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J Kemmink

Publications and source records attributed to J Kemmink.

27 records · Page 2Linked to original sources
PubMed1993

Local conformations of peptides representing the entire sequence of bovine pancreatic trypsin inhibitor and their roles in folding.

The conformational properties of seven overlapping peptides, 9 to 16 residues long, that comprise the entire primary structure of bovine pancreatic trypsin inhibitor (BPTI) have been characterized by circular dichroism and 1H nuclear magnetic resonance. The peptides are largely disordered, although apparently with somewhat different average conformational propensities of the polypeptide backbone, similar to those indicated by methods to predict secondary structure. Reduced BPTI appears to be approximately the sum of the individual peptides. Several local interactions involving aromatic rings of side-chains interacting with groups nearby in the primary structure have been identified and verified by replacing the responsible side-chains. The roles of these interactions in folding of reduced BPTI could be determined, as the conformational and nuclear magnetic resonance properties of all the major disulphide intermediates are known. Two of these local interactions contribute to the folding process and to stability of the fully folded conformation, whereas the other two do not.

Amino Acid Sequence↗
PubMed1993

On the biosynthesis of bovine pancreatic trypsin inhibitor (BPTI). Structure, processing, folding and disulphide bond formation of the precursor in vitro and in microsomes.

The natural gene for bovine pancreatic trypsin inhibitor (BPTI) was expressed by in vitro transcription/translation systems as the 100-residue pre-proBPTI, with a signal peptide for translocation into the endoplasmic reticulum. Expression in the presence of microsomes defined the site of co-translational cleavage of the signal peptide. The resulting proBPTI in the microsomes consists of the 58 residues of mature BPTI, plus an additional 13 residues at the N terminus, including a cysteine residue at position -10, and seven residues at the C terminus. ProBPTI remained in the unfolded, reduced form within microsomes when synthesized under reducing conditions, but folded and formed disulphide bonds rapidly when the disulphide form of glutathione was added. Complete folding could occur within about one minute, even when residue Cys10 was replaced by Ser. The structure of proBPTI was determined by circular dichroism and two-dimensional NMR and found to be that of mature BPTI with flexible extensions on both termini. Its inhibition of the activity of alpha-chymotrypsin was indistinguishable from that of the mature protein. The extensions of the precursor appeared to play only very minor roles in refolding in vitro under conditions where folding and disulphide bond formation are coupled. Under pH and redox conditions thought to reflect those in vivo, complete folding and disulphide bond formation required several hours. Addition of protein disulphide isomerase to in vitro folding experiments caused substantial and similar increases in the rate of formation of the fully folded state for both mature BPTI and proBPTI; the half time for folding to the native state was reduced to approximately two minutes, which is comparable to that occurring in microsomes. The absence of substantial effects of the N and C-terminal extensions on the protein structure, inhibitor activity and refolding leaves their functional roles to be discovered.

Amino Acid Sequence↗
PubMed1993

Local structure due to an aromatic-amide interaction observed by 1H-nuclear magnetic resonance spectroscopy in peptides related to the N terminus of bovine pancreatic trypsin inhibitor.

A synthetic peptide corresponding to the 15 N-terminal residues of bovine pancreatic trypsin inhibitor, with serine replacing the two cysteine residues, has been characterized by 1H-nuclear magnetic resonance spectroscopy. This peptide has a very disordered conformation that is essentially the same when it is part of the analogue of the (30-51) one-disulphide intermediate in folding. This confirms the conclusion of a previous paper, that the (30-51) intermediate is partially folded, with the N-terminal segment disordered. Local elements of non-random conformation were observed in the peptide. Especially prominent was an apparently electrostatic interaction between the face of the aromatic ring of Tyr10 and the amide group of Gly12, which caused the latter to have a very anomalous chemical shift. A similar interaction was observed in shorter peptides, especially in tetrapeptides with the sequences Tyr/Phe-X-Gly-Y. The local nature of this interaction indicates that it should be a general feature in peptides and in unfolded proteins with such a sequence.

