A cross-linked ribonuclease A derivative, Lys7-dinitrophenylene-Lys41-ribonuclease A, has been prepared and characterized for ultimate use in protein-folding experiments. Immunochemical assays and spectroscopic measurements demonstrated that the introduction of this artificial cross-link does not perturb the native conformation of ribonuclease A. The cross-linked protein exhibited a reversible thermal transition with Tm = 53 degrees C at pH 2.0, which is 25 degrees C higher than that of unmodified ribonuclease A under the same conditions. The denatured form of the cross-linked ribonuclease A has a conformational chemical potential that is 4.9 kcal/mol higher than that of the denatured form of unmodified ribonuclease A at 40 degrees C and pH 2.0, assuming that the cross-linked and the unmodified proteins have the same conformational chemical potential in the native conformation. This is in good agreement with a theoretical value of 5.2 kcal/mol, calculated from the reduction of chain entropy of the denatured form upon introduction of the extrinsic cross-link. Thus, it is concluded that the extrinsic cross-link between Lys7 and Lys41, formed by the dinitrophenylene group, does not affect the native conformation of ribonuclease A but destabilizes the denatured conformation, probably by decreasing its chain entropy.

The folding kinetics of hen egg white lysozyme and of a three-disulfide derivative of lysozyme [carboxymethyl(Cys6,Cys127)-hen egg white lysozyme] have been studied by absorbance- and fluorescence-detected stopped-flow techniques. A "very-fast" phase with a time constant in the millisecond range has been observed by both absorbance and fluorescence when unfolded lysozyme in 4 M guanidine hydrochloride, 100 mM phosphate buffer, and pH 2.0 is refolded at 0.5 M guanidine hydrochloride, 100 mM phosphate, and pH 6.7. Data obtained from fluorescence-detected refolding studies show that a transient intermediate is formed during the very-fast refolding phase. This intermediate is characterized by substantial quenching of tryptophan fluorescence. In addition, analysis of the fluorescence data indicates the presence of an additional "burst" phase that occurs within the dead time of the instrument, < 3 ms. The very-fast phase is not observed during the refolding of the three-disulfide derivative. In addition, the three-disulfide derivative re-attains the final native folded conformation more rapidly than the unmodified protein over the range of temperatures studied (10-20 degrees C). We conclude that, not only does the presence of the disulfide bond between Cys6 and Cys127 slow down the overall folding process of lysozyme, but it also directs the folding of lysozyme through a pathway characterized by a non-native tertiary interaction(s).

The galactoside-binding sites of ricin B chain can be blocked by affinity-directed chemical modification using a reactive ligand derived from asialoglycopeptides containing triantennary N-linked oligosaccharides. The terminal galactosyl residue of one branch of the triantennary oligosaccharide is modified to contain a reactive dichlorotriazine moiety. Two separate galactoside-binding sites have been clearly established in the ricin B chain by X-ray crystallography [Rutenber, E., and Robertus, J. D. (1991) Proteins 10, 260-269], and it is necessary to covalently attach two such reactive ligands to the B chain to block its binding to galactoside affinity matrixes. A method was developed using thiol-specific labeling of the ligand combined with subsequent immunoaffinity chromatography which allowed the isolation of ricin B chain peptides covalently linked to the ligand from proteolytic digests of purified blocked ricin. The sites of covalent attachment of the two ligands in blocked ricin were inferred from sequence analysis to be Lys 62 in domain 1 of the B chain and Tyr 148 in domain 2. A minor species of blocked ricin contains a third covalently attached ligand. From the analysis of peptides derived from blocked ricin enriched in this species, it is inferred that Tyr 67 in domain 1 is the specific site on the ricin B chain where a third reactive ligand becomes covalently linked to the protein. These results are interpreted as providing support for the notion that the ricin B chain has three oligosaccharide binding sites.

A three-disulfide form of hen egg white lysozyme with Cys6 and Cys127 blocked by carboxymethyl groups was prepared, purified, and characterized for eventual use in protein folding experiments. Trypsin digestion followed by proline-specific endopeptidase digestion facilitated the unambiguous assignment of the disulfide bond pairings and the modified residues in this derivative. 3SS-lysozyme demonstrated nearly full enzymatic activity at its pH optimum, pH 5.5. The 3SS-lysozyme derivative and unmodified lysozyme were shown to be identical by CD spectroscopy at pH 3.6. Immunochemical binding assays demonstrated that the conformation of lysozyme was perturbed predominantly only locally by breaking and blocking the disulfide bond between Cys6 and Cys127. Both 3SS-lysozyme and unmodified lysozyme exhibited reversible thermally induced transitions at pH 2.0, but the Tm of 3SS-lysozyme, 18.9 degrees C, was found to be 34 degrees lower than that of native lysozyme under the same conditions. The conformational chemical potential of the denatured form of unmodified lysozyme was determined from the transition curves to be approximately 6.7 kcal/mol higher than that of the denatured form of 3SS-lysozyme, at pH 2.0 and 35 degrees C, if the conformational chemical potential for the folded forms of both 3SS-lysozyme and unmodified lysozyme is arbitrarily assumed to be 0.0 kcal/mol. A calculation of the increase in the theoretical loop entropy of denatured 3SS-lysozyme resulting from the cleavage of the Cys6-Cys127 disulfide bond, however, yielded a value of only 5.4 kcal/mol for the difference in conformational chemical potential. This suggests that, in addition to the entropic component, there is also an enthalpic contribution to the difference in the conformational chemical potential corresponding to approximately 1.3 kcal/mol. Thus, it is concluded that the reduction and blocking of the disulfide bond between Cys6 and Cys127 destabilizes 3SS-lysozyme relative to unmodified lysozyme predominantly by stabilizing the denatured conformation by increasing its chain entropy.

The availability of a large number of expressed sequence tags (ESTs) has facilitated the development of molecular markers in members of the grass family. As these markers are derived from coding sequences, cross-species amplification and transferability is higher than for markers designed from genomic DNA sequences. In this study, 919 EST-based primers developed from seven grass species were assessed for their amplification across a diverse panel of 16 grass species including cereal, turf and forage crops. Out of the 919 primers tested, 89 successfully amplified DNA from one or more species and 340 primers generated PCR amplicons from at least half of the species in the panel. Only 5.2% of the primers tested produced clear amplicons in all 16 species. The majority of the primers (66.9%) were developed from tall fescue and rice and these two species showed amplification rate of 41.6% and 19.0% across the panel, respectively. The highest amplification rate was found for conserved-intron scanning primers (CISP) developed from pearl millet (91%) and sorghum (75%) EST sequences that aligned to rice sequences. The primers with successful amplification identified in this study showed promise in other grass species as demonstrated in differentiating a set of 13 clones of reed canary grass, a species for which very little genomic research has been done. Sequences from the amplified PCR fragments indicated the potential for the transferable CISP markers for comparative mapping purposes. These primer sets can be immediately used for within and across species mapping and will be especially useful for minor grass species with few or no available molecular markers.