In a previous paper, a hypothesis for protein folding was proposed in which the native structure is formed by a three-step mechanism: (A) formation of ordered backbone structures by short-range interactions, (B) formation of small contact regions by medium-range interactions, and (C) association of the small contact regions into the native structure by long-range interactions. In this paper the empirical interaction parameters, used as a measure of the medium- and long-range interactions (the standard free energy, deltaGdegrees k,l, of formation of a contact between amino acids of species k and l) that include the role of the solvent (water) and determine the conformation of a protein in steps B and C, are evaluated from the frequency of contacts in the x-ray structures of native proteins. The numerical values of deltaG degrees k,l for all possible pairs of the 20 naturally occurring amino acids are presented. Contacts between highly nonpolar side chains of amino acids such as Ile, Phe, Trp, and Leu are shown quantitatively to be stable. On the contrary, contacts involving polar side chains of amino acids such as Ser, Asp, Lys, and Glu are significantly less stable. While this implies, in a quantitative manner, that it is generally more favorable for nonpolar groups to lie in the interior of the protein molecule and for the polar side chains to be exposed to the solvent (water) rather than to form contacts with other amino acids, many exceptions to this generalization are observed.
A hypothesis for protein folding is proposed, in which the native structure is formed by a three-step mechanism: (A) formation of ordered backbone structures by short-range interactions, (B) formation of small contact regions by medium-range interactions, and (C) association of the small contact regions into the native structure by longrange interactions. Empirical interaction parameters (free energy of formation of a contact) between amino-acid residues were evaluated from the frequency of contacts in the x-ray structures of native proteins. On the basis of this mechanism, a Monte Carlo simulation of protein folding (with an accompanying decrease in the total contact free energy) was carried out for bovine pancreatic trypsin inhibitor. The predicted three-dimensional structure is in fairly good agreement with the experimental one. In order to predict the three-dimensional structure of a protein, it is necessary to circumvent the multiple-minimum problem and overcome the difficulties of treating the longrange interactions (1). For this purpose, a starting conformation was obtained from empirical prediction algorithms, and then its total conformational energy was minimized (2). As an alternative approach, the conformations of the unfolded protein can be obtained by statistical mechanical procedures involving only short-range interactions,t and altered (to a minimum-free-energy structure) by a Monte Carlo procedure involving short-, medium-, and long-range interactions. This paper describes a model for protein folding, and the results obtained for bovine pancreatic trypsin inhibitor (BPTI) by a Monte Carlo simulation of the model. A full description of the procedure and results will be published elsewhere. § I. Hypothetical mechanism of protein folding The folding of a polypeptide chain to the native structure of a protein in a given medium is assumed to occur in three steps (which may proceed simultaneously). (A) Because of short-range interactions, orderedl backbone structures, such as a-helical, extended, and chain-reversal conformations, are formed in a system at equilibrium under given physical conditions. (B) When these physical conditions are changed, so as to introduce medium-range interactions, the equilibrium is shifted, and small contact regions (defined in section II) are nucleated among the residues both in the ordered and in the unordered structures. In this step, the ordered backbone structures formed in step A may be rearranged to some exAbbreviation: BPTI, bovine pancreatic trypsin inhibitor. * From Kyoto University, 1972University, -1975 Infinite repulsion arises (excluded volume effect), when rAB < rA(w) + r-(w).[2][3]
A three-step mechanism of protein folding, proposed in our previous paper, is applied here to postulate the nature of the intermediates in the folding of rubredoxin, ferricytochrome c, and lysozyme. Contact maps are calculated for these three proteins, and it is shown that they contain much information (such as the polarity of residues in contact regions) about the structure of the native protein. Elementary processes are described for the formation of contact regions. Based on these concepts, details of the pathways of folding these three proteins from the unfolded to the native structure are postulated, focusing on the formation of ordered backbone structures (such as alpha-helical, extended, and chain-reversal conformations) in step A of the three-step mechanism and on the formation of contact regions in response to medium-and long-range interactions in steps B and C. It was found that chain reversals can often play an important role in forming contact regions in step A (short-range interactions) and in step B (medium-range interactions) but not in step C.
Colibactin is a polyketide-nonribosomal peptide hybrid secondary metabolite that can form interstrand cross-links in double-stranded DNA. Colibactin-producing Escherichia coli has also been linked to colorectal oncogenesis. Thus, there is a strong interest in understanding the role colibactin may play in oncogenesis. Here, using the high-colibactin-producing wild-type E. coli strain we isolated from a clinical sample with the activitybased fluorescent probe we developed earlier, we were able to identify colibactin 770, which was recently identified and proposed as the complete form of colibactin, along with colibactin 788, 406, 416, 420, and 430 derived from colibactin 770 through structural rearrangements and solvolysis. Furthermore, we were able to trap the degrading mature colibactin species by converting the diketone moiety into quinoxaline in situ in the crude culture extract to form colibactin 860 at milligram scale. This allowed us to determine the stereochemically complex structure of the rearranged form of an intact colibactin, colibactin 788, in detail. Furthermore, our study suggested that we were capturing only a few percent of the actual colibactin produced by the microbe, providing a crude quantitative insight into the inherent instability of this compound. Through the structural assignment of colibactins and their degradative products by the combination of LC-HRMS and NMR spectroscopies, we were able to elucidate further the fate of inherently unstable colibactin, which could help acquire a more complete picture of colibactin metabolism and identify key DNA adducts and biomarkers for diagnosing colorectal cancer.
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