Mutational analysis of the BPTI folding pathway: I. Effects of aromatic → leucine substitutions on the distribution of folding intermediates

Zhang, Jianxin; Goldenberg, David P.

doi:10.1002/pro.5560060719

Cited by 28 publications

(30 citation statements)

References 74 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We suggest that Tyr-116, perhaps through resonance effects, facilitates formation of the Cys-59/ Sec-498 intermediate selenenylsulfide by weakening the C-terminal selenenylsulfide bond in its oxidized state. It is a well known phenomenon that tyrosine residues can affect the formation and/or reactivity of dithiol/disulfide motifs, such as in the active sites of glutaredoxins (66) or during formation of folding intermediates (67). In other words, we propose that the interaction of the side chain of Tyr-116 with Sec in the selenenylsulfide of the oxidized C terminus may weaken this bond (increase its inherently low redox potential), so that its reduction by the N-terminal dithiol/chargetransfer complex becomes facilitated.…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

Cheng

Sandalova

Lindqvist

et al. 2009

Journal of Biological Chemistry

179

183

View full text Add to dashboard Cite

Selenoproteins contain a highly reactive 21st amino acid selenocysteine (Sec) encoded by recoding of a predefined UGA codon. Because of a lack of selenoprotein supply, high chemical reactivity of Sec, and intricate translation machineries, selenoprotein crystal structures are difficult to obtain. Structural prerequisites for Sec involvement in enzyme catalysis are therefore sparsely known. Here we present the crystal structure of catalytically active rat thioredoxin reductase 1 (TrxR1), revealing surprises at the C-terminal Sec-containing active site in view of previous literature. The oxidized enzyme presents a selenenylsulfide motif in trans-configuration, with the selenium atom of Sec-498 positioned beneath the side chain of Tyr-116, thereby located far from the redox active moieties proposed to be involved in electron transport to the Sec-containing active site. Upon reduction to a selenolthiol motif, the Sec residue moved toward solvent exposure, consistent with its presumed role in reduction of TrxR1 substrates or as target of electrophilic agents inhibiting the enzyme. A Y116I mutation lowered catalytic efficiency in reduction of thioredoxin, but surprisingly increased turnover using 5-hydroxy-1,4-naphthoquinone (juglone) as substrate. The same mutation also decreased sensitivity to inhibition by cisplatin. The results suggest that Tyr-116 plays an important role for catalysis of TrxR1 by interacting with the selenenylsulfide of oxidized TrxR1, thereby facilitating its reduction in the reductive half-reaction of the enzyme. The interaction of a selenenylsulfide with the phenyl ring of a tyrosine, affecting turnover, switch of substrate specificity, and modulation of sensitivity to electrophilic agents, gives important clues into the mechanism of TrxR1, which is a selenoprotein that plays a major role for mammalian cell fate and function. The results also demonstrate that a recombinant selenoprotein TrxR can be produced in high amount and sufficient purity to enable crystal structure determination, which suggests that additional structural studies of these types of proteins are feasible.

show abstract

Section: Discussionmentioning

confidence: 99%

Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

Cheng

Sandalova

Lindqvist

et al. 2009

Journal of Biological Chemistry

179

183

View full text Add to dashboard Cite

show abstract

“…Aprotinin was dissolved in the same buffer solution. The Y35G BPTI protein was produced by heterologous expression in Escherichiacoli and purified as described previously (61 Crystals were soaked briefly in reservoir solution plus 20% (v/v) ethylene glycol as cryoprotectant prior to freezing in liquid propane at 100 K. During data collection, crystals were maintained at 100 K in a stream of evaporated nitrogen (Oxford Cryosystems). Data were collected with use of a CCD detector and a rotating anode FR591 generator (Nonius Delft, Netherlands).…”

Section: Crystallographymentioning

confidence: 99%

Rigidification of a Flexible Protease Inhibitor Variant upon Binding to Trypsin

Hanson

Domek

Horvath

et al. 2007

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

The Tyr35→Gly replacement in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance the flexibility of the trypsin-binding region of the free inhibitor and to destabilize the interaction with the protease by about 3 kcal/mol. The effects of this replacement on the enzyme-inhibitor interaction were further studied here by x-ray crystallography and isothermal titration calorimetry. The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 Å resolution x-ray diffraction data collected from cryopreserved crystals, and a new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62 Å data. These structures reveal that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical to that of the wild-type protein, with a water-filled cavity in place of the missing Tyr side chain. The crystallographic temperature factors for the two complexes indicate that the mutant inhibitor is nearly as rigid as the wild-type protein when bound to trypsin. Calorimetric measurements show that the change in enthalpy upon dissociation of the complex is 2.5 kcal/mol less favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor. Thus, the destabilization of the complex resulting from the Y35G replacement is due to a more favorable change in entropy upon dissociation. The heat capacity changes for dissociation of the mutant and wild-type complexes were very similar, suggesting that the entropic effects probably do not arise from solvation effects, but are more likely due to an increase in protein conformational entropy upon dissociation of the mutant inhibitor. These results define the biophysical role of a highly conserved core residue located outside of a protein-binding interface, demonstrating that Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structure of the free protein so as to maximize binding affinity. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Abbreviations used: BPTI, bovine pancreatic trypsin inhibitor; Amino acid replacements are indicated by the wild-type residue type (using the one letter code for the 20 standard amino acid residues), followed by the residue number and the mutant residue type; Elsewhere, amino acid residue types are indicated by the standard 3-letter code. IAS, iso-aspartate; HEPES, N -2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid; Tris, tris(hydroxymethyl)-aminomethane; NMR, nuclear magnetic resonance.

