Circular permutation and deletion studies of myoglobin indicate that the correct position of its N-terminus is required for native stability and solubility but not for native-like heme binding and folding

We studied the effect of deleted and circularly permuted mutations in sperm whale myoglobin and present here results on three classes of mutants: (i) a deletion mutant, Mb(1)(-)(99), in which the C-terminal helices, G and H, were removed; (ii) two circular permutations, Mb-B_GHA, in which helix B is N-terminal and helix A is C-terminal, and Mb-C_GHAB, in which helix C is N-terminal and helices A and B are C-terminal; and (iii) a deleted circular permutation, Mb-HAB_F, in which helix H is N-terminal, helix F is C-terminal, and helix G is deleted. The conformational characteristics of the apo and holo forms of these mutants were determined at neutral pH, by spectroscopic and hydrodynamic methods. The apo form of the deleted and permuted mutants exhibited a stronger tendency to aggregate and had lower ellipticity than the wild type. The mutants retained the ability to bind heme, but only the circularly permuted holoproteins had native-like heme binding and folding. These results agree with the theory that myoglobin has a central core that is able to bind heme, but also indicate that the presence of N- and C-terminal helices is necessary for native-like heme pocket formation. Because the holopermuteins were less stable than the wild-type protein and aggregated, we propose that the native position of the N-terminus is important for the precise structural architecture of myoglobin.     

Role of the B helix in early folding events in apomyoglobin: evidence from site-directed mutagenesis for native-like long range interactions

The folding pathways of four mutants in which bulky hydrophobic residues in the B helix of apomyoglobin (ApoMb) are replaced by alanine (I28A, L29A, I30A, and L32A) have been analyzed using equilibrium and kinetic methods employing NMR, CD, fluorescence and mass spectrometry. Hydrogen exchange pulse-labeling followed by mass spectrometry reveals detectable intermediates in the kinetic folding pathways of each of these mutants. Comparison of the quench-flow data analyzed by NMR for the wild-type protein and the mutants showed that the substitutions I28A, L29A and L32A lead to destabilization of the B helix in the burst phase kinetic intermediate, relative to wild-type apomyoglobin. In contrast, the I30A mutation apparently has a slight stabilizing effect on the B helix in the burst phase intermediate; under weak labeling conditions, residues in the C helix region were also relatively stabilized in the mutant compared to the wild-type protein. This suggests that native-like helix B/helix C packing interactions occur in the folding intermediate. The L32A mutant showed significantly lower proton occupancies in the burst phase for several residues in the G helix, specifically F106, I107, E109 and A110, which are in close proximity to L32 in the X-ray structure of myoglobin, providing direct evidence that native-like helix B/helix G contacts are formed in the apomyoglobin burst phase intermediate. The L29A mutation resulted in an increase in burst phase proton occupancies for several residues in the E helix. Since these regions of the B and E helices are not in contact in the native myoglobin structure, these effects suggest the possibility of non-native B/E packing interactions in the kinetic intermediate. The differing effects of these B helix mutations on the apomyoglobin folding process suggests that each side-chain plays a different and important role in forming stable structure in the burst phase intermediate, and points to a role for both native-like and non-native contacts in stabilization of the folding intermediate.     

A misfolded form of 5S rRNA is complexed with the Ro and La autoantigens.

In both vertebrate and invertebrate cells, the 60-kDa Ro autoantigen is bound to small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the 60-kDa Ro protein is also complexed with a class of 5S rRNA precursors that contain internal mutations. Because these 5S rRNA precursors are processed inefficiently and degraded eventually, the Ro protein may function in a quality control pathway for 5S rRNA biosynthesis. We have investigated the sequence and secondary structure determinants in the mutant 5S rRNAs that confer binding by the 60-kDa Ro protein. The mutant 5S rRNAs fold to form an alternative helix that is required for recognition by the 60-kDa Ro protein. Mutations that disrupt the alternative helix eliminate Ro protein binding, whereas compensatory changes that restore the helix are bound efficiently by the Ro protein. When the structure of the mutant RNA was probed using dimethylsulfate and oligonucleotide-directed RNase H cleavage, the results were consistent with the formation of the alternative structure. The La protein, which is also complexed with the mutant 5S rRNA precursors, protects similar sequences from nuclease digestion as does the 60-kDa Ro protein. Thus, the binding sites for these two proteins are either nearby on the RNA, or the two proteins may be complexed through protein-protein interactions. When the human Ro protein is expressed in the yeast Saccharomyces cerevisiae, the protein binds wild-type 5S rRNA precursors, suggesting that a population of wild-type precursors also folds into the alternative structure.     

