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PubMed2006

Identification of carboxyl-terminal peptide fragments of parathyroid hormone in human plasma at low-picomolar levels by mass spectrometry.

For decades, researchers have tried to identify the primary structures of circulating carboxyl-terminal parathyroid hormone (C-PTH) peptide fragments that may be present at only picomolar levels in human plasma. Although immunoassays and radiosequencing techniques have provided valuable fragment characterizations, no analysis has successfully determined their exact primary structures. In this work, for the first time, four human C-PTH peptide fragments, hPTH(34-84), hPTH(37-84), hPTH(38-84), and hPTH(45-84), have been identified from human plasma using MS-based methods. C-PTH peptide fragments were isolated from plasma samples by immunoaffinity extraction. The eluate was analyzed by capillary LC fractionation followed by MALDI-TOF-MS or by on-line coupling of nano-LC with ESI-TOF-MS. Both the MALDI- and the ESI-based approaches were capable of detecting C-PTH peptide fragments in human plasma at <10 pmol/L. The MALDI-TOF approach was effective in preliminary searches for C-PTH peptide fragments, but the use of high laser power limited the resolution necessary for accurate C-PTH peptide identification. The high mass resolution (10,000) and accuracy (10 ppm) attained by the ESI-TOF approach enabled unambiguous identification of these peptides. The four C-PTH peptide fragments identified in plasma samples from patients with chronic renal insufficiency were also found in the plasma of healthy women receiving recombinant human PTH either by subcutaneous injection or by intravenous infusion. This newly developed analytical capability should greatly enhance the understanding of PTH metabolism and parathyroid gland function.

Humans↗
PubMed1995

Core domain of hirudin from the leech Hirudinaria manillensis: chemical synthesis, purification, and characterization of a Trp3 analog of fragment 1-47.

Hirudin is a small (approximately 7 kDa) disulfide-cross-linked polypeptide known as the most potent and specific thrombin inhibitor. We have previously shown that the N-terminal proteolytic fragment 1-47 of hirudin HM2 from Hirudinaria manillensis maintains inhibitory action toward thrombin [Vindigni, A., et al. (1994) Eur. J. Biochem. 226, 323-333]. Here we report the solid-phase chemical synthesis of an analog of fragment 1-47 bearing a Tyr3-->Trp exchange (Y3W analog). The crude, reduced peptide was purified by reverse-phase HPLC and subjected to oxidative folding to the disulfide-cross-linked species. The folding process of the Y3W analog was slower than that of the natural fragment 1-47, but nevertheless still occurred almost quantitatively as the natural species. The overall final yield of the synthetic product was approximately 35%, and its identity and homogeneity was established by a number of analytical techniques, including electrospray mass spectometry. The unique alignment of the three disulfide bridges of the Y3W analog was established by peptide mapping as Cys6-Cys14, Cys16-Cys28, and Cys22-Cys37 and shown to be identical to that of the natural fragment. The results of far- and near-ultraviolet circular dichroism and fluorescence emission measurements provided evidence that the Y3W analog retains the structural features of the natural species. The thermodynamic quantities (delta GD, delta Hm, delta Sm, and delta Cp) characterizing the reversible and cooperative thermal unfolding processes of the Y3W analog (Tm = 60.5 degrees C) and the natural fragment species (Tm = 62.5 degrees C) were evaluated. Despite the relatively high Tm values, the stability of both fragment species at 37 degrees C was only approximately 10 kJ mol-1, well below the average 50 kJ mol-1 typical of single-domain globular proteins. The synthetic Y3W species was found to be approximately 5-fold more active (KI = 30 +/- 5 nM) than the natural fragment 1-47 (KI = 150 +/- 20 nM) in inhibiting thrombin. Of interest was that the difference in the free energies of binding to thrombin at 37 degrees C, delta delta Gb, between the Y3W analog and natural species (4.2 kJ mol-1) was that expected for the difference in hydrophobicity between the two polypeptides resulting from the Tyr-->Trp exchange. The results of this study indicate that solid-phase chemical synthesis represents a convenient and high-yield procedure to prepare analogs of the biologically active, N-terminal core domain of hirudin with improved functional properties.

Amino Acid Sequence↗
PubMed1992

Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli.

