Native and Non-native Structure in a Protein-folding Intermediate: Spectroscopic Studies of Partially Reduced IGF-I and an Engineered Alanine Model

Hua, Qing; Narhi, Linda O.; Jia, Wenhua; Arakawa, Tsutomu; Rosenfeld, Robert; Hawkins, Nessa; Miller, James A.; Weiss, Michael A.

doi:10.1006/jmbi.1996.0320

Cited by 68 publications

(96 citation statements)

References 54 publications

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“…The number of restraints used in the refinement of the structure of Long-[Arg 3 ]IGF-I is significantly greater than used for the original IGF-I structures (9,10) and is comparable to the number used for subsequent work with IGF-II (13) and analogs of IGF-I (25,26). The solution structures of Long- [Arg 3 ]IGF-I display good convergence to a single fold in the hydrophobic core region containing the three ␣-helices (Fig.…”

Section: Structure Determination Of Long-[arg 3 ]Igf-i-mentioning

confidence: 99%

Solution Structure and Backbone Dynamics of Long-[Arg3]insulin-like Growth Factor-I

Laajoki¹,

Francis²,

Wallace³

et al. 2000

Journal of Biological Chemistry

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Section: Structure Determination Of Long-[arg 3 ]Igf-i-mentioning

confidence: 99%

Solution Structure and Backbone Dynamics of Long-[Arg3]insulin-like Growth Factor-I

Laajoki¹,

Francis²,

Wallace³

et al. 2000

Journal of Biological Chemistry

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“…The three disulfide bonds are important in maintaining the native conformation and biological activities of the insulin molecular. The contributions of these bridges to the structure, stability, and activity of hormone have been investigated in analogues lacking selected disulfide bridges (7)(8)(9)(10)(11)(12). Internal cystine A6 -A11 is close to the hydrophobic core and its substitution with Ser or Ala resulted in the unfolding of helix 2 (10,11).…”

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confidence: 99%

In Vitro Refolding of Human Proinsulin

Qiao

Min

Hua

et al. 2003

Journal of Biological Chemistry

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Human insulin is a double-chain peptide that is synthesized in vivo as a single-chain human proinsulin (HPI). We have investigated the disulfide-forming pathway of a single-chain porcine insulin precursor (PIP). Here we further studied the folding pathway of HPI in vitro. While the oxidized refolding process of HPI was quenched, four obvious intermediates (namely P1, P2, P3, and P4, respectively) with three disulfide bridges were isolated and characterized. Contrary to the folding pathway of PIP, no intermediates with one-or two-disulfide bonds could be captured under different refolding conditions. CD analysis showed that P1, P2, and P3 retained partially structural conformations, whereas P4 contained little secondary structure. Based on the timedependent distribution, disulfide pair analysis, and disulfide-reshuffling process of the intermediates, we have proposed that the folding pathway of HPI is significantly different from that of PIP. These differences reveal that the C-peptide not only facilitates the folding of HPI but also governs its kinetic folding pathway of HPI. Detailed analysis of the molecular folding process reveals that there are some similar folding mechanisms between PIP and HPI. These similarities imply that the initiation site for the folding of PIP/HPI may reside in the central ␣-helix of the B-chain. The formation of disulfide A20 -B19 may guide the transfer of the folding information from the B-chain template to the unstructured A-chain. Furthermore, the implications of this in vitro refolding study on the in vivo folding process of HPI have been discussed.

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“…The architecture of insulin and IGF-1 mainly consists of three R-helical segments (A2-A8, A13-A19, and B9-B19 in insulin; [8][9][10][11][12][13][14][15][16][17][18][42][43][44][45][46][47][48][49], and 54-61 in IGF-1) in the A-and B-chain/domains ( Figure 1A,B). The three R-helical segments are stabilized by the three disulfides (A6-A11, A7-B7, A20-B19 in insulin; 47-52, 6-48, 18-61 in IGF-1) (8)(9)(10). The conformation of the C-and D-domains of IGF-1 is highly flexible.…”

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The Different Energetic State of the Intra A-Chain/Domain Disulfide of Insulin and Insulin-like Growth Factor 1 Is Mainly Controlled by Their B-Chain/Domain

2002

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Insulin and insulin-like growth factor 1 (IGF-1) share homologous sequence, similar threedimensional structure, and weakly overlapping biological activity, but different folding information is stored in their homologous sequences: the sequence of insulin encodes one unique thermodynamically stable three-dimensional structure while that of IGF-1 encodes two disulfide isomers with different threedimensional structure but similar thermodynamic stability. Their different folding behavior probably resulted from the different energetic state of the intra A-chain/domain disulfide: the intra A-chain disulfide of insulin is a stable bond while that of IGF-1 is a strained bond with high energy. To find out the sequence determinant of the different energetic state of their intra A-chain/domain disulfide, the following experiments were carried out. First, a local chimeric single-chain insulin (PIP) with the A8-A10 residues replaced by the corresponding residues of IGF-1 was prepared. Second, the disulfide stability of two global hybrids of insulin and IGF-1, Ins(A)/IGF-1(B) and Ins(B)/IGF-1(A), was investigated. The local segment swap had no effect on the fidelity of disulfide pairing and the disulfide stability of PIP molecule although the swapped segment is close to the intra A-chain/domain disulfide. In redox buffer which favors the disulfide formation for most proteins, Ins(A)/IGF-1(B) cannot form and maintain its native disulfides just like that of IGF-1, while the disulfides of Ins(B)/IGF-1(A) are stable in the same condition. One major equilibrium intermediate with two disulfides of Ins(A)/IGF-1(B) was purified and characterized. V8 endoproteinase cleavage and circular dichroism analysis suggested that the intra A-chain/domain disulfide was reduced in the intermediate. Our present results suggested that the energetic state of the intra A-chain/domain disulfide of insulin and IGF-1 was not controlled by the A-chain/domain sequence close to this disulfide but was mainly controlled by the sequence of the B-chain/domain.Insulin is an extensively studied small globular protein with A-and B-chains linked by three disulfides (one intrachain bond, A6-A11; two interchain bonds, A7-B7 and A20-B19). Its three-dimensional structure has been well-defined by X-ray crystallography (1, 2) and NMR (3-5) since the 1970s. IGF-1 1 is an insulin-like 70-residue singlechain protein composed of B-, C-, A-, and D-domains (6). The B-and A-domains of IGF-1 are homologous to the Band A-chains of insulin, respectively; the C-domain is analogous to the C-peptide of proinsulin, but they share no sequence homology; the D-domain has no counterparts in insulins. The three-dimensional structure of IGF-1 is very similar with that of insulin (7). The architecture of insulin and IGF-1 mainly consists of three R-helical segments (A2-A8, A13-A19, and B9-B19 in insulin; 8-18, 42-49, and 54-61 in IGF-1) in the A-and B-chain/domains ( Figure 1A,B). The three R-helical segments are stabilized by the three disulfides (A6-A11, A7-B7, A20-B19 in insul...

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Native and Non-native Structure in a Protein-folding Intermediate: Spectroscopic Studies of Partially Reduced IGF-I and an Engineered Alanine Model

Cited by 68 publications

References 54 publications

Solution Structure and Backbone Dynamics of Long-[Arg3]insulin-like Growth Factor-I

Solution Structure and Backbone Dynamics of Long-[Arg3]insulin-like Growth Factor-I

In Vitro Refolding of Human Proinsulin

The Different Energetic State of the Intra A-Chain/Domain Disulfide of Insulin and Insulin-like Growth Factor 1 Is Mainly Controlled by Their B-Chain/Domain

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