Conformational and dynamic characterization of the molten globule state of an apomyoglobin mutant with an altered folding pathway.

Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding.     

Inversion of axial coordination in myoglobin to create a "proximal" ligand binding pocket

A ligand binding pocket has been created on the proximal side of the heme in porcine myoglobin by site-directed mutagenesis. Our starting point was the H64V/V68H double mutant which has been shown to have bis-histidine (His68 and His93) heme coordination [Dou, Y., Admiraal, S. J., Ikeda-Saito, M., Krzywda, S., Wilkinson, A. J., Li, T., Olson, J. S., Prince, R. C., Pickering, I. J., George, G. N. (1995) J. Biol. Chem. 270, 15993-16001]. The replacement of the proximal His93 ligand by noncoordinating Ala (H64V/V68H/H93A) or Gly (H64V/V68H/H93G) residues resulted unexpectedly in a six-coordinate low-spin species in both ferric and ferrous states. To test the hypothesis that the sixth coordinating ligand in the triple mutants was the imidazole of His97, this residue was mutated to Phe, in the quadruple mutants, H64V/V68H/H93A/H97F and H64V/V68H/H93G/H97F. The ferric quadruple mutants show a clear water/hydroxide alkaline transition and high cyanide and CO affinities, characteristics similar to those of wild-type myoglobin. The nu(Fe-CO) and nu(C-O) stretching frequencies in the ferrous-CO state of the quadruple mutants indicate that the "proximal" ligand binding heme pocket is less polar than the distal pocket in the wild-type protein. Thus, we conclude that the proximal heme pocket in the quadruple mutants has a similar affinity for exogenous ligands to the distal pocket of wild-type myoglobin but that the two pockets have different polarities. The quadruple mutants open up new approaches for developing heme chemistry on the myoglobin scaffold.     

Folding propensities of peptide fragments of myoglobin.

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.     

Location of the Zn(2+)-binding site on S100B as determined by NMR spectroscopy and site-directed mutagenesis

In addition to binding Ca(2+), the S100 protein S100B binds Zn(2+) with relatively high affinity as confirmed using isothermal titration calorimetry (ITC; K(d) = 94 +/- 17 nM). The Zn(2+)-binding site on Ca(2+)-bound S100B was examined further using NMR spectroscopy and site-directed mutagenesis. Specifically, ITC measurements of S100B mutants (helix 1, H15A and H25A; helix 4, C84A, H85A, and H90A) were found to bind Zn(2+) with lower affinity than wild-type S100B (from 2- to >25-fold). Thus, His-15, His-25, Cys-84, His-85, and perhaps His-90 of S100B are involved in coordinating Zn(2+), which was confirmed by NMR spectroscopy. Previous studies indicate that the binding of Zn(2+) enhances calcium and target protein-binding affinities, which may contribute to its biological function. Thus, chemical shift perturbations observed here for residues in both EF-hand domains of S100B during Zn(2+) titrations could be detecting structural changes in the Ca(2+)-binding domains of S100B that are pertinent to its increase in Ca(2+)-binding affinity in the presence of Zn(2+). Furthermore, Zn(2+) binding causes helix 4 to extend by one full turn when compared to Ca(2+)-bound S100B. This change in secondary structure likely contributes to the increased binding affinity that S100B has for target peptides (i.e., TRTK peptide) in the presence of Zn(2+).     

Conformational changes induced by a single amino acid substitution in the trans-membrane domain of Vpu: implications for HIV-1 susceptibility to channel blocking drugs

The channel-forming trans-membrane domain of Vpu (Vpu TM) from HIV-1 is known to enhance virion release from the infected cells and is a potential target for ion-channel blockers. The substitution of alanine at position 18 by a histidine (A18H) has been shown to render HIV-1 infections susceptible to rimantadine, a channel blocker of M2 protein from the influenza virus. In order to describe the influence of the mutation on the structure and rimantadine susceptibility of Vpu, we determined the structure of A18H Vpu TM, and compared it to those of wild-type Vpu TM and M2 TM. Both isotropic and orientationally dependent NMR frequencies of the backbone amide resonance of His18 were perturbed by rimantadine, and those of Ile15 and Trp22 were also affected, suggesting that His18 is the key residue for rimantadine binding and that residues located on the same face of the TM helix are also involved. A18H Vpu TM has an ideal, straight alpha-helix spanning residues 6-27 with an average tilt angle of 41 degrees in C14 phospholipid bicelles, indicating that the tilt angle is increased by 11 degrees compared to that of wild-type Vpu TM. The longer helix formed by the A18H mutation has a larger tilt angle to compensate for the hydrophobic mismatch with the length of the phospholipids in the bilayer. These results demonstrate that the local change of the primary structure plays an important role in secondary and tertiary structures of Vpu TM in lipid bilayers and affects its ability to interact with channel blockers.     

