Mean Residue Ellipticity Research Articles

Conformational destabilization of Bacillus licheniformis α-amylase induced by lysine modification and calcium depletion.

Bacillus licheniformis α-amylase (BLA) was chemically modified using 100-fold molar excess of succinic anhydride over protein or 0.66 M potassium cyanate to obtain 42 % succinylated and 81 % carbamylated BLAs. Size and charge homogeneity of modified preparations was established by Sephacryl S-200 HR gel chromatography and polyacrylamide gel electrophoresis. Conformational alteration in these preparations was evident by the larger Stokes radii (3.40 nm for carbamylated and 3.34 nm for succinylated BLAs) compared to 2.43 nm obtained for native BLA. Urea denaturation results using mean residue ellipticity (MRE) as a probe also showed conformational destabilization based on the early start of transition as well as ΔG(D)(H(2)O) values obtained for both modified derivatives and Ca-depleted BLA. Decrease in ΔG(D)(H(2)O) value from 5,930 cal/mol (for native BLA) to 3,957 cal/mol (for succinylated BLA), 3,336 cal/mol (for carbamylated BLA) and 3,430 cal/mol for Ca-depleted BLA suggested reduced conformational stability upon modification of amino groups of BLA or depletion of calcium. Since both succinylation and carbamylation reactions abolish the positive charge on amino groups (both α- and ε- amino), the decrease in conformational stability can be ascribed to the disruption of salt bridges present in the protein which might have released the intrinsic calcium from its binding site.

A Synthetic S6 Segment Derived from KvAP Channel Self-assembles, Permeabilizes Lipid Vesicles, and Exhibits Ion Channel Activity in Bilayer Lipid Membrane

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.

Designed Coiled Coils Promote Folding of a Recombinant Bacterial Collagen

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.

Combination Treatment with HER-2 and VEGF Peptide Mimics Induces Potent Anti-tumor and Anti-angiogenic Responses in Vitro and in Vivo

HER-2 is a member of the EGF receptor family and is overexpressed in 20-30% of breast cancers. HER-2 overexpression causes increased expression of VEGF at both the RNA and protein levels. HER-2 and VEGF are therefore considered good targets for cancer treatment, which has led to the development of two humanized monoclonal antibodies (mAb) pertuzumab and bevacizumab. Although passive immunotherapy with these Abs are approved for treatment of advanced breast cancer, a number of concerns exist. Treatment is expensive, has a limited duration of action, and is usually accompanied by serious side effects. We hypothesized that therapy with conformational peptide mimics aimed at blocking receptor-ligand interaction is potentially safer with little toxicity, cheaper with a longer half-life, and has greater penetrating abilities than mAbs. We designed and synthesized peptides based on the binding of HER-2 with pertuzumab and VEGF with VEGFR2. We show that treatment with the peptide mimics induces potent anti-tumor responses in vitro as determined by cell viability, proliferation, and HER2 phosphorylation assays. We also demonstrate in a transplantable BALB/c mouse tumor model that treatment with the peptide mimics resulted in a greater delay in tumor growth and development. Similarly, treatment with the peptide mimics inhibited angiogenesis in vivo as assessed by a Matrigel plug assay. To address the problem of degradability of L-amino acid peptides in vivo, we synthesized the retro-inverso D-peptide mimics that resulted in higher efficacy in treatment. Our study shows that combination treatment with HER-2 and VEGF peptide mimics provides greater efficacy than individual treatments.

