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Transient misfolding dominates multidomain protein folding.

Borgia A, Kemplen KR, Borgia MB, Soranno A, Shammas S, Wunderlich B, Nettels D, Best RB, Clarke J, Schuler B - Nat Commun (2015)

Bottom Line: Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies.However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours.We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

ABSTRACT
Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations. Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies. However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours. We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated.

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Ensemble kinetics of I27 tandem constructs.(a) Ensemble refolding and unfolding kinetics of I27 single-domain (black), two- (blue), three- (green) and eight-domain (purple) constructs. All multidomain constructs display two refolding phases (circles and diamonds), with a deviation from linearity at low GdmCl concentration (rollover) and two unfolding phases (circles and triangles). Unfolding of all ‘never unfolded' I27 tandem constructs is well described by a single-phase corresponding to native-state unfolding (yellow circles). Rate coefficients for both slow and fast unfolding phases match values for the native (red circles) and the stable misfolded state (red triangles) unfolding measured in single-molecule experiments27, respectively. (b,c) Comparison of the relative amplitudes for the misfolding phase of all constructs observed during refolding (b) and unfolding (c) of previously refolded protein. For both reactions, the proportion of misfolded species increases with the number of domains in a manner we would predict (1:1.3:1.8, equation 1 in Methods).
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f2: Ensemble kinetics of I27 tandem constructs.(a) Ensemble refolding and unfolding kinetics of I27 single-domain (black), two- (blue), three- (green) and eight-domain (purple) constructs. All multidomain constructs display two refolding phases (circles and diamonds), with a deviation from linearity at low GdmCl concentration (rollover) and two unfolding phases (circles and triangles). Unfolding of all ‘never unfolded' I27 tandem constructs is well described by a single-phase corresponding to native-state unfolding (yellow circles). Rate coefficients for both slow and fast unfolding phases match values for the native (red circles) and the stable misfolded state (red triangles) unfolding measured in single-molecule experiments27, respectively. (b,c) Comparison of the relative amplitudes for the misfolding phase of all constructs observed during refolding (b) and unfolding (c) of previously refolded protein. For both reactions, the proportion of misfolded species increases with the number of domains in a manner we would predict (1:1.3:1.8, equation 1 in Methods).

Mentions: Tandem repeats containing two, three or eight domains of I27 were unfolded in the chemical denaturant guanidinium chloride (GdmCl) and then rapidly diluted to monitor ensemble folding kinetics. Compared with isolated I27 domains, an extra folding phase was observed at low denaturant concentrations (Fig. 2a; Supplementary Fig. 2a). This new, faster phase has the same rate coefficients for all tandem proteins (Fig. 2a), and its relative amplitude increases with the number of repeats, as would be expected for interdomain interactions (Fig. 2b; Methods). Moreover, the native folding phase exhibits a distinct ‘rollover' at low GdmCl concentrations, which is independent of protein concentration (Supplementary Fig. 2d–f) and becomes more pronounced with increasing number of tandem repeats (Fig. 2a): we infer that rapid accumulation of transient misfolded states slows native folding. These results suggest that domain-swapped misfolding can be an even greater problem for multidomain proteins with a large number of repeats.


Transient misfolding dominates multidomain protein folding.

Borgia A, Kemplen KR, Borgia MB, Soranno A, Shammas S, Wunderlich B, Nettels D, Best RB, Clarke J, Schuler B - Nat Commun (2015)

Ensemble kinetics of I27 tandem constructs.(a) Ensemble refolding and unfolding kinetics of I27 single-domain (black), two- (blue), three- (green) and eight-domain (purple) constructs. All multidomain constructs display two refolding phases (circles and diamonds), with a deviation from linearity at low GdmCl concentration (rollover) and two unfolding phases (circles and triangles). Unfolding of all ‘never unfolded' I27 tandem constructs is well described by a single-phase corresponding to native-state unfolding (yellow circles). Rate coefficients for both slow and fast unfolding phases match values for the native (red circles) and the stable misfolded state (red triangles) unfolding measured in single-molecule experiments27, respectively. (b,c) Comparison of the relative amplitudes for the misfolding phase of all constructs observed during refolding (b) and unfolding (c) of previously refolded protein. For both reactions, the proportion of misfolded species increases with the number of domains in a manner we would predict (1:1.3:1.8, equation 1 in Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660218&req=5

f2: Ensemble kinetics of I27 tandem constructs.(a) Ensemble refolding and unfolding kinetics of I27 single-domain (black), two- (blue), three- (green) and eight-domain (purple) constructs. All multidomain constructs display two refolding phases (circles and diamonds), with a deviation from linearity at low GdmCl concentration (rollover) and two unfolding phases (circles and triangles). Unfolding of all ‘never unfolded' I27 tandem constructs is well described by a single-phase corresponding to native-state unfolding (yellow circles). Rate coefficients for both slow and fast unfolding phases match values for the native (red circles) and the stable misfolded state (red triangles) unfolding measured in single-molecule experiments27, respectively. (b,c) Comparison of the relative amplitudes for the misfolding phase of all constructs observed during refolding (b) and unfolding (c) of previously refolded protein. For both reactions, the proportion of misfolded species increases with the number of domains in a manner we would predict (1:1.3:1.8, equation 1 in Methods).
Mentions: Tandem repeats containing two, three or eight domains of I27 were unfolded in the chemical denaturant guanidinium chloride (GdmCl) and then rapidly diluted to monitor ensemble folding kinetics. Compared with isolated I27 domains, an extra folding phase was observed at low denaturant concentrations (Fig. 2a; Supplementary Fig. 2a). This new, faster phase has the same rate coefficients for all tandem proteins (Fig. 2a), and its relative amplitude increases with the number of repeats, as would be expected for interdomain interactions (Fig. 2b; Methods). Moreover, the native folding phase exhibits a distinct ‘rollover' at low GdmCl concentrations, which is independent of protein concentration (Supplementary Fig. 2d–f) and becomes more pronounced with increasing number of tandem repeats (Fig. 2a): we infer that rapid accumulation of transient misfolded states slows native folding. These results suggest that domain-swapped misfolding can be an even greater problem for multidomain proteins with a large number of repeats.

Bottom Line: Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies.However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours.We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

ABSTRACT
Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations. Simulations suggest that a large fraction of these misfolds resemble an intramolecular amyloid-like state reported in computational studies. However, for naturally occurring neighbours with low sequence identity, these transient misfolds disappear much more rapidly than for identical neighbours. We thus propose that evolutionary sequence divergence between domains is required to suppress the population of long-lived, potentially harmful misfolded states, whereas large populations of transient misfolded states appear to be tolerated.

Show MeSH