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Starting from the simple case of an external field acting on noninteracting particles, a formulation of the self-consistent field theory for treating proteins and unfolded protein chains with multiple interacting titratable groups is given. Electrostatic interactions between the titratable groups are approximated by a Debye-Huckel expression. Amino acid residues are treated as polarizable bodies with a single dielectric constant. Dielectric properties of protein molecules are described in terms of local dielectric constants determined by the space distribution of residue volume density around each ionized residue. Calculations are based on average charges of titratable groups, distance of separation between them, on their pKa's, residue volumes and on the local dielectric constant. A set of different residue volumes is used to analyze the influence of the permanent dipole of polar parts of the residue on calculated titration curves, electrostatic contribution to the free energy of protein stability, and pK shifts. Calculations with zero volumes--which means that charged portions of protein molecules are viewed as part of the high dielectric medium--give good agreement with experimental data. The theory was tested against most accurate approaches currently available for the calculation of the pKa's of ionizable groups based upon finite difference solutions of the Poisson-Boltzmann equation (FDPB). For 70 theoretically calculated pKa's in a total of six proteins the accuracy of the approach presented here is assessed by comparison of computed pKa's with that measured. The overall root-mean-square error is 0.79, compared to the value 0.89 obtained by FDPB approach given in the paper of Antosiewicz et al. (J. Mol. Biol. 238:415-436, 1994). The test of Debye-Huckel approximation for the electrostatic pair interactions shows that it is in excellent agreement with experimental data as well as the calculations of the FDPB and Tanford-Kirkwood methods on the pK shifts of His64 in the active site of subtilisin over the whole range of ionic strengths. (Gilson and Honig, Proteins 3:32-52, 1988; Russell et al., J.Mol.Biol. 193:803-813, 1987). The theory was also analytically and numerically tested on a simple models where the exact statistical mechanical treatment is still simple (Yang et al., Proteins 15:252-265, 1993; Bashford and karplus, J. Phys. Chem. 95:9556-9561, 1991).
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PMID:Self-consistent field approach to protein structure and stability. I: pH dependence of electrostatic contribution. 914 Nov 37

Three accessible disulphide bonds of basic trypsin-subtilisin inhibitor from marine turtle eggwhite have been reduced with 0.1M NaBH4 at 0 degree C under nitrogen atmosphere at pH9.8 and then S-carboxymethylated. The partially reduced inhibitor retains 80% of the native inhibitory activity towards trypsin and subtilisin. The S-carboxymethylated inhibitor undergoes slower refolding than the native inhibitor from its fully denatured and reduced state at pH 8.5 in the presence of oxidised and reduced glutathione. The refolding process was characterised by the attainment of the inhibitory activity towards trypsin and subtilisin. The values of the second order rate constant for the refolding reactions of the modified protein are 0.02 x 10(2)M-1sec1 and 0.033 x 10(2)M-1sec-1 for its trypsin and subtilisin inhibiting domains and their energies of activation are 20.1 Kcal/mole and 24.6 Kcal/mole. The partially modified inhibitor does not regain complete inhibitory activity even after long incubation in the oxido-shuffling buffer. From the above findings it can be concluded that the three disulphide bonds of the native inhibitor are not essential for the inhibitory activity of the trypsin-subtilisin inhibitor but they help in the correct refolding of the inhibitor by forming transient disulphide bonds with the external disulphide reagents as well as with the internal sulphydryl groups.
Biochem Mol Biol Int 1997 May
PMID:Refolding kinetics of partially reduced and S-carboxymethylated trypsin-subtilisin inhibitor from marine turtle eggwhite. 916 1