Influence of Glycine Substitutions on Designed Proline-Containing Transmembrane Peptides

Membrane-spanning WALP peptides of general sequence acetyl-GWW(LA)nLWWA-amide, in which “n” may range from about 3 to 12, have been well characterized. A kink induced by a central proline residue in WALP19-P10 (acetyl-GWWLALALAP10ALALALWWA-ethanolamide) has also been characterized (see Biochemistry 48: 11883). It was furthermore observed that the 2H-quadrupolar splittings from labeled alanine side chains in WALP19-P10 were especially sensitive to Leu-to-Ala substitutions elsewhere in the sequence. In this work, we examined the consequences of Leu-to-Gly substitutions within the proline-containing WALP19-P10, from a perspective that Leu-to-Gly substitutions could reasonably be expected to produce larger consequences than Leu-to-Ala substitutions. Circular dichroism spectra indicate small reductions in helicity for Gly substitutions in WALP19-P10, with the mean residue ellipticity values being similar for Leu-to-Gly substitutions on either side of the proline. Using 2H-alanines as probes within the sequence, we have found from deuterium NMR spectroscopy of peptides incorporated within oriented lipid bilayer samples that the segment N-terminal to proline is generally more susceptible to Leu-to-Gly substitutions on both sides of the proline. In contrast, the C-terminal domain is largely unaffected by the glycine substitutions in either the N- or C-terminal segment. Substitutions near the C-terminus have little effect on either segment.

A gel-based method for purification of apolipoprotein A-I from small volumes of plasma

We present here a gel-based method for rapid purification of apolipoprotein A-I (apoA-I) from small volumes of human plasma. After isolation of high density lipoprotein from plasma, the apoA-I protein was separated by electrophoresis and the apoA-I band excised from the gel. The apoA-I was then eluted from the gel strip, concentrated, and delipidated ready for use. The structure and function of the gel-purified apoA-I protein was compared against apoA-I purified by the traditional size-exclusion chromatography method. The α-helical content of the gel-purified apoA-I as determined by circular dichroism was similar to chromatography-purified apoA-I. The functional activity of gel-purified apoA-I, as determined by cholesterol efflux assays in primary human fibroblasts and RAW264.7 macrophages, was also comparable with chromatography-purified apoA-I. This method is a valid alternative for apoA-I purification with some advantages over traditional chromatography purification including a much reduced plasma volume requirement, less time and cost, and a higher percentage protein recovery. The method is particularly suitable for applications requiring the purification of apoA-I from multiple human or animal samples of interest.

Conformational and thermodynamic characterization of the premolten globule state occurring during unfolding of the molten globule state of cytochrome c

It has already been shown that the mutant Leu94Gly of horse cytochrome c exists in a molten globule (MG) state. We have carried out studies of reversible folding and unfolding induced by LiCl of this mutant at pH 6.0 and 25 °C by observing changes in the difference molar absorption coefficient at 402 nm, the mean residue ellipticity at 222 nm, and the difference mean residue ellipticity at 409 nm. This process is a three-state process when measured by these probes. The stable folding intermediate state has been characterized by far- and near-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalenesulfonic acid binding, and dynamic light scattering measurements, which led us to conclude that the intermediate is a premolten globule (PMG). Analysis of the reversible unfolding transition curves for the stability of different states in terms of the Gibbs free energy change at pH 6.0 and 25 °C led us to conclude that the MG state is more stable than the PMG state by 5.4 ± 0.1 kcal mol(-1), whereas the PMG state is more stable than the denatured (D) state by only 1.1 ± 0.1 kcal mol(-1). A comparison of the conformational and thermodynamic properties of the LiCl-induced PMG state at pH 6.0 with those of the PMG state induced by NaCl at pH 2.0 suggests that a similar PMG state is obtained under both denaturing conditions. Differential scanning calorimetry measurements suggest that heat induces a reversible two-state transition between MG and D states.

