Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A

Pearson, M.A.; Karplus, P.A.; Dodge, Robert; Laity, John H.; Scheraga, Harold A.

doi:10.1002/pro.5560070522

Cited by 38 publications

(41 citation statements)

References 19 publications

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“…Hence, global unfolding occurs at a negligible rate in these des species at 15°C and pH 8.0. This conclusion is consistent with other structural data, including thermal and chemical unfolding transitions, x-ray crystal B-factors, and hydrogen͞deuterium exchange data (13,(19)(20)(21)(22)(23). The alternative explanation (namely, that these des species are in a rapid reshuffling equilibrium with the 3S ensemble) can be ruled out because of the absence of an effect of 260 M DTT red (which should reduce any unstructured 3S species).…”

Section: Relative Contributions Of the Various Des Species To Regenersupporting

confidence: 88%

Structural determinants of oxidative folding in proteins

Welker

Narayan

Wedemeyer

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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156

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A method for determining the kinetic fate of structured disulfide species (i.e., whether they are preferentially oxidized or reshuffle back to an unstructured disulfide species) is introduced. The method relies on the sensitivity of unstructured disulfide species to low concentrations of reducing agents. Because a structured des species that preferentially reshuffles generally first rearranges to an unstructured species, a small concentration of reduced DTT (e.g., 260 M) suffices to distinguish on-pathway intermediates from dead-end species. We apply this method to the oxidative folding of bovine pancreatic ribonuclease A (RNase A) and show that des [40 -95] O xidative folding is the composite process by which a protein recovers both its native disulfide bonds (disulfide-bond regeneration) and its native structure (conformational folding). The course of oxidative folding is affected by three general structural factors that have been identified from in vitro oxidative folding studies and model systems, namely, the proximity, reactivity, and accessibility of the disulfide bonds and thiol groups. The proximity of two reactive groups (defined as their effective intramolecular concentration) is determined by the propensity of the backbone to bring the two groups into juxtaposition; in unfolded species, this proximity is largely determined by the loop entropy, although enthalpic interactions may contribute significantly as well. The reactivity of two reactive groups depends on the fact that most disulfide reactions occur through thiol͞ disulfide exchange and that only the thiolate (not the thiol) form is reactive; hence, changes in the local electrostatic environment may affect the rate of disulfide-bond reactions. However, the most critical factor seems to be the accessibility of the thiol groups and disulfide bonds (1). Thiol͞disulfide exchange reactions can occur only when a thiol and a disulfide bond come into contact; hence, burial of the disulfide bond or the thiol prevents their contact and blocks the reaction.Accordingly, the formation of stable tertiary structure that protects the native disulfide bonds is a key event in the oxidative folding of proteins, because it stabilizes such bonds by making them inaccessible to the redox reagent and protein thiols. Indeed, the rate of intramolecular disulfide reshuffling is sufficiently high so that no native disulfide bond would survive much longer than a few minutes in an unstructured disulfide intermediate with a free thiol group unless it is buried in protective tertiary structure (2). Thus, the rate-determining step in oxidative folding is often the formation of a disulfide intermediate with stable tertiary structure (1-5).However, the structural protection of the thiol groups is just as important as the protection of the disulfide bonds in reaching the transition state of oxidative folding. The burial of both thiol groups and disulfide bonds hinders any further progress in oxidative folding, because all of the reactive groups have been sequestered from the redo...

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Section: Relative Contributions Of the Various Des Species To Regenersupporting

confidence: 88%

Structural determinants of oxidative folding in proteins

Welker

Narayan

Wedemeyer

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

156

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“…Presumably, the total dimerization interface area is large enough to tolerate the single amino acid changes, and the cis-peptide bond configuration in P89A was retained. This retention is not completely unexpected and has been observed in several other cases (30,31). To verify SR␤-specific homodimerization, we performed the gel-filtration assay with SR␤ from three additional organisms: Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens.…”

Section: Sr␤ Homodimerization In Vitrosupporting

confidence: 54%

Homodimerization of the G protein SRβ in the nucleotide-free state involves proline cis/trans isomerization in the switch II region

Schwartz

Schmidt

Brohawn

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Protein translocation across and insertion into membranes is essential to all life forms. Signal peptide-bearing nascent polypeptide chains emerging from the ribosome are first sampled by the signal-recognition particle (SRP), then targeted to the membrane via the SRP receptor (SR), and, finally, transferred to the proteinconducting channel. In eukaryotes, this process is tightly controlled by the concerted action of three G proteins, the 54-kD subunit of SRP and the ␣-and ␤-subunits of SR. We have determined the 2.2-Å crystal structure of the nucleotide-free SR␤ domain. Unexpectedly, the structure is a homodimer with a highly intertwined interface made up of residues from the switch regions of the G domain. The remodeling of the switch regions does not resemble any of the known G protein switch mechanisms. Biochemical analysis confirms homodimerization in vitro, which is incompatible with SR␣ binding. The switch mechanism involves cis͞trans isomerization of a strictly conserved proline, potentially implying a new layer of regulation of cotranslational transport.cotranslational transport ͉ proline isomerization ͉ signal-recognition particle receptor N ascent proteins, destined for secretion or membrane insertion, are first targeted to the endoplasmic reticulum (ER) in eukaryotes or the plasma membrane in bacteria. Targeting in eukaryotes occurs primarily via the conserved signal-recognition particle (SRP) pathway (1). This cyclic pathway starts with SRP, a ribonucleoprotein complex consisting of the 300-nucleotide SRP-RNA and six proteins, recognizing a ribosome-nascent chain (RNC) complex and arresting translation. In a second step, the stalled SRP-RNC complex docks with the ER membranebound SRP receptor (SR) (2, 3), which is dynamically associated with the protein-conducting channel (PCC or Sec61 complex) (4). Third, the RNC is detached from the SRP, binds to the PCC, and releases the nascent polypeptide chain into an aqueous pore in the channel (5, 6). Protein synthesis can then continue. Concomitantly, SRP and SR dissociate, ready for a new cycle of protein targeting. GTP binding and hydrolysis by three synchronized G proteins regulate the process and ensure unidirectionality (7,8).Intense investigation of the protein targeting process by x-ray crystallography and cryoelectron microscopy has lead to the structural characterization of domains and interactions (9, 10). As a result of these advances, several key steps are now relatively well understood. The recent cryoelectron microscopic structure of the SRP-RNC complex shows how the elongated SRP wraps around the large ribosomal subunit, binding the N-terminal signal sequence of the nascent chain at the exit tunnel via SRP54 at one end of the molecule and arresting translation by blocking the elongation-factor-binding site with its other end (11). SRP54, the only SRP protein with a bacterial homologue, has two functional domains; the C-terminal M domain is the signal sequence-binding interface, and the N-terminal composite NG domain has regulatory GTPase acti...

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“…13 ONC 23 and RNase A 32 have similar threedimensional structures but, because ONC lacks the (65 -72) disulfide bond of RNase A, the folding/unfolding pathways probably differ. Here, therefore, we have examined the reductive unfolding pathway of ONC, and compared it with those of RNase A, of its P93A mutant (whose threedimensional structure is known 33,34 ) and of its Y92F 35 and Y92A mutants. This study provides information about important inter-residue interactions in the two variants, and their impact on the respective reductive unfolding pathways of the two proteins.…”

Section: Introductionmentioning

confidence: 99%