We introduce a method for finding weak structural similarities in a set of protein structures. Proteins are considered at their secondary structure level. The method uses a rigorous graph-theoretical algorithm which finds all structural similarities. Protein structures are modelled as undirected labelled graphs, the so-called protein graphs. We suggest that for detecting the similarities between two protein structures it is sufficient to find similarities in the protein core which consists of tightly packed secondary structure elements. Therefore, we can restrict ourselves to solving the maximal common connected subgraph problem instead of the maximal common subgraph problem. We have modified the algorithm by Bron and Kerbosch for solving that problem. The speed of the algorithm increases drastically. After calculating all maximal common connected substructures for all pairwise comparisons in a set of protein graphs the common substructure in all proteins can be calculated by intersecting them. In this paper we characterize the method briefly and explain the modelling of the protein structure in detail. For the pairwise alignment the similarity of porin (1OMF) with bacteriochlorophyll a (3BCL) and BirA protein (1BIB) with DNA polymerase III (2POL) will be discussed. In the case of the multiple structure alignment the similarity in variants of four phosphatases and in subtilisin Carlsberg, carboxypeptidase, elongation factor Tu, and flavodoxin will be represented. Our first experiments show that the method works correctly and fast. The method can be used for arbitrary graphs. Thus, different graph-theoretical models of protein structures can be examined.
Proc Int Conf Intell Syst Mol Biol 1997
PMID:Detection of distant structural similarities in a set of proteins using a fast graph-based method. 932 32

The temperature induced unfolding/dissociation of the dimeric subtilisin inhibitor from Streptomyces and its mutant D83C having an S-S crosslink between the subunits has been studied calorimetrically. Comparison of the entropies measured at different concentrations of dimer showed that the entropy cost of crosslinking is small. Its value at the standard concentration of 1 M is of the order of -(5+/-4) cal/K.mol, i.e. it is more than one order of magnitude smaller than the values of translational entropies calculated on the base of statistical thermodynamics, using in particular the Sackur-Tetrode equation, and is close to the cratic entropy value suggested by classical mixing theory.
J Mol Biol 1997 Nov 14
PMID:The entropy cost of protein association. 936 90

The propeptide of subtilisin BPN', located between a signal peptide and the mature region of the protease, is known to exhibit inhibitory activity toward subtilisin BPN', in addition to its activity as an intramolecular chaperone that facilitates folding of subtilisin BPN'. Another unique feature is that although the isolated propeptide is in a random-coil state, it forms a defined tertiary structure when it is bound to subtilisin BPN'. In this study, amino acid replacements likely to increase the hydrophobicity of the propeptide have been introduced so that the isolated propeptide forms a defined tertiary structure. By successive replacements of Ala47 by Phe, Gly13 by Ile and Val65 by Ile, the propeptide was found to form a tertiary structure in addition to an increase in its secondary structure content, which were identified by circular dichoism spectra measurements. Concurrently, the propeptide, which is a temporary inhibitor in its wild-type form, became resistant to proteolytic digestion by subtilisin BPN'. These results show not only the close relationship between tertiary structure formation in the propeptide and its function as a protease inhibitor but also the ability of a random-coil protein to form a tertiary structure after a limited number of well-designed amino acid replacements.
J Mol Biol 1998 Apr 17
PMID:Tertiary structure formation in the propeptide of subtilisin BPN' by successive amino acid replacements and its close relation to function. 957 Oct 18