Novel Recombinant Engineered gp41 N-terminal Heptad Repeat Trimers and Their Potential as Anti-HIV-1 Therapeutics or Microbicides

Peptides derived from N-terminal heptad repeat (NHR) of the HIV-1 gp41 are generally poor inhibitors of HIV-1 entry, because they tend to aggregate and do not form a trimeric coiled-coil. In this study, we have fused portions of gp41 NHR, e.g. N36 or N28, to the T4 fibritin trimerization domain, Foldon (Fd), thus constructing novel NHR trimers, designated N36Fd or N28Fd, which could be expressed in Escherichia coli cells. The purified N36Fd and N28Fd exhibited SDS-resistant trimeric coiled-coil conformation with improved alpha-helicity compared with the corresponding N-peptides. They could interact with a C-peptide (e.g. C34) to form stable six-helix bundle and possessed potent anti-HIV-1 activity against a broad spectrum of HIV-1 strains. N28Fd was effective against T20-resistant HIV-1 variants and more resistant to proteinase K compared with T20 (enfuvirtide), a C-peptide-based HIV fusion inhibitor. Therefore, N28Fd trimer has great potentials for further development as an affordable therapeutic or microbicide for treatment and prevention of HIV-1 infection.

Imidazolium Bromide-Based Ionic Liquid Assisted Improved Activity of Trypsin in Cationic Reverse Micelles

The present work reports the imidazolium-based ionic liquids (ILs) assisted enhancement in activity of water-pool solubilized enzyme trypsin in cationic reverse micelles of CTAB. A set of imidazolium ILs (1-alkyl-3-methyl imidazolium bromides) were prepared with varying lengths of their side arm which results in the differential location of these organic salts in the reverse micelles. The different ILs offered varied activating effects on the biocatalyst. The activity of trypsin improved approximately 30-300% in the presence of 0.1-10 mM of different ILs in reverse micelles of CTAB. Trypsin showed approximately 300% (4-fold) increment in its activity in the presence of IL 2 (1-ethyl-3-methyl imidazolium bromide, EMIMBr) compared to that observed in the absence of IL in CTAB reverse micelles. The imidazolium moiety of the IL, resembling the histidine amino acid component of the catalytic triad of hydrolases and its Br(-) counterion, presumably increases the nucleophilicity of water in the vicinity of the enzyme by forming a hydrogen bond that facilitates the enzyme-catalyzed hydrolysis of the ester. However, the ILs with increasing amphiphilic character had little to no effect on the activity of trypsin due to their increased distance from the biocatalyst, as they tend to get localized toward the interfacial region of the aggregates. Dynamic light scattering experimentation was carried out in the presence of ILs to find a possible correlation between the trypsin activity and the size of the aggregates. In concurrence with the observed highest activity in the presence of IL 2, the circular dichroism (CD) spectrum of trypsin in CTAB reverse micelles doped with IL 2 exhibited the lowest mean residue ellipticity (MRE), which is closest to that of the native protein in aqueous buffer.

Structure and Mode of Action of Microplusin, a Copper II-chelating Antimicrobial Peptide from the Cattle Tick Rhipicephalus (Boophilus) microplus

Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single alpha-helical globular domain, microplusin consists of five alpha-helices: alpha1 (residues Gly-9 to Arg-21), alpha2 (residues Glu-27 to Asn-40), alpha3 (residues Arg-44 to Thr-54), alpha4 (residues Leu-57 to Tyr-64), and alpha5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

Compact acid-induced state of Clitoria ternatea agglutinin retains its biological activity

The effects of pH on Clitoria ternatea agglutinin (CTA) were studied by spectroscopy, size-exclusion chromatography, and by measuring carbohydrate specificity. At pH 2.6, CTA lacks well-defined tertiary structure, as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of 50% native-like secondary structure. The mean residue ellipticity at 217 nm plotted against pH showed a transition around pH 4.0 with loss of secondary structure leading to the formation of an acid-unfolded state. This state is relatively less denatured than the state induced by 6 M guanidine hydrochloride. With a further decrease in pH, this unfolded state regains ~75% secondary structure at pH 1.2, leading to the formation of the A-state with native-like near-UV CD spectral features. Enhanced 8-anilino-1-naphthalene-sulfonate binding was observed in A-state, indicating a "molten-globule" like conformation with exposed hydrophobic residues. Acrylamide quenching data exhibit reduced accessibility of quencher to tryptophan, suggesting a compact conformation at low pH. Size-exclusion chromatography shows the presence of a compact intermediate with hydrodynamic size corresponding to a monomer. Thermal denaturation of the native state was cooperative single-step transition and of the A-state was non-cooperative two-step transition. A-State regains 72% of the carbohydrate-binding activity.