Changes in the apomyoglobin folding pathway caused by mutation of the distal histidine residue

Factors governing the folding pathways and the stability of apomyoglobin have been examined by replacing the distal histidine at position 64 with phenylalanine (H64F). Acid and urea-induced unfolding experiments using CD and fluorescence techniques reveal that the mutant H64F apoprotein is significantly more stable than wild-type apoMb. Kinetic refolding studies of this variant also show a significant difference from wild-type apoMb. The amplitude of the burst phase ellipticity in stopped-flow CD measurements is increased over that of wild-type, an indication that the secondary structure content of the earliest kinetic intermediate is greater in the mutant than in the wild-type protein. In addition, the overall rate of folding is markedly increased. Hydrogen exchange pulse labeling was used to establish the structure of the initial intermediate formed during the burst phase of the H64F mutant. NMR analysis of the samples obtained at different refolding times indicates that the burst phase intermediate contains a stabilized E helix as well as the A, G, and H helices previously found in the wild-type kinetic intermediate. Replacement of the polar distal histidine residue with a nonpolar residue of similar size and shape appears to stabilize the E helix in the early stages of folding due to improved hydrophobic packing. The presence of a hydrophilic histidine at position 64 thus exacts a price in the stability and folding efficiency of the apoprotein, but this residue is nevertheless highly conserved among myoglobins due to its importance in function.     

Extrusion of the C-terminal helix in navel orangeworm moth pheromone-binding protein (AtraPBP1) controls pheromone binding

The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male–female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present a mutational analysis on a PBP from A. transitella (AtraPBP1) to evaluate how the C-terminal helix in this protein controls pheromone binding as a function of pH. Pheromone binds tightly to AtraPBP1 at neutral pH, but the binding is much weaker at pH below 5. Deletion of the entire C-terminal helix (residues 129–142) causes more than 100-fold increase in pheromone-binding affinity at pH 5 and only a 1.5-fold increase at pH 7. A similar pH-dependent increase in pheromone binding is also seen for the H80A/H95A double mutant that promotes extrusion of the C-terminal helix by disabling salt bridges at each end of the helix. The single mutants (H80A and H95A) also exhibit pheromone binding at pH below 5, but with ∼2-fold weaker affinity. NMR and circular dichroism data demonstrate a large overall structural change in each of these mutants at pH 4.5, indicating an extrusion of the C-terminal helix that profoundly affects the overall structure of the low pH form. Our results confirm that sequestration of the C-terminal helix at low pH as seen in the recent NMR structure may serve to block pheromone binding. We propose that extrusion of these C-terminal residues at neutral pH (or by the mutations in this study) exposes a hydrophobic cleft that promotes high affinity pheromone binding.     

Permuteins of interleukin 1 beta--a simplified approach for the construction of permutated proteins having new termini.

A technique for the rapid and simple generation of permutated versions of the interleukin-1 beta (IL-1 beta) gene is described. In this method, the human IL-1 beta cDNA is twice amplified by the polymerase chain reaction (PCR) and the resulting DNA fragments are ligated in tandem. Between the two genes, the DNA sequence encodes a short four amino acid loop to link the native N- and C-terminal ends of the IL-1 beta protein. By using PCR amplification from this starting template, a new version of the IL-1 beta cDNA was obtained that encodes a permutated form of the IL-1 beta protein where the new N- and C-terminal amino acids correspond to residues 65 and 64 of the native IL-1 beta sequence, respectively. The name 'permutein' is proposed to describe proteins generated by this technology. The molecular profile (IL-1 receptor binding, biologic activity and solution properties) of the IL-1 permutein produced by this technology, permutein 65/64, is shown to be identical to that of native IL-1 beta. The approach should be useful to define further the structural features of this protein that are important for its function.     

