Oxidative refolding of IGF-1 yields two products of similar thermodynamic stability: A bifurcating protein-folding pathway

Miller, James A.; Narhi, Linda O.; Hua, Qing; Rosenfeld, Robert; Arakawa, Tsutomu; Rohde, Michael F.; Prestrelski, Steven J.; Lauren, Scott L.; Stoney, Kendall; Tsai, Larry B.

doi:10.1021/bi00070a032

Cited by 107 publications

(187 citation statements)

References 64 publications

Supporting

Mentioning

180

Contrasting

Order By: Relevance

“…This notion is supported further by the results of recent research on native and recombinant insulin-like growth factor (IGF-I) (Axelsson et al, 1992;Narhi et al, 1993;Miller et al, 1993 ;Yan & Erickson, 1994), which in aqueous solution folds into a spatial structure similar to that of insulin (Sato et al, 1993). Axelsson et al (1992) reported that correct disulfide arrangement is important for the full biological activity of IGF-I.…”

Section: Figmentioning

confidence: 91%

Paired natural cysteine mutation mapping: Aid to constraining models of protein tertiary structure

1995

View full text Add to dashboard Cite

This paper discusses the benefit of mapping paired cysteine mutation patterns as a guide to identifying the positions of protein disulfide bonds. This information can facilitate the computer modeling of protein tertiary structure. First, a simple, paired natural-cysteine-mutation map is presented that identifies the positions of putative disulfide bonds in protein families. The method is based on the observation that if, during the process of evolution, a disulfide-bonded cysteine residue is not conserved, then it is likely that its counterpart will also be mutated. For each target protein, protein databases were searched for the primary amino acid sequences of all known members of distinct protein families. Primary sequence alignment was carried out using Pileup algorithms in the GCG package. To search for correlated mutations, we listed only the positions where cysteine residues were highly conserved and emphasized the mutated residues. In proteins of known three-dimensional structure, a striking pattern of paired cysteine mutations correlated with the positions of known disulfide bridges. For proteins of unknown architecture, the mutation maps showed several positions where disulfide bridging might occur.

show abstract

Section: Figmentioning

confidence: 91%

Paired natural cysteine mutation mapping: Aid to constraining models of protein tertiary structure

1995

View full text Add to dashboard Cite

show abstract

“…The only study to have yielded kinetic data from an NMR experiment shows how to extract time constants for the development of native chemical shifts in hen egg white lysozyme (23). On the other hand the structures of several oxidative folding intermediates and their variants have been partially or fully elucidated with NMR (19, 24 -33), x-ray crystallography (29), circular dichroism (34 -37), fluorescence, Fourier transform infrared, and, in one case, also photo-CIDNP (25).…”

mentioning

confidence: 99%

Oxidative Folding of Amaranthus α-Amylase Inhibitor

Cemazar

Zahariev

Pongor

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Oxidative folding is the fusion of native disulfide bond formation with conformational folding. This complex process is guided by two types of interactions: first, covalent interactions between cysteine residues, which transform into native disulfide bridges, and second, non-covalent interactions giving rise to secondary and tertiary protein structure. The aim of this work is to understand both types of interactions in the oxidative folding of Amaranthus ␣-amylase inhibitor (AAI) by providing information both at the level of individual disulfide species and at the level of amino acid residue conformation. The cystine-knot disulfides of AAI protein are stabilized in an interdependent manner, and the oxidative folding is characterized by a high heterogeneity of one-, two-, and three-disulfide intermediates. The formation of the most abundant species, the main folding intermediate, is favored over other species even in the absence of non-covalent sequential preferences. Time-resolved NMR and photochemically induced dynamic nuclear polarization spectroscopies were used to follow the oxidative folding at the level of amino acid residue conformation. Because this is the first time that a complete oxidative folding process has been monitored with these two techniques, their results were compared with those obtained at the level of an individual disulfide species. The techniques proved to be valuable for the study of conformational developments and aromatic accessibility changes along oxidative folding pathways. A detailed picture of the oxidative folding of AAI provides a model study that combines different biochemical and biophysical techniques for a fuller understanding of a complex process.Cracking the code for folding a string of amino acid residues into a biologically active three-dimensional structure is still a major challenge of both chemical and biological interest. The information that determines the folding of a protein is contained solely in its amino acid residues (1). In proteins that contain cysteine residues the formation of disulfide bonds is an additional degree of freedom in the folding process. In these proteins a fusion between the recovery of the native tertiary structure (conformational folding) and the regeneration of native disulfide bonds is coupled in the oxidative folding process (2).Disulfide formation is important for in vivo folding of a considerable number of eukaryotic proteins and is a key posttranslational modification for the stabilization of folded structures. The study of oxidative folding in vitro can provide a detailed structural description in terms of the disulfide intermediate species present along the pathway of the complex folding process. A thorough account of an oxidative folding reaction should combine a study of the formation of covalently stable intermediate species and their disulfide bond connectivity and an analysis of the conformational assembly of these species. An established method for elucidation of the exact role that disulfides play in the process of achieving...