The recent cloning and sequencing of several insect vitellogenins (Vg), the major yolk protein precursor of most oviparous animals, and the mosquito Vg receptor (VgR) has brought the study of insect vitellogenesis to a new plane. Insect Vgs are homologous to nematode and vertebrate Vgs. All but one of the insect Vgs for which we know the primary structure are cleaved into two subunits at a site [(R/K)X(R/K)R or RXXR with an adjacent beta-turn] recognized by subtilisin-like proprotein convertases. In four of the Vgs, the cleavage site is near the N-terminus, but in one insect species, it is near the C-terminus of the Vg precursor. Multiple alignments of these Vg sequences indicate that the variation in cleavage location has not arisen through exon shuffling, but through local modifications of the amino acid sequences. A wasp Vg precursor is not cleaved, apparently because the sequence at the presumed ancestral cleavage site has been mutated from RXRR to LYRR and is no longer recognized by convertases. Some insect Vgs contain polyserine domains which are reminiscent of, but not homologous to, the phosvitin domain in vertebrate Vgs. The sequence of the mosquito VgR revealed that it is a member of the low-density lipoprotein receptor (LDLR) family. Though resembling chicken and frog VgRs, which are also members of the LDLR family, it is twice as big, carrying two clusters of cysteine-rich complement-type (Class A) repeats (implicated in ligand-binding) instead of one like vertebrate VgRs and LDLRs. It is very similar in sequence and domain arrangement to the Drosophila yolk protein receptor (YPR), despite a non-vitellogenin ligand for the latter. Though vertebrate VgRs, insect VgR/YPRs, and LDLR-related proteins/megalins all accommodate one cluster of eight Class A repeats, fingerprint analysis of the repeats in these clusters indicate they are not directly homologous with one another, but have undergone differing histories of duplications, deletions, and exon shuffling so that their apparent similarity is superficial. The so-called epidermal growth factor precursor region contains two types of motifs (cysteine-rich Class B repeats and YWXD repeats) which occur independently of one another in diverse proteins, and are often involved in protein-protein interactions, suggesting that they potentially are involved in dimerization of VgRs and other LDLR-family proteins. Like the LDLR, but unlike vertebrate VgRs and the Drosophila YPR, the mosquito VgR contains a putative O-linked sugar region on the extra-cellular side of the transmembrane domain. Its function is unclear, but may protect the receptor from membrane-bound proteases. The cytoplasmic tail of insect VgR/YPRs contains a di-leucine (or leucine-isoleucine) internalization signal, unlike the tight-turn tyrosine motif of other LDLR-family proteins. The importance of understanding the details of yolk protein uptake by oocytes lies in its potential for exploitation in novel insect control strategies, and the molecular characterization of the proteins involved has made the development of such strategies a realistic possibility.
Insect Biochem Mol Biol
PMID:Molecular characteristics of insect vitellogenins and vitellogenin receptors. 969 32

The thermodynamics and kinetics of actin interaction with Arabidopsis thaliana actin-depolymerizing factor (ADF)1, human ADF, and S6D mutant ADF1 protein mimicking phosphorylated (inactive) ADF are examined comparatively. ADFs interact with ADP.G-actin in rapid equilibrium (k+ = 155 microM-1.s-1 and k- = 16 s-1 at 4 degreesC under physiological ionic conditions). The kinetics of interaction of plant and human ADFs with F-actin are slower and exhibit kinetic cooperativity, consistent with a scheme in which the initial binding of ADF to two adjacent subunits of the filament nucleates a structural change that propagates along the filament, allowing faster binding of ADF in a "zipper" mode. ADF binds in a non-cooperative faster process to gelsolin-capped filaments or to subtilisin-cleaved F-actin, which are structurally different from standard filaments (Orlova, A., Prochniewicz, E., and Egelman, E. H. (1995) J. Mol. Biol. 245, 598-607). In contrast, the binding of phalloidin to F-actin cooperatively inhibits its interaction with ADF. The ADF-facilitated nucleation of ADP.actin self-assembly indicates that ADF stabilizes lateral interactions in the filament. Plant and human ADFs cause only partial depolymerization of F-actin at pH 8, consistent with identical functions in enhancing F-actin dynamics. Phosphorylation does not affect ADF activity per se, but decreases its affinity for actin by 20-fold.
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PMID:Kinetic analysis of the interaction of actin-depolymerizing factor (ADF)/cofilin with G- and F-actins. Comparison of plant and human ADFs and effect of phosphorylation. 969 36

