Folding of a large protein at high structural resolution

Abstract

Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways.SAXS | HDX | protein collapse | denatured state ensemble F ifty years after Anfinsen's seminal demonstration that an unfolded protein can refold spontaneously when placed under native conditions, major questions concerning the folding process remain unanswered (1, 2). What is the unfolded state like, its degree of compaction, the reality and character of residual structure before folding begins, and its possible role in guiding the folding process (3-7)? Analogous questions relate to folding intermediates and the folding pathway itself. Do proteins fold through many alternative independent pathways as earlier theoretical investigations have suggested (8-12), or do they fold through necessary intermediates in a distinct pathway (13), as a growing list of experimental observations indicate (14, 15)? To answer these questions, it will be necessary to define experimentally the intermediate forms that proteins move through on their way to the native state. The problem has been that these transient states are beyond the reach of the usual high-resolution crystallographic and NMR structural methods. Most experimental folding studies have therefore relied on low-resolution optical methods that can follow folding in real time but rarely provide the structural information necessary to resolve the basic mechanistic questions.Recent work...

Folding and assembly of the large molecular machine Hsp90 studied in single-molecule experiments

“…S5. Structurally similarly heterogeneous burstphase intermediates have been inferred from bulk studies of other large proteins like maltose-binding protein (MBP) and Tim barrel protein (6,21).…”

“…Although the in vitro folding of small and mediumsized proteins is relatively well understood (1)(2)(3)(4)(5), very limited information exists about the complete folding process of such large proteins (6). In general, larger proteins often exhibit a multitude of intermediate and aggregation-prone misfolded states (4,7).…”

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

“…The analysis of deuterium content involved the use of previously described custom Python scripts (64,66,67). Significant differences in deuterium uptake levels between WT and mutant were identified by Student's t test as having a P value < 0.05, as described previously (68).…”

The nature of protein folding pathways

“…3) (29). This condition might be expected to spawn multiple folding routes as in the new view model, but it does not.…”

“…These experiments largely avoided the back-unfolding HX labeling artifact by using a short labeling pulse (12 ms). Longer pulses (up to 42 ms) allowed the back-unfolding of the weakly protected regions in the initially collapsed form to be studied (29). Higher protection seems to correlate with the amphipathic nature of different segments and their tendency to form helical structure.…”