We have designed dimeric antibody fragments that assemble in Escherichia coli. They are based on single-chain FV fragments, with a flexible hinge region from mouse IgG3 and an amphiphilic helix fused to the C-terminus of the antibody fragment. The sequence of the helix was taken either from that of a previously reported four-helix bundle design or from a leucine zipper, optionally extended with a short cysteine-containing peptide. The bivalent fragments associate in vivo, either with covalent linkage or with a monomer-dimer equilibrium, and results from ultracentrifugation sedimentation studies and SDS-PAGE are consistent with dimers. All constructs are able to bind to surface-bound antigen under conditions in which only bivalent but not monovalent antibody fragments bind. The covalent bundle helix construct shows binding characteristics nearly identical to those of the much larger whole mouse antibody, resulting in substantially more stable immunoglobulin-antigen complexes than in the case of monovalent fragments. This modular design of natural and engineered protein domains directly leads to a boost of avidity, and it allows the construction of bispecific antibody fragments in functional form in E. coli.

Amino Acid Sequence↗
PubMed1992

Structure of the proteolytic fragment F34 of calmodulin in the absence and presence of mastoparan as revealed by solution X-ray scattering.

The solution X-ray scattering technique has been applied to examine the conformations of the proteolytic fragment F34 (78Asp-148Lys) of calmodulin in the absence of both Ca2+ and mastoparan, in the presence of Ca2+ only, and in the presence of both Ca2+ and mastoparan. The radius of gyration and the molecular weight for the F34 fragment increased by 1.1 +/- 0.3 A and 19%, respectively, upon binding of both 2 mol of Ca2+/mol to the F34 fragment and mastoparan to form the tertiary complex. A smaller change was found for the Ca(2+)-saturated F34 fragment in the absence of mastoparan (0.3 +/- 0.3 A) without any change of the molecular weight. The analysis based on the small-angle scattering data showed that the F34 fragment in the presence of Ca2+ alone preserved the tertiary structure of the globular domain in the crystal to a great extent. Further analyses based on a two-domain model showed that the center-to-center distance between F34 and mastoparan is about 12.7 A, if the structure of the F34 fragment in the presence of mastoparan resembles that in the absence of mastoparan and if mastoparan in the complex retains an alpha-helical conformation. The modeling studies using their crystal structure coordinates have been made on the basis of the solution X-ray scattering data. The combined results support a model proposed by Persechini and Kretsinger [Persechini, A., & Kretsinger, R. H. (1988) J. Cardiovasc. Pharmacol. 12 (Suppl. 5), S1-S12], although the center-to-center distance between mastoparan and the F34 fragment is shorter by about 5 A than that in their model.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Attenuated total reflectance Fourier transform infrared studies of the interaction of melittin, two fragments of melittin, and delta-hemolysin with phosphatidylcholines.

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) has been used to monitor alterations in phospholipid organization in thin layers of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), induced by the membrane lytic peptide melittin, its fragments 1-15 (hydrophobic fragment) and 16-26 (hydrophilic fragment), and delta-hemolysin. In addition, the secondary structures of the peptides and the orientation of helical fragments were determined with respect to the bilayer. The insertion of melittin into POPC caused large perturbations in the order and increased rates of motion of the acyl chains, as monitored by the frequency and half-width of the symmetric CH2 stretching vibration near 2850 cm-1, as well as by the ATR dichroic ratio for this mode. Changes in DPPC organization were less and were consistent with peptide-induced static disordering (gauche rotamer formation) in the acyl chains. Melittin adopted primarily an alpha-helical secondary structure, although varying small proportions of beta and/or aggregated forms were noted. The helical segments were preferentially oriented perpendicular to the bilayer plane. Several modes of melittin/lipid interaction were considered in an attempt to semiquantitatively understand the observed dichroic ratios. By considering the peptide as a bent rigid rod, a plausible model for its lytic properties has been developed. The hydrophilic fragment in DPPC showed a secondary structure with little alpha-helix present. As judged by its effect on phospholipid acyl chain organizational parameters, the fragment did not penetrate the bilayer substantially. The hydrophobic fragment in DPPC gave amide I spectral patterns consistent with a mixture of predominantly beta-antiparallel pleated sheet with a smaller fraction of alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1989

Cross-linking of a monomeric 39/34-kDa dispase fragment of von Willebrand factor (Leu-480/Val-481-Gly-718) to the N-terminal region of the alpha-chain of membrane glycoprotein Ib on intact platelets with bis(sulfosuccinimidyl) suberate.