Role of helix B residues in interfacial activation of a bacterial phosphatidylinositol-specific phospholipase C

The Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), an interfacial enzyme associated with prokaryotic infectivity, is activated by binding to zwitterionic surfaces, particularly phosphatidycholine (PC). Two tryptophan residues (Trp47 in the two-turn helix B and Trp242 in a disordered loop) at the rim of the barrel structure are critical for this interaction. The helix B region (Ile43 to Gly48) in wild-type PI-PLC orients the side chains of Ile43 and Trp47 so that they pack together and form a hydrophobic protrusion from the protein surface that likely facilitates initial membrane binding. In previous studies we reported that in the crystal structure of the dimeric W47A/W242A mutant, which is unable to bind to PC, the helix B region has been reorganized by the mutation into an extended loop. Here we report the construction and characterization (catalytic activity, fluorescence, and NMR studies) of a series of PI-PLC mutants targeting helix B residues and surrounding regions to explore what is needed to stabilize the "membrane-active" conformation of the helix B region. Results strongly suggest that, while hydrophobic groups and presumably an intact helix B are critical for the initial binding of PI-PLC to membranes, disruption of helix B to allow enzyme dimerization is what leads to the activated PI-PLC conformation.     

Oxidation of tryptophans in an interhelical hydrophobic cluster of myoglobin alters the thermodynamics of the denaturation transition

Model folding studies of sperm whale myoglobin have illustrated the presence of hydrophobic interfacial regions between elements of secondary structure. The specific oxidation of two tryptophan residues, in the A-H helix contact of sperm whale myoglobin, to the less hydrophobic oxindolylalanine residues is utilized to probe the contribution of hydrophobic packing density in this contact region. The acid denaturation of the modified protein is no longer a simple two-state process exhibiting the presence of stable intermediates. The relative stability of the intermediate is shown to be +5.3 kcal/mol less stable than native myoglobin. This value is consistent with the predicted relative stability, based upon electrostatic model calculations, of the docking of the A helix with a des-A helix myoglobin. The presence of stable intermediate structures in the denaturation pathway of the modified protein is consistent with the proposed role of hydrophobic interactions in damping structural fluctuations and statistical mechanical models of noncooperative protein unfolding. These results demonstrate the relationship between large-scale fluctuations and the frictional forces governing small-scale motions within the protein core.     

Structure of Ala24/Asp61 --> Asp24/Asn61 substituted subunit c of Escherichia coli ATP synthase: implications for the mechanism of proton transport and rotary movement in the F0 complex

The structure of the A24D/D61N substituted subunit c of Escherichia coli ATP synthase, in which the essential carboxylate has been switched from residue 61 of the second transmembrane helix (TMH) to residue 24 of the first TMH, has been determined by heteronuclear multidimensional NMR in a monophasic chloroform/methanol/water (4:4:1) solvent mixture. As in the case of the wild-type protein, A24D/D61N substituted subunit c forms a hairpin of two extended alpha-helices (residues 5-39 and 46-78), with residues 40-45 forming a connecting loop at the center of the protein. The structure was determined at pH 5, where Asp24 is fully protonated. The relative orientation of the two extended helices in the A24D/D61N structure is different from that in the protonated form of the wild-type protein, also determined at pH 5. The C-terminal helix is rotated by 150 degrees relative to the wild-type structure, and the N-terminal helix is rotated such that the essential Asp24 carboxyl group packs on the same side of the molecule as Asp61 in the wild-type protein. The changes in helix-helix orientation lead to a structure that is quite similar to that of the deprotonated form of wild-type subunit c, determined at pH 8. When a decameric ring of c subunits was modeled from the new structure, the Asp24 carboxyl group was found to pack in a cavity at the interface between two subunits that is similar to the cavity in which Asp61 of the wild-type protein is predicted to pack. The interacting faces of the packed subunits in this model are also similar to those in the wild-type model. The results provide further evidence that subunit c is likely to fold in at least two conformational states differing most notably in the orientation of the C-terminal helix. Based upon the structure, a mechanistic model is discussed that indicates how the wild-type and A24D/D61N subunits could utilize similar helical movements during H(+) transport-coupled rotation of the decameric c ring.     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

Distal pocket polarity in ligand binding to myoglobin: structural and functional characterization of a threonine68(E11) mutant

Site-directed mutagenesis studies have confirmed that the distal histidine in myoglobin stabilizes bound O2 by hydrogen bonding and have suggested that it is the polar character of the imidazole side chain rather than its size that limits the rate of ligand entry into the protein. We constructed an isosteric Val68 to Thr replacement in pig myoglobin (i) to investigate whether the O2 affinity could be increased by the introduction of a second hydrogen-bonding group into the distal heme pocket and (ii) to examine the influence of polarity on the ligand binding rates more rigorously. The 1.9-A crystal structure of Thr68 aquometmyoglobin confirms that the mutant and wild-type proteins are essentially isostructural and reveals that the beta-OH group of Thr68 is in a position to form hydrogen-bonding interactions both with the coordinated water molecule and with the main chain greater than C=O of residue 64. The rate of azide binding to the ferric form of the Thr68 mutant was 60-fold lower than that for the wild-type protein, consistent with the proposed stabilization of the coordinated water molecule. However, bound O2 is destabilized in the ferrous form of the mutant protein. The observed 17-fold lowering of the O2 affinity may be a consequence of the hydrogen-bonding interaction made between the Thr68 beta-OH group and the carbonyl oxygen of residue 64. Overall association rate constants for O2, NO, and alkyl isocyanide binding to ferrous pig myoglobin were 3-10-fold lower for the mutant compared to the wild-type protein, whereas that for CO binding was little affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR structure of the alpha-hemoglobin stabilizing protein: insights into conformational heterogeneity and binding

