely immobilized and inaccessible in comparison to the virion and micelle-solubilized forms. The overall results are interpreted in terms of the structural changes accompanying disassembly and insertion of the coat protein into the Escherichia coli inner membrane.

Alkylation↗

PubMed1995

Packing of coat protein amphipathic and transmembrane helices in filamentous bacteriophage M13: role of small residues in protein oligomerization.

Filamentous bacteriophage M13, an important cloning and phage display vector, is encapsulated by ca 2700 copies of its 50-residue major coat protein (gene 8). This protein occurs as a membrane protein while stably inserted into its E. coli host inner membrane, and as a coat protein upon assembly and packing onto phage DNA in the lipid-free virion. To examine the specific protein-protein interactions underlying these processes, we used a combination of randomized and saturation mutagenesis of the entire gene 8 to assess the susceptibility of each position to mutation. In the resulting library of ca 100 viable M13 mutants, "small" residues (Ala,Gly,Ser), which constitute the non-polar face of the N-terminal amphipathic helical segment, and a face of the hydrophobic (effective transmembrane) helical segment, were found to be highly conserved. These results support a model in which coat protein packing is stabilized by the presence within each protein subunit of two "oligomerization segments", i.e. specific helical regions with faces rich in small residues which function to promote the close approach of alpha-helices.

Amino Acid Sequence↗

PubMed1995

An engineered disulfide bridge in the transmembrane region of phage M13 coat protein stabilizes the alpha-helical dimer.

A single Cys-residue (Cys24) was introduced into the 50-amino acid major coat protein of M13 bacteriophage as part of a two-site substitution (Y24C-V31A) within the effective transmembrane (TM) segment (Tyr21 to Ile39) of the coat protein. Mutant Y24C-V31A was able to complete the phage life cycle and was shown to contain free sulfhydryls in the intact virus, as evidenced by susceptibility of Y24C-V31A phage to alkylation by Cys-specific 14C-iodoacetamide (14C-IAN). In contrast, the protein solubilized in deoxycholate micelles was resistant to 14C-IAN modification and was virtually inert to a transition from a characteristic alpha-helical oligomeric state to an aggregated beta-sheet structure relative to WT and V31A coat proteins, as shown by circular dichroism spectroscopy and SDS-PAGE. Reduction of mainly dimeric Y24C-V31A protein using beta-mercaptoethanol (beta-ME) generated monomeric species and resulted in a loss of helical thermostability. The overall results indicated that solubilization of Y24C-V31A coat protein into micelles resulted in formation of thermostable disulfide-bridged helical dimers. The disulfide bridge is deduced to be positioned along the stripe of residues involved in hydrophobic packing of TM parallel helical dimers.

Amino Acid Sequence↗

PubMed1995

Manipulation of peptide conformations by fine-tuning of the environment and/or the primary sequence.

The widely observed phenomenon that peptides are capable of adopting multiple conformations in different environments suggests that secondary structure formation in a peptide segment is a process involving not only the peptide itself but also the surrounding solvent media. The influence of the primary sequence and the molecular environment on peptide conformations are now investigated using synthetic peptides of amino acid sequence H2N-(Ser-Lys)2-Ala-X-Gly-Ala-X-Gly-Trp-Ala-X-Gly-(Lys-Ser)3-OH, where X = Ile or Val. These two peptides, namely 3I (X = Ile) and 3V (X = Val), are found to lack defined secondary structure in aqueous buffer. However, discrete conformational states, e.g., alpha-helices and beta-sheets, are readily generated and interconverted for both peptides when the buffer is modulated with the addition of methanol, sodium dodecyl sulfate (SDS) micelles, or phospholipid vesicles. The role of the primary sequence in affecting peptide conformations is manifested in that peptides 3I and 3V, which differ respectively in their content of beta-branched Ile or Val residues, differ in their secondary structures at monomeric concentrations in 2 mM SDS and in mixed lipid vesicles of phosphatidic acid and phosphatidylcholine. The overall results suggest that peptide segments can be conformationally flexible entities poised to react to minor modulation in either the molecular environment or the primary sequence, a circumstance that may be relevant to protein functioning and folding.

Amino Acid Sequence↗

PubMed1995

Peptides in membranes: helicity and hydrophobicity.