A 39/34-kilodalton (kDa) monomeric dispase fragment of von Willebrand factor (vWF) has been purified by heparin affinity chromatography. Detailed structural analysis of the individual 39- and 34-kDa fragments indicated that they had identical amino acid sequences extending from Leu-480/Val-481 to Gly-718 with an intramolecular disulfide bond between Cys-509 and Cys-695. In addition to the binding site for heparin, the 39/34-kDa fragment also contained binding sites for collagen and for platelet membrane glycoprotein (GP) Ib. Unlike native vWF, the 39/34-kDa fragment bound to GP Ib without the requirement for a modulator but showed increased binding in the presence of botrocetin. The 39/34-kDa vWF fragment was cross-linked to intact human platelets by using the membrane-impermeable, homobifunctional cross-linking reagent bis(sulfosuccinimidyl) suberate. Two distinct cross-linked species of similar molecular weight (220/200 kDa, nonreduced; 190/175 kDa, reduced) were identified by SDS-polyacrylamide gel electrophoresis and autoradiography, consistent with the cross-linking of the 125I-labeled 39/34-kDa vWF fragment to GP Ib. The formation of these cross-linked species was enhanced 1.5-2.5-fold in the presence of the modulator botrocetin. The platelet membrane protein involved in cross-linking was shown unequivocally to be GP Ib since (i) neither cross-linked species was formed with Bernard-Soulier syndrome platelets, which genetically lack the GP Ib-IX complex, (ii) both cross-linked species were specifically immunoprecipitated by anti-GP Ib polyclonal and monoclonal antibodies, and (iii) the formation of the cross-linked species was completely inhibited only by those anti-GP Ib-IX complex monoclonal antibodies that inhibited vWF-GP Ib-IX complex interaction. Proteolysis of cross-linked platelets with endoproteinase Lys-C, which preferentially cleaves off the N-terminal peptide domain on the alpha-chain of GP Ib, indicated that the 39/34-kDa vWF fragment was cross-linked exclusively to this region of the GP Ib-IX complex.

Amino Acid Sequence↗
PubMed1989

Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study.

The contribution of the various regions of the mitochondrial precursor protein apocytochrome c to the interaction of the protein with phosphatidylserine dispersions has been studied with chemically and enzymatically prepared fragments of horse heart apocytochrome c and phospholipids spin-labeled at different positions of the sn-2 chain. Three amino-terminal heme-less peptides, two heme-containing amino-terminal fragments, one central fragment, and three carboxy-terminal fragments were studied. The electron spin resonance spectra of phospholipids spin-labeled at the C5 position of the fatty acid chain indicate that both amino-terminal and carboxy-terminal fragments of the apocytochrome c molecule cause a restriction of motion of the lipids, whereas the heme-containing peptides and protein have less effect. In addition, a second motionally more restricted lipid component, which is observed for apocytochrome c interacting with phosphatidylserine dispersions containing lipids spin-labeled at the C12 or C14 position [Görrissen, H., Marsh, D., Rietveld, A., & de Kruijff, B. (1986) Biochemistry 25, 2904-2910], was observed both on binding the carboxy-terminal fragments and on binding of the amino-terminal fragments of the precursor protein. Interestingly, even a small water-soluble peptide consisting of the 24 carboxy-terminal residues gave rise to a two-component spectrum, with an outer hyperfine splitting of the restricted lipid component of 59 G, indicating a considerable restriction of the chain motion. This suggests that both the carboxy- and amino-terminal parts of the protein penetrate into the center of the bilayer and cause a strong perturbation of the fatty acyl chain motion. The implications of these findings for the mechanism of apocytochrome c translocation across membranes are discussed.

Animals↗
PubMed1990

A comparison of strategies to stabilize immunoglobulin Fv-fragments.

Fv-Fragments of antibodies may dissociate at low protein concentrations and are too unstable for many applications at physiological temperatures. To stabilize Fv-fragments against dissociation, we have tested and compared three different strategies on the Fv-fragment of the well-characterized phosphocholine binding antibody McPC603 expressed and secreted in Escherichia coli: chemical cross-linking of the variable domains, introduction of an intermolecular disulfide bond, and construction of a peptide linker to produce a "single-chain" Fv-fragment. All the linked fragments show hapten affinities nearly identical with that of the whole antibody independent of protein concentration and are significantly (up to 60-fold) stabilized against irreversible thermal denaturation. All genetically engineered linked Fv-fragments can be obtained in native conformation in E. coli. The reported strategies for generating Fv-fragments with improved physicochemical properties may extend their usefulness in biotechnology as well as in therapeutic and diagnostic applications.

