What is the role of non-native intermediates of β-lactoglobulin in protein folding?

Abstract

The mechanism of ␣3␤ transition in folding of ␤-lactoglobulin is discussed based on free energy landscape analysis of a long lattice model. It is found that helical propensity of ␤-lactoglobulin is driven by conformational entropy and is intrinsically coded in its native structure. We propose a view on a role of folding intermediate, which is ''on-pathway'' but rich in non-native structures. The present results suggest that the native structure topology plays an important role in ␣3␤ transition.T ransitions from ␣-helix to ␤-sheet have been observed in folding processes of some mainly ␤-sheet proteins, such as ␤-lactoglobulin (␤LG) (1) and plasminogen activator inhibitor type I (2). In these cases, ␣-rich structures appear as folding intermediates. Such folding processes via non-native intermediates are rather unusual and thus their mechanism has been attracting much attention. From the theoretical point of view, ␣3␤ transitions during the folding processes are of great interest because they seem not to be described by the funnel picture, although it successfully describes folding mechanism of some small proteins (3-10). To get more general insight into folding mechanism, it is important to understand the mechanism of the ␣3␤ transition.In this paper, we discuss the folding mechanism of ␤LG as a prototype of the ␣3␤ transition. ␤LG has the following properties: (i) it has a predominantly ␤-sheet native structure [upand-down ␤ barrel (UD␤) topology] (11), and (ii) the refolding intermediate contains a significantly large amount of non-native ␣-helical structures. In other words, the folding process consists of two processes, namely, a fast process U3I(␣) followed by a slow process I(␣)3N, where U and N are the fully unfolded state and the native state, respectively, and I(␣) is the highly helical intermediate (1), and (iii) accumulation of ␣-helical intermediate also is observed in the equilibrium unfolding experiment using guanidine hydrochloride ref. 13).Why does the folding intermediate of ␤LG contain non-native highly ␣-helical structures? And what is their role? To understand the nature of ␣3␤ transition, we study a simple lattice protein model that has properties similar to those of ␤LG. The ModelThe model we study here is the modified HP model (13) in which a protein consists of a self-avoiding chain on a three-dimensional cubic lattice with two types of amino acids: H (hydrophobic) and P (polar). The energy (or solvent averaged free energy) E of a chain conformation is determined by the number of HϪ-H contact n h and that of PϪ-P contact n p as E ϭ Ϫ (n h ϩ n p ), where Ͼ 0 is a constant (we measure the energy in the unit of hereafter). The chain length is 80 and the sequence isThis sequence was originally designed by O'Toole and Panagiotopoulos (13) so as to have a four-helix bundle-like native structure (shown in figure 4 of ref. 14), whose energy E is Ϫ94. In the following sections, we will show that this lattice model is indeed a good model of ␤LG for the purpose of qualitative discussions on ␣3␤ tra...

Simulation of Top7-CFr: A transient helix extension guides folding

“…in experimental (13)(14)(15) as well as computational studies using simplified approaches (16,17). Our results are obtained using first-principle all-atom Monte Carlo simulations.…”

β-Lactoglobulin Assembles into Amyloid through Sequential Aggregated Intermediates

“…46 β-LGa has structural elements that are conformationally labile. [48][49][50][51][52][53] β-LGa can exist in an equilibrium between folded, partially structured, and unfolded states. 13,37,[48][49][50][51][52][53][54][55][56][57][58] β-LGa has been reported to form nonnative α-helices prior to complete folding.…”

“…13,37,[48][49][50][51][52][53][54][55][56][57][58] β-LGa has been reported to form nonnative α-helices prior to complete folding. 13,[49][50][51][52][53][54][55][56] NMR has shown that these α-helices must melt and form a β-strand to complete the native-state LGa. Hydrophobic amino acid residues are in slate; hydrophilic residues are in brick.…”

Glycerol-Induced Aggregation of the Oligomeric L-Asparaginase II from E. coli Monitored with ATR-FTIR

“…From the excellent works on mechanisms of aggregation [9,57,58] it can be concluded that the precursor of all types of aggregations are the partially folded intermediates with native protein like topology. Since changes of standard free energy caused by the stabilising effect of glycerol on protein conformation is too small (~0.1 kcal mol -1 of protein and added glycerol [59,60]) for simple kinetic intermediates to overcome the free energy barrier between two states [13,61,62], while the molten globule like states are energetically closely related to the native state and in some cases are more stable then the native one [63], the glycerol should be considered as a compound able to selectively affect and to accumulate equilibrium molten globule like intermediates. For the asparaginase, we observed nearly identical profiles of amide I, II, and III in the aggregated states, no shift along the x-axis was observed between the three precipitates of the protein, and since the FTIR spectra are very sensitive to the changes of dihedral angles ϕ and ψ [64], based on the curvefit data (table 1) and the above mentioned studies, we can conclude that there is only one structure, partially folded molten globule like on the pathway of L-asparaginase folding, stabilised by glycerol and followed by aggregation upon refolding.…”