Spontaneous refolding of the large multidomain protein malate synthase G proceeds through misfolding traps

Abstract

Most protein folding studies until now focus on single domain or truncated proteins. Although great insights in the folding of such systems has been accumulated, very little is known regarding the proteins containing multiple domains. It has been shown that the high stability of domains, in conjunction with inter-domain interactions, manifests as a frustrated energy landscape, causing complexity in the global folding pathway. However, multidomain proteins despite containing independently foldable, loosely cooperative sections can fold into native states with amazing speed and accuracy. To understand the complexity in mechanism, studies were conducted previously on the multidomain protein malate synthase G (MSG), an enzyme of the glyoxylate pathway with four distinct and adjacent domains. It was shown that the protein refolds to a functionally active intermediate state at a fast rate, which slowly produces the native state. Although experiments decoded the nature of the intermediate, a full description of the folding pathway was not elucidated. In this study, we use a battery of biophysical techniques to examine the protein's folding pathway. By using multiprobe kinetics studies and comparison with the equilibrium behavior of protein against urea, we demonstrate that the unfolded polypeptide undergoes conformational compaction to a misfolded intermediate within milliseconds of refolding. The misfolded product appears to be stabilized under moderate denaturant concentrations. Further folding of the protein produces a stable intermediate, which undergoes partial unfolding-assisted large segmental rearrangements to achieve the native state. This study reveals an evolved folding pathway of the multidomain protein MSG, which involves surpassing the multiple misfolding traps during refolding.

Conformational catalysis of cataract-associated aggregation in a human eye lens crystallin occurs via interface stealing

“…Beyond the native and fully unfolded states, most proteins can adopt partially unfolded or misfolded intermediates. Intermediates are especially important for multidomain proteins, which comprise most eukaryotic proteins yet only a small minority of biophysical studies to-date (1)(2)(3). These intermediates are often rare or transient and thus challenging to investigate, yet they have large effects on the protein's stability, evolution, and function.…”

“…Beyond the native and fully unfolded states, most proteins can adopt partially unfolded or misfolded intermediates. Intermediates are especially important for multidomain proteins, which comprise most eukaryotic proteins yet only a small minority of biophysical studies to-date (1)(2)(3).…”

Hidden kinetic traps in multidomain folding highlight the presence of a misfolded but functionally competent intermediate

“…However, the possibility that the interaction between these structural subunits may play a critical role in the formation of the native structure, as well as in dictating misfolding events, cannot be excluded ( 2 , 3 ). Consequently, recent years have seen a considerable increase in efforts to understand the folding of more complex multidomain systems ( 2 , 4 – 12 ).…”

A novel hotspot of gelsolin instability triggers an alternative mechanism of amyloid aggregation

“…Pressure effects were not promptly and completely reversible for all the proteins analyzed: after 12 h the recovery of the fluorescence characteristics was 70–80% for WT and less (about 50%) for the mutants, both without (1 mM EDTA) and with Ca 2+ (50 mM): The delayed recovery suggests that after decompression there is slow, continuous reorganization of the loose structure. Folding/unfolding transitions of multidomain proteins are usually characterized by a few partially folded equilibrium intermediates in which folded and unfolded domains coexist [54] , [55] . Usually these intermediate states are produced fast, and slowly evolve to the native one [54] .…”

“…Folding/unfolding transitions of multidomain proteins are usually characterized by a few partially folded equilibrium intermediates in which folded and unfolded domains coexist [54] , [55] . Usually these intermediate states are produced fast, and slowly evolve to the native one [54] . At each pressure, the spectral changes in protein fluorescence emission were quantified by determining the center of spectral mass, defined as ν P = (Σ ν i F i )/ F T ) where F i is the fluorescence intensity at the wavenumber ν i , and F T is the total fluorescence emitted from the protein.…”