Chemical Phenomena↗
PubMed1978

Metal ion induced conformational transitions of prothrombin and prothrombin fragment 1.

Circular dichroism experiments indicate that prothrombin fragment 1 undergoes essentially the same secondary structural change whether in the presence of Ca(2+), Mg(2+), or Mn(2+). Titration with any of these metal ions results in a sigmoidal titration curve indicative of cooperative binding. Mg(2+) and Ca(2+) have nearly identical transition midpoints, while that for Mn(2+) is an order of magnitude less. These results correlate well with the results of previous metal ion intrinsic fluorescence quenching experiments. Fragment 1 has previously been shown to undergo a second transition corresponding to dimerization at high calcium concentrations. The present circular dichroism experiments show that this transition does not result in a gross alteration of secondary structure in the fragment 1 molecule. Studies with prothrombin, similar to those with fragment 1, indicate a similar metal ion dependent conformational change but of smaller magnitude. As apparently only the fragment 1 portion of the molecule undergoes the transition, it would appear that the covalently linked fragment 1 is constrained from attaining the same conformation as the purified entity. This suggests that caution must be used in interpreting the results of metal ion binding studies using fragment 1 as an analogue for prothrombin.

Animals↗
PubMed1975

Interaction of histone f2al fragments with deoxyribonucleic acid. Circular dichroism and thermal denaturation studies.

The glycine-arginine-rich histone, f2al (IV) (102 amino acids), from calf thymus was cleaved at residue 84 with cyanogen bromide. Complexes containing homologous DNA and each f2al fragment were reconstituted by means of Gdn-HC1 gradient dialysis. The circular dichroic (CD) spectra of these complexes were all examined in 0.14 M NaC1. The CD spectra of the DNA-f2al fragment complexes did not differ appreciably from that of DNA alone in the wavelength region above 240 nm. However, intact f2al-DNA complexes yield CD spectra which differ significantly (enhanced, blue-shifted, 273-nm band) from that of native DNA (Shih and Fasman, 1971). The small C-terminal fragment (85-102) was bound weakly to DNA under the conditions used. However, the large basic N-terminal fragment (1-83) was bound as well to DNA as was whole f2al, but produced no CD distortion. The conformation of the N-terminal fragment, unlike intact f2al, was not changed upon increasing the ionic strength to 0.14 M NaF. These results complement previous studies on f2al and its N-terminal CNBr fragment (Ziccardi and Schumaker, 1973). Thermal denaturation of the complexes in 2.5 X 10(-4) M EDTA was monitored simultaneously by changes in the absorption and CD spectra. All complexes showed a thermal transition at 45 degrees (Tml), attributable to the melting of free, double-stranded DNA. In addition, f2al-DNA and N fragment-DNA complexes displayed melting phenomena at 88 and 78 degrees (Tm2), respectively, caused by the denaturation of the histone-bound DNA. This difference in Tm2 constitutes further evidence that loss of the 18-amino-acid carboxyl end segment of f2al prohibits the unique type of interaction which occurs between DNA and the intact histone.

Animals↗
PubMed2004

Nicked apomyoglobin: a noncovalent complex of two polypeptide fragments comprising the entire protein chain.

