The role of conformational flexibility in prion propagation and maintenance for Sup35p

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The role of conformational flexibility in prion propagation and maintenance for Sup35p
2001
Thomas Scheibel & Susan L. Lindquist
Nature Structural Biology
Howard Hughes Medical Institute and Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois 60637, USA.
Correspondence should be addressed to Susan L. Lindquist lindquist@wi.mit.edu
The [PSI+] factor of Saccharomyces cerevisiae is a protein-based genetic element (prion) comprised of a heritable altered conformation of the cytosolic translation termination factor Sup35p. In vitro, the prion-determining region (NM) of Sup35p undergoes conformational conversion from a highly flexible soluble state to structured amyloid fibers, with a rate that is greatly accelerated by preformed NM fiber nuclei. Nucleated conformational conversion is the molecular basis of the genetic inheritance of [PSI+] and provides a new model for studying amyloidogenesis. Here we investigate the importance of structure and structural flexibility in soluble NM. Elevated temperatures, chemical chaperones and certain mutations in NM increase or change its structural content and inhibit or enhance nucleated conformational conversion. We propose that the structural flexibility of NM is particularly suited to allowing heritable protein-based changes in cellular behavior.
The [PSI+] factor, a protein-based genetic element (prion) of Saccharomyces cerevisiae, represents a newly discovered type of epigenetic inheritance in which changes in phenotype are transmitted through self-perpetuating, conformationally altered forms of cellular proteins1, 2. The inheritance of [PSI+] from mother to daughter cells is based on the transmission of conformational information from ordered nonfunctional Sup35p aggregates to the soluble, functional Sup35p, a subunit of the polypeptide chain release complex that is essential for translation termination3, 4, 5. This molecular mechanism is reminiscent of the self-promoted conformational conversion proposed for mammalian prion diseases4, 5, 6, but in yeast it produces heritable changes in metabolism rather than disease. In vitro, the prion-determining region of Sup35, NM, has the unusual property of remaining as a random coil-rich protein in solution for hours before it converts to a structure with all the characteristics of amyloid fibers7, 8. The contribution of structural content in the soluble protein prior to conformational conversion is a major unanswered question in prion biology. Here we show that factors influencing conformational flexibility of Sup35/NM influence amyloid propagation in vitro. Together with other analyses in vivo, our data suggest that the unusual structural properties and the conformational flexibility of the NM-region of Sup35 contribute to the mechanism by which it serves as a protein-based element of genetic inheritance.
NM purification with and without denaturant
Because NM tends to polymerize during purification, previous in vitro studies used protein purified in denaturant, followed by dilution into physiological buffer, to investigate conformational conversion7, 8, 9. The random coil content of such resolubilized NM is unusually high, emphasizing the importance of ensuring that the structure and behavior of NM purified under these conditions reflect that of NM unexposed to denaturant. We purified a GST-NM fusion protein under nondenaturing conditions, removed the GST domain by proteolytic cleavage and compared it to the standard NM preparation for secondary structure content, amyloid assembly kinetics and amyloid fiber morphology. NM in 8 M guanidinium chloride showed a CD spectrum indicative of a random coil with no detectable secondary structure (Fig. 1a). In physiological buffer, NM displayed a far-UV CD signal representative of a molecule rich in random coil but partially structured, regardless of whether it had been purified under denaturing or nondenaturing conditions (Fig. 1a). The rates of conformational conversion determined by Congo red (CR) binding10 for both unnucleated and nucleated reactions were indistinguishable for NM prepared under both conditions (Fig. 1b; data not shown). Fiber morphology was indistinguishable when visualized by either electron (EM) or atomic force (AFM) microscopy (data not shown; refs 7,8). More important, NM prepared under both conditions showed the same changes in behavior when exposed to elevated temperature or chemical chaperones (see below). Because these criteria detected no differences between the two types of preparation, further studies employed NM purified under denaturing conditions due to higher yields and greater ease of handling.
Figure 1. Comparison of NMwt purified under denaturing and nondenaturing conditions.

