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  • Both the cis-trans equilibrium and isomerizationdynamics of a single proline amide modulate2-microglobulin amyloid assemblyVladimir Yu. Torbeev and Donald Hilvert1

    Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland

    Edited by Ronald T. Raines, University of WisconsinMadison, Madison, WI, and accepted by the Editorial Board October 29, 2013 (received for reviewJune 1, 2013)

    The human protein 2-microglobulin (2m) aggregates as amyloidfibrils in patients undergoing long-term hemodialysis. Isomeriza-tion of Pro32 from its native cis to a nonnative trans conformationis thought to trigger 2m misfolding and subsequent amyloid as-sembly. To examine this hypothesis, we systematically varied thefree-energy profile of proline cis-trans isomerization by replacingPro32 with a series of 4-fluoroprolines via total chemical synthesis.We show that 2ms stability, (un)folding, and aggregation prop-erties are all influenced by the rate and equilibrium of Pro32 cis-trans isomerization. As anticipated, the 2m monomer was eitherstabilized or destabilized by respective incorporation of (2S,4S)-fluoroproline, which favors the native cis amide bond, or the ste-reoisomeric (2S,4R)-fluoroproline, which disfavors this conforma-tion. However, substitution of Pro32 with 4,4-difluoroproline,which has nearly the same cis-trans preference as proline but anenhanced isomerization rate, caused pronounced destabilizationof the protein and increased oligomerization at neutral pH. Moreremarkably, these subtle alterations in chemical compositionincorporation of one or two fluorine atoms into a single prolineresidue in the 99 amino acid long proteinmodulated the aggre-gation properties of 2m, inducing the formation of polymorphi-cally distinct amyloid fibrils. These results highlight the importanceof conformational dynamics for molecular assembly of an amyloidcross- structure and provide insights into mechanistic aspects ofPro32 cis-trans isomerism in 2m aggregation.

    protein conformation | polymorphism | amyloidogenesis |native chemical ligation

    Human 2-microglobulin (2m) is a component of the class Imajor histocompatibility complex (1). Although the WTprotein is generally stable at neutral pH, in kidney diseasepatients undergoing long-term hemodialysis, it forms insolubleamyloid deposits causing a condition known as dialysis-relatedamyloidosis (2). Collagen and glycosaminoglycans facilitate fibrilformation at neutral pH (3, 4), as do organic solvents (5), di-valent metal ions (Cu2+) (6, 7), and surfactants (8). Amyloids canalso be induced at low pH (99.9%) than proline, does not result in enhancedamyloid formation (11). Other factors, such as protein confor-mational dynamics (11), are probably important for the initiationand assembly processes.In this paper, we report a robust chemical synthesis of the 99-

    residue human 2m protein that allows us to probe the role ofPro32 cis-to-trans isomerization in amyloid formation with mech-anistically more informative substitutions than can be achievedusing conventional site-directed mutagenesis. Specifically, wegenerated three 2m variants in which Pro32 was replaced with(2S, 4S)-4-fluoroproline (4S-fpr), (2S, 4R)-4-fluoroproline (4R-Fpr),and 4,4-difluoroproline (F2Pro), respectively (16) (Fig. 1C).Fluorinated proline analogs have been used extensively to probethe structure and stability of many proteins (1720). They arenearly isosteric to the natural amino acid and preserve the uniqueconformational properties of the five-membered pyrrolidine ringbut exhibit altered cis-trans amide isomer ratios. For example, theconfiguration at C4 in 4R-Fpr stabilizes the exo ring pucker viaa gauche effect, which, in turn, leads to higher stability of thetrans-prolyl conformer [6.7:1 versus 4.6:1 for proline in the modelcompound Ac-Xaa-OMe (20)] (Fig. 1D). The opposite is true for4S-fpr, which favors the endo ring pucker, reducing the prefer-ence for the trans-prolyl conformer to a factor of 2.5:1 (20). F2Prohas similar cis-trans peptide bond propensities as native proline


    2-Microglobulin is an abundant and normally soluble protein.In patients undergoing chronic dialysis, however, it forms in-soluble amyloid plaques, leading to medical complications. Ithas been suggested that the conformational transformation ofsoluble protein monomers into polymeric amyloids is mediatedby isomerization of a single amino acid, namely, proline 32. Inthis study, we probed the role of this amino acid by chemicallysynthesizing uniquely tailored protein analogs containingnoncanonical amino acids at position 32. Our results show thatboth the chemical equilibrium and rate of cis-trans isomeriza-tion of proline 32 are critical for the solubility of 2-micro-globulin and its self-assembly into morphologically distinctamyloid fibrils. These insights may aid ongoing efforts toprovide remedies against dialysis-related amyloidosis.