Limited proteolysis of the 153-residue chain of horse apomyoglobin (apoMb) by thermolysin results in the selective cleavage of the peptide bond Pro88-Leu89. The N-terminal (residues 1-88) and C-terminal (residues 89-153) fragments of apoMb were isolated to homogeneity and their conformational and association properties investigated in detail. Far-UV circular dichroism (CD) measurements revealed that both fragments in isolation acquire a high content of helical secondary structure, while near-UV CD indicated the absence of tertiary structure. A 1:1 mixture of the fragments leads to a tight noncovalent protein complex (1-88/89-153, nicked apoMb), characterized by secondary and tertiary structures similar to those of intact apoMb. The apoMb complex binds heme in a nativelike manner, as given by CD measurements in the Soret region. Second-derivative absorption spectra in the 250-300 nm region provided evidence that the degree of exposure of Tyr residues in the nicked species is similar to that of the intact protein at neutral pH. Also, the microenvironment of Trp residues, located in positions 7 and 14 of the 153-residue chain of the protein, is similar in both protein species, as given by fluorescence emission data. Moreover, in analogy to intact apoMb, the nicked protein binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonate (ANS). Taken together, our results indicate that the two proteolytic fragments 1-88 and 89-153 of apoMb adopt partly folded states characterized by sufficiently nativelike conformational features that promote their specific association and mutual stabilization into a nicked protein species much resembling in its structural features intact apoMb. It is suggested that the formation of a noncovalent complex upon fragment complementation can mimic the protein folding process of the entire protein chain, with the difference that the folding of the complementary fragments is an intermolecular process. In particular, this study emphasizes the importance of interactions between marginally stable elements of secondary structure in promoting the tertiary contacts of a native protein. Considering that apoMb has been extensively used as a paradigm in protein folding studies for the past few decades, the novel fragment complementing system of apoMb here described appears to be very useful for investigating the initial as well as late events in protein folding.

Animals↗
PubMed1996

Delineation of an active fragment and poly(L-proline) II conformation for candidacidal activity of bactenecin 5.

Bactenecin 5 and its fragments [BN22 (1-22), BN16 (7-22), and BC24 (20-43)] were synthesized by solid-phase methods. Their antifungal activities on Candida albicans have been studied and compared with those of the native bactenecin 5. The conformational preferences of these peptides in aqueous and nonaqueous solutions and in lipid vesicles were examined by circular dichroism. The highly active N-terminal fragment (BN16) was examined in aqueous solution using 500 MHz two-dimensional NMR. Bactenecin 5 and its fragments are potent candidacidal agents against C. albicans. The N-terminal fragments (BN22 and BN16) of bactenecin 5 are relatively more active than the C-terminal fragment BC24, especially at lower concentrations. The N-terminal region (7-22) which retains the activity of the whole molecule appears to be the functional domain for candidacidal activity. The CD spectra of bactenecin 5 and its fragments are reminiscent of the CD spectrum of poly(L-proline) type II structure in aqueous and nonaqueous solutions and also in lipid vesicles. The temperature dependence of NH chemical shifts and 1H/2H exchange effect on amide resonances suggest the absence of intramolecularly hydrogen-bonded NH groups. The coupling constant (JNH-CalphaH) values, conformational restriction offered by the Pro residues (phi = -60 degrees +/- 15 degrees), the set of medium- and short-range nuclear Overhauser effects observed for the active N-terminal fragment (BN16), and the restrained structure calculation using DIANA suggest that poly(L-proline) type II conformers of the peptide molecules could be significantly populated in aqueous solution. The ability of bactenecin peptides to induce disruption of lipid vesicles correlates well with their activity. Our results suggest that poly(L-proline) type II structure may, indeed, be the biologically active conformation for candidacidal activity of bactenecin peptides.

Amino Acid Sequence↗
PubMed1996

Factor XIIIa-catalyzed cross-linking of recombinant alpha C fragments of human fibrinogen.

Direct measurements of the structure and function of the COOH-terminal portion of the A alpha chain (residues 220-610) of human fibrinogen have been hampered by the difficulty of isolating intact fragments derived from this protease-sensitive region. Here, we overcame this problem by expressing two fragments, alpha C45K (A alpha 221-610) and a truncated version of it, alpha C30K (A alpha 368-610), in Escherichia coli. Both proteins were purified to homogeneous state, and their integrity was confirmed at protein level by sequencing. Upon treatment with factor XIIIa, the alpha C45K fragment but not the alpha C30K fragment formed polymers similar to those derived from fibrin clots. Sequence analysis of cross-linked alpha C45K polymers revealed involvement in the cross-linking reaction of at least three Gln residues (221, 237, 328) in the NH2-terminal region of the fragment and four Lys residues (539, 556, 580, 601) located in the COOH-terminal part of the molecule. In addition, a fraction of alpha C45K fragment was found in an intramolecular cross-linked form, suggesting a high level of flexibility of its polypeptide chain and consistent with the location of its donor and acceptor residues in clusters near the ends of the molecule. The alpha C30K fragment, lacking the NH2-terminal Gln residues, was not able to form polymers or internally cross-linked monomers. Thus, the C-terminal part of the A alpha chain comprises an autonomous, functionally active, and flexible region that plays a key role in alpha polymer formation and stabilization of fibrin clots by factor XIIIa.