a, Secondary structure of resolubilized NMwt (10 M) and nondenatured NMwt (0.5 M, the low concentration is due to technical difficulties in concentrating) by far-UV CD. The higher signal noise for nondenatured NMwt is due to a lower protein concentration. Shown are NMwt diluted out of denaturant (solid black line); nondenatured NMwt (dotted gray line); and NMwt in 8 M guanidinium chloride (dotted black line). b, Unseeded, unrotated conformational conversion of resolubilized and nondenatured protein (0.5 M each) was monitored by CR binding. NMwt diluted out of denaturant is represented by a circle; nondenatured NMwt by triangle.
Full Figure and legend (19K)
Contribution of partially structured NM to conversion
Many proteins reach their final stable structures by progressing through partially folded intermediates. This is also true of amyloid polymerization, where mildly denaturating conditions, such as low pH or elevated temperatures, produce partially unfolded intermediates that provide the basis for conformational conversion11, 12, 13. To determine whether the partially structured state that naturally exists in the soluble form of NM contributes to nucleated conformational conversion, we tested the effects of elevated temperatures, chemical chaperones and certain NM mutants. All of the conditions described influenced neither the quaternary structure of soluble NM nor that of assembled amyloid fibers as detected by static and dynamic light scattering or AFM (see below; data not shown).
Temperature-induced gain of structure
With increasing temperature, NM underwent a conformational transition from a random coil-dominated structure to one with high secondary structure content that remained distinct from that of -sheet-rich amyloid fibers (Fig. 2a). The amplitude for the mean residue ellipiticity at []222nm (reflecting structural content) increased, whereas the amplitude for the mean residue ellipiticity at []208nm (reflecting random coil content) decreased. Because of the large impact of the random-coil structure on the CD signal of NM at 218 nm, which is commonly used to monitor -sheet secondary structure, 222 nm was chosen as a more reliable indicator of secondary structure gain. Above 60 ?C, structural content remained stable up to 98 ?C (Fig. 2b; data not shown), even with prolonged incubation (90 min) (data not shown).

Figure 2. Influences of temperature and chemical chaperones on NMwt secondary structure.

a, Temperature-induced NMwt (10 M) secondary structure changes by far-UV CD. NMwt remained at each temperature for 10 min before the spectrum was taken. For comparison, a far UV-CD spectrum of NMwt fibers is shown. b, The mean residue ellipiticities of NMwt increased at []222 nm (filled circle) and decreased at []208nm (filled square) as a function of temperature. The transition is thermodynamically reversible in a heating-cooling experiment (closed versus open symbols). c, Time course of temperature-induced gain (solid black line) and loss (dotted gray line) of NMwt (5 M) secondary structure monitored at 222 nm. The reaction is concentration-independent between 0.5 M and 65 M. NMwt was incubated at each temperature for 30 min before shifting to the final temperature. The kinetics could be fit with a single exponential, with rate constants kgain = 1.7 10-2 s-1 and kloss = 1.9 10-2 s-1. d, NMwt secondary structure by far-UV CD. Buffer is standard buffer (solid black line); TMAO, standard buffer plus 2.5 M TMAO (dotted dark gray line); and glycerol, standard buffer plus 3 M glycerol (25 %) (dotted light gray line).
Full Figure and legend (40K)

For proteins from mesophilic organisms, the gain of stable soluble structure with increasing temperature is very unusual because these proteins usually denature and aggregate at high temperatures14. A small number of proteins, including certain amyloidogenic polypeptides, are notable exceptions15, 16, 17, 18. In contrast to the one other amyloidogenic peptide tested15, the temperature-induced structural gain in NM was thermodynamically reversible and exhibited no hysteresis, suggesting that it gains and loses structure through similar pathways (Fig. 2b). The rates of the structural gain and loss were kinetically indistinguishable, with rate constants of kgain = 1.7 ( 0.1) 10-2 s-1 and kloss = 1.9 ( 0.1) 10-2 s-1 (Fig. 2c). Thus, NM behavior is unique in many respects, even from other proteins with high structural flexibility.
Effect of osmolytes on secondary structure
Osmolytes have been shown to accelerate conformational conversion of an amyloidogenic polypeptide19. Therefore, they may influence the partially structured state of soluble NM. Increasing trimethylamine N-oxide (TMAO) to 2.5 M or glycerol to 3 M (25% glycerol) led to a gain of NM secondary structure with no further changes at higher concentrations (Fig. 2d; data not shown). Because of the large impact of random-coil structure on the CD signal of NM, the changes appear small. However, in the presence of glycerol, the signal increased 60% and in the presence of TMAO it increased 100% at 222 nm. These effects of TMAO and glycerol were specific and not simply based on enhancing local concen