show abstract

“…In the case of IGF-1, when the disulfide 18 -61 (corresponding to the disulfide A20-B19 of insulin) is deleted, the mutant IGF-1 cannot be expressed, but the IGF-1 mutant with the single disulfide 18 -61 acquires a compact partially folded conformation (35). Moreover, during in vitro refolding, the disulfide 18 -61 is formed first (23)(24)(25)(26)(27). And this disulfide bond was retained in all of the intermediates identified.…”

Section: Reversible In Vitro Refolding/unfolding Pathways and Identicalmentioning

confidence: 91%

“…Nonspecific formation of secondary disulfide was also noticeable in both the folding pathway of insulin/PIP and IGF-1 (22)(23)(24)(25)(26)(27). Previously, we have analyzed four two-disulfide PIP models that retain A20-B19 disulfide (36), and we found that all of the model peptides adopted a partially folded conformation; the refolding rates of the four model peptides were similar, but their refolding yields were different.…”

Section: Reversible In Vitro Refolding/unfolding Pathways and Identicalmentioning

confidence: 94%

“…Breaking either disulfide results in unfolding of helix 2 in both insulin and IGF-1. Although folding studies of insulin and IGF-1 revealed that they share some common features in their folding pathways (20 -27), their folding behaviors are significantly different; insulin/PIP folds into one thermodynamic structure (20 -22), whereas IGF-1 folds into two isomers (native and swap) with different disulfide linkages but similar thermodynamic stability (23)(24)(25)(26)(27).…”

mentioning

confidence: 99%

See 1 more Smart Citation

In Vitro Refolding/Unfolding Pathways of Amphioxus Insulin-like Peptide

Chen

Jin

Dong

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Amphioxus insulin-like peptide (AILP) belongs to the insulin superfamily and is proposed as the common ancestor of insulin and insulin-like growth factor 1. Herein, the studies on oxidative refolding and reductive unfolding of AILP are reported. During the refolding process, four major intermediates, P1, P2, P3, and P4, were captured, which were almost identical to those intermediates, U1, U2, U3, and U4, captured during the AILP unfolding process. P4 (U4) has the native disulfide A20-B19; P1 (U1), P2 (U2), and P3 (U3) have two disulfide bonds, which include A20-B19. Based on the analysis of the time course distribution and properties of the intermediates, we proposed that fully reduced AILP refolded through 1SS, 2SS, and 3SS intermediate stages to the native form; native AILP unfolded through 2SS and 1SS intermediate stages to the full reduced form. A schematic flow chart of major oxidative refolding and reductive unfolding pathways of AILP was proposed. Implication for the folding behavior of insulin family proteins was discussed. There may be seen three common folding features in the insulin superfamily: 1) A20-B19 disulfide is most important and formed during the initial stage of folding process; 2) the second disulfide is nonspecifically formed, which then rearranged to native disulfide; 3) in vitro refolding and unfolding pathways may share some common folding intermediates but flow in opposite directions. Furthermore, although swap AILP is a thermodynamically stable final product, a refolding study of swap AILP demonstrated that it is also a productive intermediate of native AILP during refolding.Since Anfinsen and co-workers (1) first demonstrated in the 1960s that the amino acid sequence of a protein determines its three-dimensional structure, significant advances have been made in the understanding of protein folding through experimental and theoretical approaches (2-4). However, the folding mechanism, such as how a linear polypeptide strand starts and walks along what pathway to fold into a protein molecule, is still poorly understood. In the investigations of the protein folding pathway, the proteins with disulfide linkages are frequently chosen as models, because the formation of disulfide bridges is always coupled with folding and assembly, which can be used as a unique probe for the study of protein folding and assembly (5-7). Oxidative folding studies on the disulfide-coupled folding of some small globular proteins, such as bovine pancreatic trypsin inhibitor (8, 9), RNase A (10), hirudin (11), etc., have revealed the sequences of preferred kinetic intermediates of such proteins, by which the in vitro folding pathway of the proteins was defined.The insulin superfamily members are good model proteins for the folding studies of disulfide-containing proteins. Insulin and insulin-like growth factor 1 (IGF-1) 1 are two extensively studied members of the superfamily. They have similar threedimensional structure and identical disulfide pairing (12, 13). Both proteins mainly consist of three ␣-helical se...

show abstract

Oxidative refolding of IGF-1 yields two products of similar thermodynamic stability: A bifurcating protein-folding pathway

Cited by 107 publications

References 64 publications

Paired natural cysteine mutation mapping: Aid to constraining models of protein tertiary structure

Paired natural cysteine mutation mapping: Aid to constraining models of protein tertiary structure

Oxidative Folding of Amaranthus α-Amylase Inhibitor

In Vitro Refolding/Unfolding Pathways of Amphioxus Insulin-like Peptide

Contact Info

Product

Resources

About