Synthetic model membrane-interactive peptides--both of natural and designed sequence--have become convenient and systematic tools for determination of how the membrane-spanning segments within integral membrane proteins confer protein structure and biology. Conformational studies on these peptides demonstrate that the alpha-helix is the natural choice of conformation for a peptide segment in a membrane, and that a helical conformation will arise "automatically" in a peptide above a threshold hydrophobicity that allows it to associate stably with the membrane. Environmental and sequential contexts thus impart conformational versatility to many of the amino acids, thereby providing a mechanism for producing the diverse structural and functional properties of proteins.

Amino Acid Sequence↗

PubMed1994

An irregularity in the transmembrane domain helix correlates with the rate of insulin receptor internalization.

Internalization of insulin and its receptor via receptor-mediated endocytosis is an important step in insulin-induced signal transduction. To investigate the structural determinants underlying the enhanced internalization rate observed for the insulin receptor transmembrane mutant Gly933-Pro934-->Ala-Ala (GP-->AA), we have designed and chemically synthesized two peptides, IR(TM)-GP and IR-(TM)-AA, corresponding respectively to the N-terminal portion of the wild-type and the mutant insulin receptor TM segment containing these sites. Conformational studies by circular dichroism (CD) spectroscopy on these two peptides in their monomeric states revealed that peptide IR(TM)-GP forms an irregular helix in the membrane-mimetic environments of sodium dodecyl sulfate (SDS) micelles with a possible "kink" in the helix imposed by its Gly-Pro sequence, while peptide IR(TM)-AA assumes largely classical alpha-helical structure under corresponding conditions. The helical pattern of peptide IR(TM)-AA was maintained at elevated temperatures, while the shape of the CD curve for peptide IR(TM)-GP was found to alter as a function of temperature. At higher concentrations, both peptides formed high molecular weight aggregates in SDS micelles, as demonstrated by SDS-PAGE gels, but peptide IR(TM)-AA was shown to aggregate more readily and more extensively than peptide IR(TM)-GP. Fluorescent dye-leakage experiments indicated that peptide IR(TM)-GP produces an enhanced disruption of the membrane bilayer in phosphatidylglycerol vesicles vs that induced by IR(TM)-AA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Transmembrane aromatic amino acid distribution in P-glycoprotein. A functional role in broad substrate specificity.

Multidrug resistance (MDR) in cancer cells is associated with overexpression of P-glycoprotein (Pgp), a membrane protein which interacts with structurally diverse hydrophobic molecules of high membrane affinity. In an analysis of the molecular basis for this broad range of substrate specificity, we found that the transmembrane (TM) regions of Pgp are rich in highly conserved aromatic amino acid residues. Computer-generated three-dimensional model structures showed that a typical substrate, rhodamine 123, can intercalate between three to four phenylalanine side-chains in any of several Pgp TM helices with minimal protrusion of the drug into bulk lipid, and that five to six (of the 12 Pgp putative TM segments) helices can facilitate transport through creation of a sterically compatible pore. In contrast to the case for proteins involved in the transport of membrane-impermeable, relatively polar substrates, the "transport path" for Pgp substrates need not be polar, and may involve either an internal channel occupied largely by aromatic side-chains, or external gaps along TM helix-lipid interfaces. Weakly polar interactions between drug cationic sites and Pgp aromatic residues contribute additionally to overall protein/drug binding. The ability of Pgp to recognize and efflux structurally diverse molecules suggests that rather than a unique structure, the Pgp channel may maintain the intrinsic capacity to undergo wide-ranging drug-dependent dynamic reorganization.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

A measure of helical propensity for amino acids in membrane environments.

The frequent occurrence of beta-sheet promoting residues such as Ile, Val, and Thr in the alpha-helical transmembrane segments of most integral membrane proteins suggests that the helical propensities of these residues are altered in the hydrophobic environment of the lipid bilayer. Systematic studies of model peptides by circular dichroism models spectroscopy in various micellar/vesicular media allow the establishment of a ranking order of helical propensity for uncharged amino acids in the membrane environment. In contrast to their conformational preferences in water, the helical proclivity of amino acids in membranes is shown to be governed by their side chain hydrophobicity, and by the hydropathy of the local peptide segments in which the residues reside [corrected].