The structure of alpha-hemoglobin stabilizing protein (AHSP), a molecular chaperone for free alpha-hemoglobin, has been determined using NMR spectroscopy. The protein native state shows conformational heterogeneity attributable to the isomerization of the peptide bond preceding a conserved proline residue. The two equally populated cis and trans forms both adopt an elongated antiparallel three alpha-helix bundle fold but display major differences in the loop between the first two helices and at the C terminus of helix 3. Proline to alanine single point mutation of the residue Pro-30 prevents the cis/trans isomerization. The structure of the P30A mutant is similar to the structure of the trans form of AHSP in the loop 1 region. Both the wild-type AHSP and the P30A mutant bind to alpha-hemoglobin, and the wild-type conformational heterogeneity is quenched upon complex formation, suggesting that just one conformation is the active form. Changes in chemical shift observed upon complex formation identify a binding interface comprising the C terminus of helix 1, the loop 1, and the N terminus of helix 2, with the exposed residues Phe-47 and Tyr-51 being attractive targets for molecular recognition. The characteristics of this interface suggest that AHSP binds at the intradimer alpha1beta1 interface in tetrameric HbA.     

Defining the structural requirements for a helix in 23 S ribosomal RNA that confers erythromycin resistance

The helix spanning nucleotides 1198 to 1247 (helix 1200-1250) in Escherichia coli 23 S ribosomal RNA (rRNA) is functionally important in protein synthesis, and deletions in this region confer erythromycin resistance. In order to define the structural requirements for resistance, we have dissected this region using in vitro mutagenesis. Erythromycin resistance is established after a minimal deletion of three bases, CAU1231 or AUG1232. The maximum deletion observed to confer resistance is 25 bases. The level of erythromycin resistance conferred by intermediate sized deletions is variable and some deletion mutants show a sensitive phenotype. Deletions that extend into the base-pairing between GCC1208 and GGU1240 result in non-functional 23 S RNAs, which consequently do not confer resistance. A number of phylogenetically conserved nucleotides have been shown to be non-essential for 23 S RNA function. However, removal of either these or non-conserved nucleotides from helix 1200-1250 measurably reduces the efficiency of 23 S RNA in forming functional ribosomes. We have used chemical probing and a modified primer extension method to investigate erythromycin binding to wild-type and resistant ribosomes with a 12-base deletion in 23 S RNA. Erythromycin interacts as strongly with mutant 23 S RNA as with wild-type 23 S RNA. Deletions in the 1200-1250 helix do not therefore confer resistance by reducing erythromycin binding, but by suppressing the effects of the drug at the level of its mechanism of action.     

Membrane partitioning and lipid selectivity of the N-terminal amphipathic H0 helices of endophilin isoforms

Endophilin is an N-BAR protein, which is characterized by a crescent-shaped BAR domain and an amphipathic helix that contributes to the membrane binding of these proteins. The exact function of that H0 helix has been a topic of debate. In mammals, there are five different endophilin isoforms, grouped into A (three members) and B (two members) subclasses, which have been described to differ in their subcellular localization and function. We asked to what extent molecular properties of the H0 helices of these members affect their membrane targeting behavior.We found that all H0 helices of the endophilin isoforms display a two-state equilibrium between disordered and α-helical states in which the helical secondary structure can be stabilized through trifluoroethanol. The helicities in high TFE were strikingly different among the H0 peptides. We investigated H0-membrane partitioning by the monitoring of secondary structure changes via CD spectroscopy. We found that the presence of anionic phospholipids is critical for all H0 helices partitioning into membranes. Membrane partitioning is found to be sensitive to variations in membrane complexity. Overall, the H0 B subfamily displays stronger membrane partitioning than the H0 A subfamily. The H0 A peptide-membrane binding occurs predominantly through electrostatic interactions. Variation among the H0 A subfamily may be attributed to slight alterations in the amino acid sequence. Meanwhile, the H0 B subfamily displays greater specificity for certain membrane compositions, and this may link H0 B peptide binding to endophilin B's cellular function.