show abstract

“…These substitutions were distributed throughout the sequence of the protein and have been found to cause a variety of effects on the stability of the native protein or the major one-disulfide intermediate, @30-51# Goldenberg et al, 1989Goldenberg et al, , 1992, 1997a, 1997b!. The reaction rates for these proteins are compared in Fig-ure 3B.…”

Section: Reactivities Of Cysteine Thiols In Reduced Bptimentioning

confidence: 99%

“…These experiments revealed that only a fraction of the possible disulfidebonded intermediates accumulate to significant levels during refolding, thus providing some of the first experimental evidence to support the view that proteins fold via specific pathways. In subsequent studies, a variety of techniques, including NMR spectroscopy and mutational analysis, have been used to analyze the conformations of the intermediates and the interactions that stabilize them~Goldenberg et al, 1989;van Mierlo et al, 1991avan Mierlo et al, , 1991bvan Mierlo et al, , 1993van Mierlo et al, , 1994Staley & Kim, 1992Yu et al, 1995;Zhang & Goldenberg, 1997a, 1997b!. Although there have been significant disagreements about the relative importance of intermediates with native and non-native disulfides, there is general agreement that the steady-state distribution of intermediates is dominated by species with only native disulfides, but that intermediates with nonnative bonds also play important kinetic roles in the process of forming the native protein~Weissman & Kim, 1991Creighton, 1992;Darby et al, 1995!. The initial steps in the refolding of reduced BPTI quickly lead to a steady-state distribution of one-disulfide intermediates, and this distribution is dominated by one or two species~depending on Reprint requests to: David P. Goldenberg, Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, Utah 84112-0840; e-mail: goldenberg@biology.utah.edu.…”

mentioning

confidence: 99%

Early events in the disulfide‐coupled folding of BPTI

Bułaj

Goldenberg

1999

Protein Science

Self Cite

View full text Add to dashboard Cite

Recent studies of the refolding of reduced bovine pancreatic trypsin inhibitor~BPTI! have shown that a previously unidentified intermediate with a single disulfide is formed much more rapidly than any other one-disulfide species. This intermediate contains a disulfide that is present in the native protein~between Cys14 and 38!, but it is thermodynamically less stable than the other two intermediates with single native disulfides. To characterize the role of the @14-38# intermediate and the factors that favor its formation, detailed kinetic and mutational analyses of the early disulfideformation steps were carried out. The results of these studies indicate that the formation of @14-38# from the fully reduced protein is favored by both local electrostatic effects, which enhance the reactivities of the Cys14 and 38 thiols, and conformational tendencies that are diminished by the addition of urea and are enhanced at lower temperatures. At 25 8C and pH 7.3, approximately 35% of the reduced molecules were found to initially form the 14-38 disulfide, but the majority of these molecules then undergo intramolecular rearrangements to generate non-native disulfides, and subsequently the more stable intermediates with native disulfides. Amino acid replacements, other than those involving Cys residues, were generally found to have only small effects on either the rate of forming @14-38# or its thermodynamic stability, even though many of the same substitutions greatly destabilized the native protein and other disulfide-bonded intermediates. In addition, those replacements that did decrease the steady-state concentration of @14-38# did not adversely affect further folding and disulfide formation. These results suggest that the weak and transient interactions that are often detected in unfolded proteins and early folding intermediates may, in some cases, not persist or promote subsequent folding steps.Keywords: @14-38# intermediate; bovine pancreatic trypsin inhibitor; disulfide bonds; protein folding; unfolded proteins For more than two decades, bovine pancreatic trypsin inhibitor BPTI! has served as an important model for both theoretical and experimental studies of protein folding~Levitt & Warshel, 1975;Creighton, 1978;Goldenberg et al., 1989;Weissman & Kim, 1991;Goldenberg, 1992;Daggett & Levitt, 1993;Darby et al., 1995!. Pioneering work by Creighton~1978! demonstrated that the folded structure of BPTI depends on its three disulfide bonds and that these disulfides could be used as experimental probes of conformation during oxidative refolding of the reduced protein. These experiments revealed that only a fraction of the possible disulfidebonded intermediates accumulate to significant levels during refolding, thus providing some of the first experimental evidence to support the view that proteins fold via specific pathways. In subsequent studies, a variety of techniques, including NMR spectroscopy and mutational analysis, have been used to analyze the conformations of the intermediates and the interactions that stab...

show abstract

Mutational analysis of the BPTI folding pathway: I. Effects of aromatic → leucine substitutions on the distribution of folding intermediates

Cited by 28 publications

References 74 publications

Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

Rigidification of a Flexible Protease Inhibitor Variant upon Binding to Trypsin

Early events in the disulfide‐coupled folding of BPTI

Contact Info

Product

Resources

About