Amino Acid Sequence↗

PubMed1994

A measure of helical propensity for amino acids in membrane environments.

The frequent occurrence of beta-sheet promoting residues such as Ile, Val, and Thr in the alpha-helical transmembrane segments of most integral membrane proteins suggests that the helical propensities of these residues are altered in the hydrophobic environment of the lipid bilayer. Systematic studies of model peptides by circular dichroism spectroscopy in various micellar/vesicular media allow the establishment of a ranking order of helical propensity for uncharged amino acids in the membrane environment. In contrast to their conformational preferences in water, the helical proclivity of amino acids in membranes is shown to be governed by their side chain hydrophobicity, and by the hydropathy of the local peptide segments in which the residues reside.

Amino Acid Sequence↗

PubMed1993

Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.

Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins.

Alanine↗

PubMed1993

Peptide environment specifies conformation. Helicity of hydrophobic segments compared in aqueous, organic, and membrane environments.

Transmembrane segments in integral membrane proteins exist characteristically as helices in lipid bilayers, yet are often rich in residues considered helix-destabilizing (Val, Ile, Gly) in soluble proteins. We propose that helicity of a transmembrane segment is likely to be affected by factors other than the "intrinsic" helical propensities of its component amino acids. This hypothesis is tested by comparing the conformation(s) in aqueous, organic, membrane-mimetic (micellar), and membrane (bilayer) environments of designed model peptides with systematically altered helical propensity and/or segmental hydrophobicity. Peptides of prototypic sequence NH2-(Ser-Lys)2-Ala5-Leu6-Ala7-Ala8-Leu9-Ala10-++ +Trp11-Ala12-Leu13-Ala14- (Lys-Ser)3-OH were synthesized, which incorporate a hydrophobic core "guest" segment (residues 5-14) into a water-soluble hydrophilic host matrix. Related peptides featured substitution of Leu6,9,13-->Gly, Leu6,9,13-->Ala, and Ala7,10,14-->Gly. Circular dichroism spectra revealed that algorithms for soluble proteins correctly predicted peptide helical proclivities in aqueous solutions, but peptide helicity in organic (trifluoroethanol) solvents, membrane-mimetic SDS micelles, and negatively charged lipid bilayer vesicles, was found to be governed almost exclusively by the segmental hydrophobicity of the peptide mid-hydrophobic core segment. In related Trp fluorescence studies, peptide-membrane association was similarly correlated with extent of hydrophobic interaction.

Amino Acid Sequence↗

PubMed1993

Mutagenesis of bacteriophage IKe major coat protein transmembrane domain: role of an interfacial proline residue.

The transmembrane (TM) domain of the 53-residue major coat protein of the M13-related bacteriophage IKe (residues 24-42: LISQTWPVVTTVVVAGVLI) has been subjected to randomized mutagenesis to probe the conformation and stability of the TM domain, as well as the effect of structurally-important residues such as proline. TM mutants were obtained by the Eckstein method of site-directed mutagenesis using the IKe genome as template so as to eliminate the need for subcloning. Over 40 single- and double-site viable mutants of bacteriophage IKe were isolated. Every residue in the TM segment, except the highly conserved Trp29, could be mutated to at least one other residue; polar and charged mutations occurred in the TM segment adjacent to the N-terminal domain (residues 24-28), while non-polar substitutions predominated in the C-terminal portion (residues 30-42). The Pro30 locus tolerated four mutations-Ala, Gly, Cys, and Ser- which represent the four side chains of least volume. Mutant coat proteins obtained directly from the phage in milligram quantities were studied by circular dichroism spectroscopy and SDS-PAGE gels. Wild type IKe coat protein solubilized in sodium deoxycholate micelles was found to occur as an alpha-helical, monomeric species which is stable at 95 degrees C, whereas the mutant Pro30-->Gly undergoes an irreversible conformational transition at ca. 90 degrees C to an aggregated beta-sheet structure. The result that Pro30 stabilizes the TM helix in the micellar membrane suggests a sterically-restricted location for the wild type Pro pyrrolidine side chain in the bulky Trp-Pro-Val triad, where it may be positioned to direct the initiation of the subsequent TM core domain helix.

Amino Acid Sequence↗

PubMed1993

Conformational states of mutant M13 coat proteins are regulated by transmembrane residues.

