Further evidence suggesting that the slow phase in protein unfolding and refolding is due to proline isomerization: a kinetic study of carp parvalbumins

Molecular dynamics simulations have been performed (AMBER version 3.1) on solvated residues 1-65 of bovine prothrombin fragment 1 (BF1) by using the 2.8-A resolution crystallographic coordinates as the starting conformation for understanding calcium ion-induced conformational changes that precede experimentally observable phospholipid binding. Simulations were performed on the non-metal-bound crystal structure, the form resulting from addition of eight calcium ions to the 1-65 region of the crystal structure, the form resulting from removal of calcium ions after 107 ps and continuing the simulation, and an isolated hexapeptide loop (residues 18-23). In all cases, the 100-ps time scale seemed adequate to sample an ensemble of solution conformers within a particular region of conformation space. The non-metalcontaining BF1 did not unfold appreciably during a 106-ps simulation starting from the crystallographic geometry. The calcium ion-containing structure (Ca-BF1) underwent an interesting conformational reorganization during its evolution from the crystal structure: during the time course of a 107-ps simulation, Ca-BF1 experienced a trans -* cis isomerization of the y-carboxyglutamic acid-21 (Gla-21)-Pro-22 peptide bond.Removal of the calcium ions from this structure followed by 114 ps of additional molecular dynamics showed significant unfolding relative to the final 20-ps average structure ofthe 107-ps simulation; however, the Gla-21-Pro-22 peptide bond remained cis. A 265-ps simulation on the termini-protected hexapeptide loop (Cys-18 to Cys-23) containing two calcium ions also did not undergo a trans -* cis isomerization. It is believed that the necessary activation energy for the transitional event observed in the Ca-BF1 simulation was largely supplied by global conformational events with a possible assist from relief of intermolecular crystal packing forces. The presence of a Gla preceding Pro-22, the inclusion of Pro-22 in a highly strained loop structure, and the formation of two long-lived salt bridges prior to isomerization may all contribute to this finding.Prothrombin, a protein of central importance in the coagulation cascade, is converted to thrombin by the action of a macromolecular complex involving the protease factor Xa bound to the membrane protein factor Va. The conversion occurs on the surface of the membrane; binding of the substrate, prothrombin, is mediated by calcium ions. Magnesium ions do not support the prothrombin-membrane interaction (1-6). Bovine fragment 1 (BF1) corresponds to the amino-terminal 156 residues of bovine prothrombin; the first 33 amino acids contain 10 y-carboxyglutamic acid (Gla) residues. These Gla residues are created by means of posttranslational modification of glutamic acid residues in the presence of a vitamin K-dependent carboxylase and endow prothrombin with its essential calcium-mediated phospholipid-binding properties (1). The Gla domain of prothrombin has classically referred to the first 48 residues of the aminoterminal region and is highly conser...

supporting

confidence: 84%

Solution conformations of the gamma-carboxyglutamic acid domain of bovine prothrombin fragment 1, residues 1-65.

Charifson

Darden

Tulinsky

et al. 1991

“…Moreover (28)(29)(30)(31)(32)(33), andthe slow phase has been attributed to praline isomerization. This assumption is supported by other investigations (34)(35)(36)(37)(38)(39)(40). Recent observations, however, suggest that praline isomerization occurs after most of the confonnational folding has taken-place (41)(42)(43).…”

supporting

confidence: 74%

Regeneration of RNase A from the reduced protein: models of regeneration pathways.

Konishi¹,

Ooi²,

Scheraga³

1982

Two models ofprotein-folding pathways are proposed on the basis ofequilibrium and Idnetic data in the literature.One is a growth-type model-.e., nucleation of the native-like structure occurs in the folding process, in the rate-limiting step(s), and subsequent folding around the nucleation sites proceeds smoothly to form the native disulfide bonds and conformation. The other is a rearrangement-type model-i.e., proper On the basis of experimental and theoretical studies, three different models have been proposed forprotein-folding pathways.In the first, the dominant near-neighbor interactions lead to the formation of nucleation sites among nearby residues in the amino-acid sequence in a rate-limiting step(s) in the initial stages of folding, this is followed by a rapid growth phase in which other parts of the chain fold around the nucleation sites '(1-19

“…An obvious possibility is the transient presence of one or more cis peptide bonds per unfolded RNase A molecule. Although the probability of any one peptide bond being cis is low (say 1%), nevertheless, there are 123 peptide bonds in RNase A and the probability of any unfolded molecule having at least one cis bond could be quite high (23). Once the cis bond isomerizes to trans, it would be trapped in the trans conformation by folding, and so the kinetic barrier presented by a cis peptide bond would be transient.…”

Section: Resultsmentioning

confidence: 99%

Early folding intermediate of ribonuclease A.

Udgaonkar

Baldwin

1990

176

139

Pulsed hydrogen exchange (2H-IH) is used to characterize the folding process of ribonuclease A (disulfide bonds intact). The results show one principal early folding intermediate (Ii), which is formed rapidly after the start of folding and whose proton-exchange properties change with the time of folding. AU probes that are hydrogen bonded within the ,8-sheet of native ribonuclease A are protected in II. Thus, the results suggest that the fl-sheet is formed rapidly and cooperatively. The initial protection factors of probes in the fl-sheet are between 10 and 100, but they increase with time of folding and exceed 1000 at 400 msec from the start offolding. Thus, the fl-sheet is only moderately stable when it is first formed, but subsequent events stabilize it, possibly through interactions involving hydrophobic side chains. The large protection factors of the f8-sheet probes in an early folding intermediate are unexpected and remarkable. Probes in the three a-helices are fewer in number and give less accurate data than the f8-strand probes. The folding kinetics expected for a simple sequential model of folding are outlined. An important difference between the observed and predicted behavior is that the early folding intermediate is not fully populated when it is first formed.A recently developed method (1, 2) of studying kinetic folding intermediates uses 2H-1H exchange to pulse label accessible peptide amide protons in folding intermediates. A preliminary study of the folding of RNase A, disulfide bonds intact, has been reported (1). Since then, the complete amide proton assignments of RNase A have been reported (3, 4), and the results, which have been extended, can now be interpreted in structural terms. Twenty-seven amide protons are used as probes of structure in this work: their locations are shown in Fig. 1. All but two are hydrogen bonded and all are located in a-helices or 8-strands; more than half are in the threestranded antiparallel 8-sheet.Our report is focused on early events in the folding process. In kinetic experiments using pulse labeling to measure protection factors, the measurable range is 1-1000. Larger protection factors cannot be quantitated, but ones measured for native proteins are as large as 109 (7). The protection factor is defined as the ratio of two exchange rate constants: k (solvent exposed)/k (observed), where the solvent-exposed rate is measured on a peptide with the same sequence or is calculated from data on model peptides (8). Pulsed hydrogen exchange shows which protons are protected in a folding intermediate and can give their protection factors, but it does not show directly whether protection occurs by hydrogen bonding and it does not reveal hydrogen-bond acceptor groups. The first step in structural interpretation is to compare the pattern of protection results with the structure of native RNase A.Significant changes in methodology have been made since our previous report (1). In that work, introduction of pulse labeling was found to be an important advance over the...