Amino Acid Sequence↗
PubMed1996

Presence of a slow dimerization equilibrium on the thermal unfolding of the 205-316 thermolysin fragment at neutral pH.

Differential scanning calorimetry and size-exclusion chromatography have been used to characterize the dimerization and unfolding of the 205-316 C-terminal fragment of thermolysin at pH 7.5. We show that the folded fragment dimerizes at low temperature with a moderate affinity and undergoes thermal unfolding according to a N(2) <==> 2N <==> 2U model. This behavior has already been observed at acid pH, where a similar dissociation equilibrium has been found [Azuaga, A., Conejero-Lara, F., Rivas G., De Filippis, V., Fontana A., & Mateo, P. L. (1995) Biochim. Biophys. Acta 1252, 95-102]. Nevertheless, at pH 7.5 the dimerization equilibrium slows down below about 30 degrees C, with virtually no interconversion between the monomeric and the dimeric states of the fragment. We have studied the kinetics of interconversion between monomer and dimer by size-exclusion chromatography experiments and have shown that a very high energy barrier (83.8 kJ/mol at 26.5 degrees C) exists between either state. A mathematical analysis of the DSC thermograms on the basis of the proposed model has allowed us to obtain the thermodynamic characterization of the dimerization and the unfolding processes of the fragment and confirms the kinetic parameters obtained in the chromatographic experiments. The thermodynamic functions for the unfolding of the fragment are compatible with some degree of disorder in the structures of both the monomer and the dimer. According to circular dichroism measurements, the dimerization of the fragment seems to be linked to some conformational change in the subunits, most probably due to a rearrangement of the existing secondary-structure elements. This fragment displays several features already observed in folding intermediates, such as the partial disorder of the polypeptidic chain, association processes, and kinetic barriers between different regions in the conformational space.

Bacillus↗
PubMed1996

The cytoplasmic fragment of the aspartate receptor displays globally dynamic behavior.

A number of cloned soluble fragments if the bacterial chemotaxis transmembrane receptors retain partial function. Prior studies of a fragment corresponding to the cytoplasmic domain (c-fragment) of the Escherichia coli aspartate receptor have correlated the signaling state of mutant receptors with the oligomerization state of the c-fragments: equilibria of smooth-swimming mutants are shifted toward oligomeric states; tumble mutants are shifted toward monomeric states [Long, D. G., & Weis, R. M. (1992) Biochemistry 31, 9904-9911]. We have applied several experimental probes of local and global structural flexibility to two signaling states, the wild-type (monomeric) and S461L smooth mutant (predominantly dimeric) c-fragments. Featureless near-UV CD spectra are observed, which indicate that the single Trp residue is in a symmetric environment (most likely averaged by fluctuations) and suggest that the C-termini of both proteins are highly mobile. Both proteins undergo extremely rapid proteolysis and enhance ANS fluorescence, which indicates that many sites are accessible to trypsin cleavage and hydrophobic sites are accessible to ANS binding. The global nature of the flexibility is demonstrated by 1H NMR studies. Lack of chemical shift dispersion suggests that fluctuations average the environments of side chains and backbone protons. Rapid exchange of 99% of the observable amide protons suggests that these fluctuations give high solvent accessibility to nearly the entire backbone. This evidence indicates that both monomeric and dimeric c-fragments are globally flexible proteins, with properties similar to "molten-globule" states. The significance of this flexibility depends on whether it is retained in functioning receptors: the c-fragment structure may lack important tertiary contacts, protein-protein interactions, or topological constraints needed to stabilize a nondynamic native structure, or the cytoplasmic domain of the native receptor may retain flexibility which may be modulated in the mechanism of transmembrane signaling.

Anilino Naphthalenesulfonates↗
PubMed1998

Specific cross-links between fragments of proteolyzed Na,K-ATPase induced by o-phthalaldehyde.

