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Heme binding by the N-terminal fragment 1-44 of human growth hormone
Full text linkFragment 1-44 of human growth hormone (hGH), prepared in vitro by limited proteolysis of the hormone with pepsin at low pH, encompasses in full the N-terminal helix of this four-helix bundle protein [Spolaore, B., Polverino de Laureto, P., Zambonin, M., and Fontana, A. (2004) Biochemistry 40, 9460-9468]. Here, we report the new and interesting observation that fragment 1-44 can bind heme. The binding property is specific for the N-terminal helix of hGH, since heme binding does not occur with fragment 45-191 or the entire protein. The spectral characteristics of Fe-protoporphyrin IX are those of a low-spin, hexacoordinated iron ligated by two imidazole rings of His residues or His and Met residues. Far-UV circular dichroism (CD) measurements revealed that fragment 1-44 acquires a helical secondary structure upon heme binding. Heme appears to be bound to the fragment in a stereospecific way, since an induced dichroic signal is observed in the Soret region of the CD spectrum. The heme-fragment complex occurs in a 1:1 molar ratio, as determined by spectrophotometric titration, as well as by electrospray-ionization mass spectrometric analysis of the complex. The fragment alone is much more susceptible to tryptic digestion than the heme complex, implying a more folded and rigid structure of this last species. It is proposed that the molecular features of fragment 1-44 determining its heme-binding property reside in the amphipathic character of the helix adopted by the fragment, as well as in the presence in its polypeptide chain of His18, His21, and Met14. These residues can act as specific ligands for the heme-iron, as observed with cytochromes
Analysis of the partly folded state of alpha-lactalbumin bound to membranes and identification of the regions of the polypeptide chain involved in this interaction.
No full textPurification and characterisation of a novel rhamnose-binding lectin from the compound ascidian Botryllus schlosseri.
No full textStructural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins
No full textThe human KRAS proto-oncogene contains a critical nuclease hypersensitive element (NHE) upstream of the major transcription initiation site. In this article, we demonstrate by primer-extension experiments, PAGE, chemical footprinting, CD, UV and FRET experiments that the G-rich strand of NHE (32R) folds into intra-molecular G-quadruplex structures. Fluorescence data show that 32R in 100 mM KCl melts with a biphasic profile, showing the formation of two distinct G-quadruplexes with T(m) of approximately 55 degrees C (Q(1)) and approximately 72 degrees C (Q(2)). DMS-footprinting and CD suggest that Q(1) can be a parallel and Q(2) a mixed parallel/antiparallel G-quadruplex. When dsNHE (32R hybridized to its complementary) is incubated with a nuclear extract from Panc-1 cells, three DNA-protein complexes are observed by EMSA. The complex of slower mobility is competed by quadruplex 32R, but not by mutant oligonucleotides, which cannot form a quadruplex structure. Using paramagnetic beads coupled with 32R, we pulled down from the Panc-1 extract proteins with affinity for quadruplex 32R. One of these is the heterogeneous nuclear ribonucleoprotein A1, which was previously reported to unfold quadruplex DNA. Our study suggests a role of quadruplex DNA in KRAS transcription and provides the basis for the rationale design of molecular strategies to inhibit the expression of KRAS
Site-specific derivatization of proteins using metal ion chelating peptides and transglutaminase
No full textLimited proteolysis of human growth hormone at low pH: isolation, characterization, and complementation of the two biologically relevant fragments 1-44 and 45-191
No full textThe limited proteolysis approach was used to analyze the conformational features of human growth hormone (hGH) under acidic solvent conditions (A-state). Pepsin was used as the proteolytic probe because of its poor substrate specificity and its activity at low pH. Limited proteolysis of hGH in its A-state results in a selective cleavage of the Phe44-Leu45 peptide bond, leading to the production of fragments 1-44 and 45-191. The two fragments were isolated in homogeneous form for studying their conformational properties by means of spectroscopic methods. Fragment 1-44 was shown to retain little secondary and tertiary structure at neutral pH, while fragment 45-191 independently folds into a highly helical secondary structure. In particular, we have shown that the two peptic fragments are able to associate into a stable and native-like hGH complex 1-44/45-191. Our proteolysis data indicate that in acid solution hGH adopts a partly folded state characterized by a local unfolding of the first minihelix (residues 38-47) encompassing the Phe44-Leu45 peptide bond. Of interest, hGH has both insulin-like and diabetogenic effects. Two fragments of hGH occur in vivo and exert these two opposite activities, namely, fragment 1-43 showing an insulin-potentiating effect and fragment 44-191 showing a diabetogenic activity. The results of this study suggest that the conformational changes of hGH induced by an acidic pH promote the generation of the two physiologically relevant fragments by proteolytic processing of the hormone. Although pepsin cannot be the enzyme responsible for the in vivo processing of the hormone, we propose that limited proteolysis of hGH at low pH is physiologically relevant, since the hormone is exposed to an acidic environment in the cell. This study reports for the first time the analysis of the conformational features of the two individual functional domains of hGH and of their complex
Identification of the major allergenic proteins and their chemical and physico-chemical modifications in a food matrix by SDS-PAGE and LC-MS.
No full textProtein interactions leading to conformational changes monitored by limited proteolysis: apo form and fragments of horse cytochrome c
No full textProteolysis experiments have been used to monitor the conformational transitions from an unfolded to a folded state occurring when the apo form of horse cytochrome c (cyt c) binds the heme moiety or when two fragments of cyt c form a native-like 1:1 complex. Proteinase K was used as a proteolytic probe, in view of the fact that the broad substrate specificity of this protease allows digestion at many sites along a polypeptide chain. The rather unfolded apo form of cyt c binds heme with a concomitant conformational transition to a folded species characterized by an enhanced content of helical secondary structure. While the holoprotein is fully resistant to proteolytic digestion and the apoprotein is digested to small peptides, the noncovalent complex of the apoprotein and heme exhibits an intermediate resistance to proteolysis, in agreement with the fact that the more folded structure of the complex makes the protein substrate more resistant to proteolysis. The noncovalent native-like complex of the two fragments 1-56 and 57-104 of cyt c, covering the entire polypeptide chain of 104 residues of the protein, is rather resistant to proteolysis, while the individual fragments are easily digested. Fragment 57-104 is fast degraded to several peptides, while fragment 1-56 is slowly degraded stepwise from its C-terminal end, leading initially mostly to fragments 1-48 and 1-40 and, at later stages of proteolysis, fragments 1-38, 1-35, 1-33, and 1-31. Thus, proteolysis data indicate that the heme containing fragment 1-56 has a rather compact core and a C-terminal flexible tail. Upon prolonged incubation of the complex of fragments 1-56 and 57-104 (nicked cyt c) with proteinase K, a chain segment is removed from the nicked protein, leading to a gapped protein complex of fragments of 1-48 and 57-104 and, on further digestion, fragments 1-40 and 57-104. Of interest, the chain segment being removed by proteolysis of the complex matches the omega-loop which is evolutionarily removed in cyt c of microbial origin. Overall, rates and/or resistance to proteolysis correlates well with the extent of folding of the protein substrates, as deduced from circular dichroism measurements. Thus, our results underscore the utility of proteolytic probes for analyzing conformational and dynamic features of proteins. Finally, a specific interest of the cyt c fragment system herewith investigated resides in the fact that the fragments are exactly the exon products of the cyt c gene
Site-specific protein conjugation with the [185/187Re(N)(PNP)]-scaffold via Transglutaminase
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