Conformational Properties of β-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrPC) to a disease-specific isomer (PrPSc), shifting from a predominantly alpha-helical conformation to one dominated by beta-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrPSc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant beta-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrPC conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a beta-rich conformation. This precursor state is almost as compact as the folded PrPC structure and, as it assembles, only residues 126-227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.

A Functional and Structural Study of Troponin C Mutations Related to Hypertrophic Cardiomyopathy

Recently four new hypertrophic cardiomyopathy mutations in cardiac troponin C (cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the Ca(2+) sensitivity of force development were evaluated (Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, D. J., Ommen, S. R., Potter, J. D., and Ackerman, M. J. (2008) J. Mol. Cell. Cardiol. 45, 281-288). We performed actomyosin ATPase and spectroscopic solution studies to investigate the molecular properties of these mutations. Actomyosin ATPase activity was measured as a function of [Ca(2+)] utilizing reconstituted thin filaments (TFs) with 50% mutant and 50% wild type (WT) and 100% mutant cardiac troponin (cTn) complexes: A8V, C84Y, and D145E increased the Ca(2+) sensitivity with only A8V demonstrating lowered Ca(2+) sensitization at the 50% ratio when compared with 100%; E134D was the same as WT at both ratios. Of these four mutants, only D145E showed increased ATPase activation in the presence of Ca(2+). None of the mutants affected ATPase inhibition or the binding of cTn to the TF measured by co-sedimentation. Only D145E increased the Ca(2+) affinity of site II measured by 2-(4'-(2''-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid fluorescence in isolated cTnC or the cTn complex. In the presence of the TF, only A8V was further sensitized to Ca(2+). Circular dichroism measurements in different metal-bound states of the isolated cTnCs showed changes in the secondary structure of A8V, C84Y, and D145E, whereas E134D was the same as WT. PyMol modeling of each cTnC mutant within the cTn complex revealed potential for local changes in the tertiary structure of A8V, C84Y, and D145E. Our results indicate that 1) three of the hypertrophic cardiomyopathy cTnC mutants increased the Ca(2+) sensitivity of the myofilament; 2) the effects of the mutations on the Ca(2+) affinity of isolated cTnC, cTn, and TF are not sufficient to explain the large Ca(2+) sensitivity changes seen in reconstituted and fiber assays; and 3) changes in the secondary structure of the cTnC mutants may contribute to modified protein-protein interactions along the sarcomere lattice disrupting the coupling between the cross-bridge and Ca(2+) binding to cTnC.

The Membrane-binding Motif of the Chloroplast Signal Recognition Particle Receptor (cpFtsY) Regulates GTPase Activity

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally. Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpSRP-dependent protein targeting. This membrane-binding motif is both necessary and sufficient to direct cpFtsY and fused passenger proteins to thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may be functionally replaced by the corresponding region from E. coli, confirming that the membrane-binding motif is conserved among organellar and prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding correlates with liposome-induced GTP hydrolysis stimulation. Mutations that debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP hydrolysis, suggesting that negative regulation is provided by the intact membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD structural studies of the N-terminal region and the analogous region in the E. coli SRP receptor revealed a conformational change in secondary structure that takes place upon lipid binding. These studies suggest that the cpFtsY membrane-binding motif plays a critical role in the intramolecular communication that regulates cpSRP receptor functions at the membrane.