Amides↗
PubMed1988

Photoreactivation of the thymine dimer containing DNA octamer d(GCGTTGCG).d(CGCAACGC) by the photoreactivating enzyme from Anacystis nidulans.

Irradiation of the double-stranded octamer d(GCGTTGCG).d(CGCAACGC) with UV light causes dimerization of the two central thymine residues. Proton NMR data reveal that this photodimer has the same chemical structure as the photodimer, which is formed upon UV irradiation of the single strand d(GCGTTGCG), a cis-syn-cyclobutane-type thymine dimer. Irradiation of the purified thymine dimer double-stranded octamer d(GCGTTGCG).d(CGCAACGC) with visible light in the presence of photoreactivating enzyme isolated from Anacystis nidulans leads to an increase in absorbance at 260 nm, which is characteristic for the repair of thymine dimers. The NMR spectrum recorded after the photoreactivating treatment indeed shows that a complete conversion to the starting octamer has occurred.

Cyanobacteria↗
PubMed1987

1H NMR study of the exchangeable protons of the duplex d(GCGTTGCG).d(CGCAACGC) containing a thymine photodimer.

A comparison is presented of the imino proton NMR spectra of the double stranded octamer d(GCGTTGCG).d(CGCAACGC) and the same octamer in which the two central thymine residues occur as a cis-syn thymine dimer. Except for the terminal base pairs all imino protons were detected and assigned in the NMR spectrum. The spectra show that in the thymine dimer duplex, contrary to common belief, all base pairs occur in a hydrogen bonded form, although the hydrogen bonds of the two central AT base pairs are substantially weakened. The melting temperature decreases about 13 degrees C on thymine dimer formation.

Imines↗
PubMed1987

Conformational changes in the oligonucleotide duplex d(GCGTTGCG) x d(CGCAACGC) induced by formation of a cis-syn thymine dimer. A two-dimensional NMR study.

In order to obtain insight into the repair mechanism of DNA containing thymine photo-dimer, the conformation of the duplex d(GCGTTGCG) x d(CGCAACGC) with a thymine dimer incorporated has been studied by proton NMR and the results are compared with NMR data of the parent octamer. Two-dimensional nuclear Overhauser enhancement (2D NOE) spectroscopy and two-dimensional homonuclear Hartmann-Hahn spectroscopy have been applied to assign all the non-exchangeable base protons and most of the deoxyribose protons of both duplexes. From these experiments it is clear that indeed a cis-syn cyclobutane-type thymine photodimer is formed by the irradiation of this oligonucleotide with ultraviolet light. Comparison of 2D NOE spectra and the 1H chemical shifts of the damaged and the intact DNA duplexes reveals that formation of a thymine dimer induces small distortions of the B-DNA structure, the main conformational change occurring at the site of the thymine dimer.

Binding Sites↗
PubMed1987

Two-dimensional 1H NMR study of two cyclobutane type photodimers of thymidylyl-(3'----5')-thymidine.

2D NMR spectroscopy and J coupling constant analysis are applied to resolve the structure of two photoproducts of thymidylyl-(3'----5')-thymidine. These products are cyclobutane type thymine dimers possessing the cis-syn (the predominant one) and trans-syn geometry. The cis-syn is formed in an ANTI-ANTI conformation about the N-glycosyl linkages and resembles the normal base-stacked configuration. The glycosidic conformation in solution of the 5' terminal fragment differs from the crystal in which the less common SYN conformation is observed. In this isomer only the sugar pucker of the 3' terminal fragment is changed substantially with respect to the dinucleotide. The trans-syn isomer is formed in a SYN-ANTI glycosidic conformation. In this isomer the sugar puckers of both deoxyribose rings are affected and a preference for a pure 2'-endo conformation is observed.

Cyclobutanes↗