    Author contributions: V.Y.T. and D.H. designed research; V.Y.T. performed research; V.Y.T.and D.H. analyzed data; and V.Y.T. and D.H. wrote the paper.

    The authors declare no conflict of interest.

    This article is a PNAS Direct Submission. R.T.R. is a guest editor invited by the EditorialBoard.1To whom correspondence should be addressed. E-mail:

    This article contains supporting information online at PNAS | December 10, 2013 | vol. 110 | no. 50 | 2005120056







  • but a significantly lower activation barrier for isomerization (16).Biochemical characterization of WT 2m and the three analogsprovides unique insight into the mechanistic consequences of per-turbing the Pro32 peptide bond on protein (un)folding and sub-sequent aggregation to give oligomers and amyloid fibrils.

    ResultsChemical Synthesis of Human 2m and Analogs by Native ChemicalLigation. WT 2m was assembled by one pot native chemicalligation of three unprotected peptide fragments (Fig. 2 and SIAppendix, Figs. S1 and S2) (21). The peptide segments weresynthesized using an in situ neutralization protocol for Boc-based solid phase peptide synthesis (22) and subsequently puri-fied to homogeneity by reverse-phase HPLC (RP-HPLC). Nativechemical ligation of the C terminal and middle fragments wasfollowed by thiazolidine (Thz) deprotection, ligation with theN-terminal peptide, and formyl-deprotection of tryptophans. AfterRP-HPLC purification, formation of the native disulfide bond byair-oxidation, and further RP-HPLC purification, the protein

    was folded by dialysis against 25 mM sodium phosphate buffer(pH 7.5). High purity of synthetic 2m was verified by analyticalRP-HPLC and high-resolution mass spectrometry (Fig. 2B). Theprotein adopts a native fold as judged by CD spectroscopy (Fig.3A) and 1H-NOESY NMR spectroscopy (SI Appendix, Fig. S8).The three 2m analogs containing the noncanonical amino

    acids 4R-Fpr, 4S-fpr, and F2Pro at position 32 were synthesizedanalogously using N-Boc-protected 4-fluoroprolines instead ofproline (see SI Appendix and Figs. S3S6 for analytical data).Near-UV CD and 1H-NOESY spectra (SI Appendix, Figs. S7S10) show that the variants adopt the same overall tertiarystructure as WT 2m. As expected for such close structuralanalogs, only small differences localized near residue 32 areobserved in the 1H-NOESY spectra (SI Appendix, Figs. S8S10).

    Fluoroprolines Modulate 2m Stability. Far-UV CD spectra andthermal denaturation curves for WT 2m and the three analogsare depicted in Fig. 3 A and B, respectively. Chemically synthe-sized 2m has a CD spectrum that is virtually identical to thepublished spectra of recombinantly expressed protein containingan additional N-terminal methionine (9, 23). Chief features area strong maximum at 203 nm and a weak minimum at 221 nm. TheCD spectra of [4S-fpr32]2m and [4R-Fpr32]2m are similar (Fig.3A). The relative stability of these three proteins was assessed bythermal denaturation. Their apparent melting temperatures (Tm)correlate with the cis-amide conformational preferences of therespective proline derivative at position 32 (Fig. 1D), decreasing inthe order [4S-fpr32]2m > WT 2m > [4R-Fpr32]2m (Table 1).The [F2Pro32]2m protein deviates from these trends. Its far-

    UV CD spectrum shows a diminished maximum at 203 nm and aslightly blue-shifted minimum at 218 nm (Fig. 3A). Interestingly,similar features were previously observed for 2m variants con-taining trans-configured Pro32, including the P5G point mutantand N6, which lacks six N-terminal amino acids (23). Like theseproteins, [F2Pro32]2m exhibits reduced thermal stability (Tm =56 C) and less cooperative thermal denaturation compared withWT 2m (Fig. 3B).Chemical denaturation (Fig. 3C and SI Appendix, Fig. S13)

    and foldingunfolding kinetics (SI Appendix, Fig. S14) of theFig. 1. Human 2-microglobulin (2m). (A) Primary amino acid sequence ofthe 99-residue 2m with Pro32 highlighted in red and the two cysteine-containing ligation sites underlined. (B) Native structure of 2m showing theBC loop (cyan) containing cis-Pro32 (red) (based on PDB ID 2YXF) (C) Struc-ture of proline and the three fluorine-containing proline analogs used in thisstudy: (2S, 4S)-4-fluoroproline (4S-fpr), (2S, 4R)-4-fluoroproline (4R-Fpr), and4,4-difluoroproline (F2Pro). (D) Stereoelectronic effects associated with theC4 substituent influence both the proline ring conformation as well as thecis-trans amide bond equilibrium (17). An electron-withdrawing group atthe 4R position stabilizes the exo ring pucker via a gauche e