Distinctions between Hydrophobic Helices in Globular Proteins and Transmembrane Segments as Factors in Protein Sorting

Transmembrane (TM) segments in proteins can be distinguished in amino acid sequences as continuous stretches of hydrophobic residues. However, examination of a data base of helical water-soluble (globular) proteins revealed that nearly one-third contained helices of sufficient length to span a bilayer (> or =19 residues) that had mean hydrophobicity > or =actual TM segments. We now report that synthetic peptides corresponding to these globular protein sequences, which we termed "delta-helices," behave like native TM sequences and readily insert into membrane mimetic environments in helical conformations. As well, certain delta-helix sequences can integrate into the membrane bilayer when placed into a membrane-targeted chimeric protein. We establish that delta-helices can be distinguished computationally from bona fide TM segments by the decreased frequency of occurrence of Ile/Val residues and by their relatively decreased solvent accessibilities (versus other globular helices) within tertiary structure. The further observations that (i) delta-helices generally contain three or more charged residues and (ii) delta-helices display relatively even distribution of these charged residues along their lengths, rather than concentration near their N and C termini as observed for TM segments, may constitute key recognition factors in diverting delta-helices from the membrane in vivo. Although a discrete biological role for delta-helices remains to be pinpointed, our overall results suggest that such segments may be required for globular protein folding and identify additional factors that may be important in the correct selection of TM segments by the cellular machinery.

ACDP– a Java application for data processing and analysis of protein circular dichroism spectra

ACDPis a Java application for processing protein circular dichroism (CD) spectra either individually or within a series. Data processing includes spectrum subtraction (`baseline correction'), conversion of the raw CD into units of mean residue ellipticity, wavelength monitoring and graphical inspection. Three different algorithms for secondary structure deconvolution have been implemented, and spectra can be analysed without the need for data reformatting, using a neural network approach, variable selection or linear combination of prototype spectra. The application is written entirely in Java and is thus portable to a wide variety of platforms, requiring only the Java Runtime Environment.

Domain Mapping of the Polycystin-2 C-terminal Tail Using de Novo Molecular Modeling and Biophysical Analysis

In polycystic kidney disease (PKD), polycystin-2 (PC2) is frequently mutated or truncated in the C-terminal cytoplasmic tail (PC2-C). The currently accepted model of PC2-C consists of an EF-hand motif overlapping with a short coiled coil; however, this model fails to explain the mechanisms by which PC2 truncations C-terminal to this region lead to PKD. Moreover, direct PC2 binding to inositol 1,4,5-trisphosphate receptor, KIF3A, and TRPC1 requires residues in PC2-C outside this region. To address these discrepancies and investigate the role of PC2-C in PC2 function, we performed de novo molecular modeling and biophysical analysis. De novo molecular modeling of PC2-C using the ROBETTA server predicts two domains as follows: an EF-hand motif (PC2-EF) connected by a linker to a previously unidentified C-terminal coiled coil (PC2-CC). This model differs substantially from the current model and correlates with limited proteolysis, matrix-assisted laser desorption/ionization mass spectroscopy, N-terminal sequencing, and improved coiled coil prediction algorithms. PC2-C is elongated and oligomerizes through PC2-CC, as measured by analytical ultracentrifugation and size exclusion chromatography, whereas PC2-EF is globular and monomeric. We show that PC2-C and PC2-EF have micromolar affinity for calcium (Ca2+) by isothermal titration calorimetry and undergo Ca2+-induced conformational changes by circular dichroism. Mutation of predicted EF-hand loop residues in PC2 to alanine abolishes Ca2+ binding. Our results suggest that PC2-CC is involved in PC2 oligomerization, and PC2-EF is a Ca2+-sensitive switch. PKD-associated PC2 mutations are located in regions that may disrupt these functions, providing structural insight into how PC2 mutations lead to disease.