We have used o-phthalaldehyde (OPA) to cross-link adjacent fragments of "19 kDa membranes", a tryptic preparation of Na,K-ATPase lacking the ATP site but retaining cation occlusion sites. Treatment with OPA of "19 kDa membranes" or detergent-solubilized membranes containing occluded Rb ions [Or, E., Goldshleger, R., Tal, D. M., and Karlish, S. J. D. (1996) Biochemistry 35, 6853-6864] yielded cross-linked products of 25 and 31 kDa. Both species contained the 19 kDa fragment of the alpha subunit (transmembrane segments M7-M10). In addition, the 25 kDa product contained the fragment including M5-M6, while the 31 kDa product contained a 16 kDa fragment of the beta subunit. Cross-linking was unaffected by the absence or presence of ligands (Na, Rb, or Mg and ouabain). Cross-linking was largely abolished in thermally inactivated "19 kDa membranes". When proteolytic digestion of the 25 and 31 kDa products was combined with antibody binding, PKA-dependent phosphorylation, and sequencing of fragments, approximate positions of the cross-links were established. In the 25 kDa product, the cross-link was located within the short cytoplasmic segment Asn831-Arg841 of the 19 kDa fragment preceding M7 and within Ala749-Ala770 preceding M5. Thus, M7 and M5 are likely to be in close proximity. In the 31 kDa product, the cross-link was located in the extracellular loop of the alpha subunit between M7 and M8, close to residues which are known to interact with the beta subunit. Functional implications of the interactions between the fragments of the alpha (M5-M6 and M7-M10) and beta subunits are discussed.

Animals↗
PubMed1999

Different equilibrium stability behavior of ScFv fragments: identification, classification, and improvement by protein engineering.

A classification of scFv fragments concerning their unfolding/refolding equilibria is proposed. It is based on the analysis of different mutants of the levan-binding A48 scFv fragment and the HER-2 binding 4D5 scFv fragment as well as a "hybrid" scFv carrying the VL domain of 4D5 and the VH domain of an A48 mutant. The denaturant-induced unfolding curves of the corresponding scFv fragments were measured and, if necessary for the classification, compared with the denaturation of the isolated domains. Depending on the relative intrinsic stabilities of the domains and the stability of the interface, the different scFv fragments were grouped into different classes. We also demonstrate with several examples how such a classification can be used to improve the stability of a given scFv fragment, by concentrating engineering efforts on the "weak part" of the particular molecule, which may either be the intrinsic stability of VL, of VH, or the stability of the interface. One of the scFv fragments obtained by this kind of approach is extremely stable, starting denaturation only at about 7 M urea. We believe that such extremely stable frameworks may be very suitable recipients in CDR grafting experiments. In addition, the thermodynamic equilibrium stabilities of seven related A48 scFv mutants covering a broad range of stabilities in urea unfolding were shown to be well correlated with thermal aggregation properties measured by light scattering and analytical gel filtration.

Chromatography, Gel↗
PubMed2006

Solution structures of human LL-37 fragments and NMR-based identification of a minimal membrane-targeting antimicrobial and anticancer region.

To understand the structure and activity relationship of human LL-37, a series of peptide fragments was designed. The N-terminal fragment, LL-37(1-12), was not active, while the C-terminal fragment, LL-37(13-37), killed Escherichia coli, as well as drug-sensitive and drug-resistant cancer cells. A 13-residue core antibacterial and anticancer peptide, corresponding to residues 17-29 of LL-37, was identified based on total correlated spectroscopy by trimming nonessential regions (TOCSY-trim). Because LL-37 acts on bacterial membranes, three-dimensional structures of its fragments were determined in micelles by NMR, including structural refinement by natural abundance 15N and 13C chemical shifts. Aromatic-aromatic interactions in the N-terminal fragment were proposed to be essential for LL-37 aggregation. The LL-37 core peptide adopts a similar structure in the micelles of SDS or dioctanoyl phosphatidylglycerol. This structure is retained in the C-terminal fragment LL-37(13-37) and very likely in intact LL-37 based on peptide-aided signal assignments. The higher antibacterial activity of the LL-37 core peptide than aurein 1.2 was attributed to additional cationic residues. To achieve selective membrane targeting, D-amino acids were incorporated into LL-37(17-32). While the D-peptide showed similar antibacterial activity to the L-diastereomer, it lost toxicity to human cells. Structural analysis revealed hydrophobic defects in the new amphipathic structure of the D-peptide, leading to a much shorter retention time on a reversed-phase HPLC column. It is proposed that hydrophobic defects as a result of incoherent hydrophobic packing provide a structural basis for the improvement in cell selectivity of the LL-37 fragment.

Amino Acid Sequence↗