The recombinational hot spot chi modulates the nuclease and helicase activities of the RecBCD enzyme, leading to generation of an early DNA intermediate for homologous recombination. Here we identify the subunit location of the nuclease active site in RecBCD. The isolated RecB protein cleaves circular single-stranded M13 phage DNA, but RecB1-929, comprising only the 100 kDa N-terminal domain of RecB, does not. We reported previously that the reconstituted RecB1-929CD enzyme also is not a nuclease, suggesting that the C-terminal 30 kDa domain of RecB is a non-specific ssDNA endonuclease. However, we were unable to detect nuclease activity with the subtilisin-generated C-terminal 30 kDa fragment of RecB. Since the subtilisin-generated fragment did not bind to a ssDNA-agarose column, we designed a chimeric enzyme by attaching the C-terminal 30 kDa domain of RecB to the gene 32 protein of T4 phage, a ssDNA binding protein that does not have strand scission ability. In addition, Asp427 in the chimeric enzyme (Asp1080 in RecB), a residue that is conserved among several RecB homologs, was substituted to alanine (the D427A mutant). The wild-type chimeric enzyme cleaves the M13 DNA and the D427A mutation abolishes the endonuclease activity of the chimeric enzyme but does not affect its DNA binding ability. This finding indicates an unusual bipartite nature in the structural organization of RecB, in which the DNA-binding function is located in the N-terminal 100 kDa domain and the nuclease catalytic domain is located in the C-terminal 30 kDa domain. The purified RecBD1080ACD mutant is a processive helicase but not a nuclease, demonstrating that RecBCD has a single nuclease active site in the C-terminal 30 kDa domain of RecB.
J Mol Biol 1998 Nov 06
PMID:Identification of the nuclease active site in the multifunctional RecBCD enzyme by creation of a chimeric enzyme. 979 Aug 41

PC2 and PC3 are neuroendocrine specific members of the eukaryotic subtilisin-like proprotein convertase (PC) family. Both are sorted via the regulated secretory pathway into secretory granules. In order to identify sequences in PC2 which are involved in targeting to the regulated secretory pathway we expressed a series of PC2 cDNAs containing mutations in the C terminal or propeptide domains in the mouse corticotrophic AtT20 cell line. Sorting of endogenous PC3 was used as a control. PC2 and PC3 were secreted with similar kinetics and sorted to secretory granules with similar efficiencies. Deletions of up to 50 amino acids from the C-terminus of proPC2 had no effect on secretion or sorting, but larger deletions completely prevented maturation or secretion. Two large deletions within the propeptide also prevented secretion. Smaller deletions between the primary and secondary cleavage sites, or of the primary cleavage site, reduced the amount of protein secreted but did not affect sorting to secretory granules. Replacement of the propeptide of PC2 with that of the endogenous PC3 also had no effect on secretion or sorting. The results indicate that targeting of proPC2 to the regulated secretory pathway is dependent on more than one region within the proPC2 molecule.
J Mol Endocrinol 1998 Oct
PMID:Sorting of PC2 to the regulated secretory pathway in AtT20 cells. 980 64

We report here the crystallographic structure determination of an autoprocessed (Ser221Cys)-subtilisin E-propeptide complex at 2.0 A resolution. The subtilisin domain sequence has a single substitution (Ser221Cys) which has been shown to block the maturation process prior to degradation of the propeptide domain (77 residues) that acts as an intramolecular chaperon. This mutation, however, did not prevent the enzyme from cleaving its propeptide domain with a 60-80% efficiency. The current determination is the first example of a subtilisin E-propeptide complex which has been autoprocessed. A previous structure determination of a BPN'-prosegment complex has been reported in which the subtilisin domain was extensively mutated and a calcium binding loop was deleted. Further, in this earlier determination, the complex was formed by the addition of separately expressed propeptide domain. The structure determination reported here provides additional information about the nature of the interaction between the subtilisin and propeptide domains in this complex.
J Mol Biol 1998 Nov 20
PMID:The crystal structure of an autoprocessed Ser221Cys-subtilisin E-propeptide complex at 2.0 A resolution. 981 47


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