Three Consecutive Arginines Are Important for the Mycobacterial Peptide Deformylase Enzyme Activity

Genes encoding the peptide deformylase enzyme (def) are present in all eubacteria and are involved in the deformylation of the N-formyl group of newly synthesized polypeptides during protein synthesis. We compared the amino acid sequences of this enzyme in different mycobacterial species and found that they are highly conserved (76% homology with 62% identity); however, when this comparison was extended to other eubacterial homologs, it emerged that the mycobacterial proteins have an insertion region containing three consecutive arginine residues (residues 77-79 in Mycobacterium tuberculosis peptide deformylase (mPDF)). Here, we demonstrate that these three arginines are important for the activity of mPDF. Circular dichroism studies of wild-type mPDF and of mPDF containing individual conservative substitutions (R77K, R78K, or R79K) or combined substitutions incorporated into a triple mutant (R77K/R78K/R79K) indicate that such mutations cause mPDF to undergo structural alterations. Molecular modeling of mPDF suggests that the three arginines are distal to the active site. Molecular dynamics simulations of wild-type and mutant mPDF structures indicate that the arginines may be involved in the stabilization of substrate binding pocket residues for their proper interaction with peptide(s). Treatment with 5'-phosphothiorate-modified antisense oligodeoxyribonucleotides directed against different regions of def from M. tuberculosis inhibits growth of Mycobacterium smegmatis in culture. Taken together, these results hold out the possibility of future design of novel mycobacteria-specific PDF inhibitors.

Mapping the Binding Site on Small Ankyrin 1 for Obscurin

Small ankyrin 1 (sAnk1), an integral protein of the sarcoplasmic reticulum encoded by the ANK1 gene, binds with nanomolar affinity to the C terminus of obscurin, a giant protein surrounding the contractile apparatus in striated muscle. We used site-directed mutagenesis to characterize the binding site on sAnk1, specifically addressing the role of two putative amphipathic, positively charged helices. We measured binding qualitatively by blot overlay assays and quantitatively by surface plasmon resonance and showed that both positively charged sequences are required for activity. We showed further that substitution of a lysine or arginine with an alanine or glutamate located at the same position along either of the two putative helices has similar inhibitory or stimulatory effects on binding and that the effects of a particular mutation depended on the position of the mutated amino acid in each helix. We modeled the structure of the binding region of sAnk1 by homology with ankyrin repeats of human Notch1, which have a similar pattern of charged and hydrophobic residues. Our modeling suggested that each of the two positively charged sequences forms pairs of amphipathic, anti-parallel alpha-helices flanked by beta-hairpin-like turns. Most of the residues in homologous positions along each helical unit have similar, though not identical, orientations. CD spectroscopy confirmed the alpha-helical content of sAnk1, approximately 33%, predicted by the model. Thus, structural and mutational studies of the binding region on sAnk1 for obscurin suggest that it consists of two ankyrin repeats with very similar structures.

Structural and Functional Characterization of Recombinant Matrilin-3 A-domain and Implications for Human Genetic Bone Diseases

Mutations in matrilin-3 result in multiple epiphyseal dysplasia, which is characterized by delayed and irregular bone growth and early onset osteoarthritis. The majority of disease-causing mutations are located within the beta-sheet of the single A-domain of matrilin-3, suggesting that they disrupt the structure and/or function of this important domain. Indeed, the expression of mutant matrilin-3 results in its intracellular retention within the rough endoplasmic reticulum of cells, where it elicits an unfolded protein response. To understand the folding characteristics of the matrilin-3 A-domain we determined its structure using CD, analytical ultracentrifugation, and dual polarization interferometry. This study defined novel structural features of the matrilin-3 A-domain and identified a conformational change induced by the presence or the absence of Zn(2+). In the presence of Zn(2+) the A-domain adopts a more stable "tighter" conformation. However, after the removal of Zn(2+) a potential structural rearrangement of the metal ion-dependent adhesion site motif occurs, which leads to a more "relaxed" conformation. Finally, to characterize the interactions of the matrilin-3 A-domain we performed binding studies on a BIAcore using type II and IX collagen and cartilage oligomeric matrix protein. We were able to demonstrate that it binds to type II and IX collagen and cartilage oligomeric matrix protein in a Zn(2+)-dependent manner. Furthermore, we have also determined that the matrilin-3 A-domain appears to bind exclusively to the COL3 domain of type IX collagen and that this binding is abolished in the presence of a disease causing mutation in type IX collagen.