Mutational and structural analysis of the 28 viable bacteriophage M13 mutants obtained by randomized mutagenesis of the effective transmembrane (TM) segment of the 50-residue major coat (gene VIII) protein (residues 21-39) demonstrated that M13 coat protein functionality, as reflected by phage viability, is incompatible with an increase in Gly + beta-branched residue content in its TM core. SDS-polyacrylamide gel electrophoresis and circular dichroism spectroscopy performed in membrane environments on purified mutant coat proteins revealed that these proteins exist in a range of state(s), identified as helical monomers and dimers and polymeric (alpha-helical and/or beta-sheet) species, of which relative populations, and thermally induced conformational transitions, were dependent uniquely upon mutation type and locus. Mutations to relatively polar residues (e.g. G23D, Y24D, Y24H, A27E, I32T, and T36S) stabilized principally monomeric species, while mutants with decreased beta-branched content in the protein TM hydrophobic core (e.g. V29A, V30A, V31A, V31L, and V33A) displayed mainly dimeric species. Mutation of Ile37-->Thr within a "Sternberg-Gullick" consensus sequence of the coat protein TM segment led to a highly oligomerized/polymerized protein. The overall results suggest that TM residues in M13 coat protein are not universal components of a hydrophobic anchor segment per se, but are further selected (i) to impart conformational flexibility to the TM segment through helix destabilization and (ii) to retain the capacity to regulate protein-protein association and packing motifs within membranes.

Amino Acid Sequence↗

PubMed1993

Non-random distribution of amino acids in the transmembrane segments of human type I single span membrane proteins.

The distribution of amino acids in the transmembrane segments and flanking regions of 115 human type I single span (amino terminus extracellular and carboxyl terminus cytosolic) plasma membrane proteins was found to be non-random. In this sample, Ile was preferentially localized to the amino-terminal region of the hydrophobic transmembrane segments, followed by Val, while Leu predominated in the carboxyl-terminal half of the segment. Although Gly residues were preferentially located in the transmembrane segment, this residue was excluded from the carboxyl-terminal and adjacent boundary regions. Aromatic residues (Tyr, Trp and Phe) occurred preferentially at the cytoplasmic boundary, with Trp also favored at the extracellular boundary. The extracellular flanking sequence amino-terminal to the transmembrane segment was enriched in residues predicted to initiate helix formation (Pro, Asn and Ser), while Arg and Lys were enriched in the cytoplasmic flank where they may function as topological determinants. The positional preferences of these particular amino acids within the transmembrane segment and flanking regions suggests that, in addition to lipid-protein interactions, these residues may participate in specific protein-protein interactions. A consensus sequence motif for type I membrane proteins is proposed and its role in the biosynthesis, folding, assembly and function of these segments is discussed.

Amino Acid Sequence↗

PubMed1993

The structure and function of central nervous system myelin.

Multiple sclerosis (MS) is characterized by the active degradation of central nervous system myelin, a multilamellar membrane system that insulates nerve axons. MS arises from complex interactions between genetic, immunological, infective, and biochemical mechanisms. Although the circumstances of MS etiology remain hypothetical, one persistent theme involves immune system recognition of myelin-specific antigens derived from myelin basic protein, the most abundant extrinsic myelin membrane protein, and/or another equally suitable myelin protein or lipid. Knowledge of the biochemical and physical-chemical properties of myelin proteins, and lipids, particularly their composition, organization, structure, and accessibility with respect to the compacted myelin multilayers, thus becomes central to understanding how and why these antigens become selected during the development of MS. This article focuses on the current understanding of the molecular basis of MS as it may relate to the protein and lipid components of myelin, which dictate myelin morphology on the basis of protein-lipid and lipid-lipid interactions, and the relationship, if any, between the protein/lipid components and the destruction of myelin in pathological situations.

Amino Acid Sequence↗

PubMed1992

Glycine and beta-branched residues support and modulate peptide helicity in membrane environments.

Transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation once inserted into the membrane, yet consist of primary sequences rich in residues known in soluble proteins as helix-breakers (Gly) and beta-sheet promoters (Ile, Val, Thr). To examine the specific 2 degrees structure propensities of such residues in membrane environments, we have designed and synthesized a series of 20-residue peptides with 'guest' hydrophobic segments--expected to provide three turns of incipient alpha-helix content--embedded in 'host' hydrophilic (Lys-Ser) matrices. Circular dichroism (CD) spectra of the model peptides in water showed that significant helical content was observed only for peptides with high Ala content; others behaved as 'random coils'. However, in the membrane-mimetic environment of sodium dodecylsulfate (SDS) micelles, it was found that Gly can be accommodated as readily as Ala, and Ile or Val as readily as Leu, in hydrophobic alpha-helices. Further subtleties of structural preferences could be observed in electrically-neutral lyso-phosphatidylcholine (LPC) micelles, where helical propensity decreased in the order Ala-Leu-rich > Gly-Leu-rich > Gly-Ile(Val)-rich hydrophobic segments. The results conjure a role of environment-dependent helix-modulation for Gly, Ile, and Val residues--and suggest that these residues may provide, in part, the structural basis for conformational transitions within or adjacent to membrane domains, such as those accompanying membrane insertion and/or required for transport or signalling functions.

Amino Acid Sequence↗

PubMed1992

Transmembrane region of wild-type and mutant M13 coat proteins. Conformational role of beta-branched residues.

Although transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation, beta-sheet promoters (Val, Ile, Thr) often account for approximately 40% of TM residue composition. We are examining the conformational role(s) of these residues, using as a model system the major coat protein of the filamentous bacteriophage M13. This 50-residue protein, which is located at the Escherichia coli host membrane during phage reproduction, contains a prototypic 19-residue hydrophobic midregion (residues 21-39: YIGYAWAMVVVIVGATIGI). Using "Eckstein" site-directed mutagenesis, we have generated several viable M13 coat protein mutants with beta-branched amino acid substitutions within their TM region. Mutant coat proteins, including Ile32----Val (I32V) and Ala27----Thr (A27T), were obtained in milligram quantities by growing M13 mutant phages in liter preparations, confirming that these coat proteins are capable of assuming their normal biological function(s) in phage reproduction. Circular dichroism spectroscopy performed in the membrane-mimetic medium of deoxycholate micelles indicated comparable alpha-helical contents of mutants I32V and A27T to wild-type protein. 13C nuclear magnetic resonance experiments with mutant A27T demonstrated that the combination of additional beta-branched content and introduction of an -OH substituent induced chemical shift and temperature-dependent changes and influenced the local protein environment at sites up to 12 residues remote from the mutation site. In contrast, mutant I32V (of which a salient feature is a mid-TM pentavaline segment) behaved very similarly to wild-type coat. These findings are interpreted in terms of the range of TM secondary structure and stability which can be accommodated by viable M13 coat protein mutants.

Amino Acid Sequence↗

PubMed1992

Minimum energy conformations of proline-containing helices.

Proline occurs frequently in transmembrane alpha-helices of transport and receptor proteins even though statistical surveys demonstrate the overwhelming preference of this residue for a non-alpha-helical, hydrophilic environment. As a result, membrane-buried proline has been proposed to be functionally important, with function arising from structural discontinuity or destabilization of the helix. Destabilization may occur by Pro-mediated conformational transitions between discrete states, and may be manifested in membrane protein systems through reversible processes such as channel opening and closing or signal transduction. In this study, computer modeling of a model transmembrane alpha-helix, (Ala)8-Leu-Pro-Phe-(Ala)8, in a medium of low polarity (dielectric = 2), is used to examine the occurrence and energetic accessibility of Pro-mediated conformational interconversions. Leu psi and chi 1, Pro psi, and Phe phi and chi 1 torsion angles were assigned random values so that a data base of 200 conformations for each of the cis and trans states was generated. The conformations were minimized and low-energy structures organized into families. This analysis demonstrated that the most populated lowest energy family is the Trans-I conformation, corresponding to proline in a kinked alpha-helix. Two additional trans structures, Trans-II and Trans-III, as well as a cis conformation, Cis-I, are also energetically competitive. Interconversions between the trans states could thus be mediated by changes at a single torsion angle, accompanied by minor local hydrogen-bonding rearrangements. This work substantiates that membrane-buried proline can provide the basis for conformational transitions between discrete alpha-helix-based structures in a nonpolar environment.

Amino Acid Sequence↗