Biophysical Journal Volume 109 September 2015 852–853
852
New and Notable What’s in a Sequence? Distinct Structures and Dynamics of Two Disordered Calcitonin Family Peptides Martin Muschol1,* 1 Department of Physics, University of South Florida, Tampa, Florida
Over the past 15 years, the study of intrinsically disordered proteins (IDPs) or protein regions (IDP regions) has undergone an explosion in research activity. Initially considered a curiosity, at best, intrinsic disorder proteins are recognized now as representing a large fraction of the proteome of eukaryotes (1). With the classical structure-function paradigm being of little use, IDPs continue to pose experimental and theoretical challenges of identifying the underlying principles that bestow this new class of proteins with their biological functions. This challenge has spawned a slew of creative experimental and theoretical approaches of how to characterize and categorize the structure(s) and dynamics of IDPs, and how to correlate those with their biological performance (for a recent review, see Habchi et al. (2)). In this issue of the Biophysical Journal, Sizemore et al. (3) apply one of those novel approaches, tryptophancysteine quenching (TCQ). Developed in the Eaton and Hofrichter lab at the National Institutes of Health, Bethesda, MD (Lapidus et al. (4)), TCQ monitors the quenching dynamics of the tryptophan triplet state by a cysteine (or cystine) located at the opposite end of a peptide. TCQ is as conceptually straightforward as it is elegant and offers multiple experimental advantages. It utilizes trypto-
Submitted June 19, 2015, and accepted for publication July 15, 2015. *Correspondence: [email protected] Editor: Elizabeth Rhoades. Ó 2015 by the Biophysical Society 0006-3495/15/09/0852/2
phan phosphorescence instead of requiring one to attach large extrinsic fluorophores. Furthermore, the experimentally observed lifetime yields two basic parameters of IDPs simultaneously. The static tryptophan-cysteine reaction rate kR is closely related to the end-to-end distribution of the peptide. This allows for comparisons with various theoretical polymer models such as the random Gaussian or the wormlike chain (5). In addition, TCQ provides information about chain dynamics via the diffusive rate kD by which the ends come into contact. Dynamic parameters are particularly important for understanding the biological functionality of IDPs, which relies on their intrinsic pliability. It will be interesting to see whether TCQ can be further extended to obtain information on both intra- and intermolecular interactions, the latter of which are of particular interest to the process of fibril formation. Here, the authors use this experimental approach to compare the mean sizes and internal chain dynamics for two closely related members of the calcitonin peptide family: calcitoningene-related peptide (CGRP) and amylin (islet amyloid polypeptide, IAPP). Both peptides are of immediate biomedical relevance. CGRP is a known trigger of migraine attacks while amylin forms the substrate for amyloid fibril growth in type-II diabetes. The sequences of both peptides have a length of 37 amino acids, have a 47% sequence homology, and share a loop structure near the N-terminus formed by a C2–C7 disulfide bond. The immediate question the authors pose is whether, despite these similarities, there are discernible differences in the solution structure and dynamics of these two closely related peptides. Their starting point is a comparison of the end-to-end separation of CGRP in a good solvent (6 M guanidine hydrochloride) to prior measurements of amylin under the same conditions (6). There, both peptides assume rather extended states, with CGRP margin-
ally more expanded than amylin. Upon transfer to aqueous solutions, both peptides collapse, with amylin being noticeably more compact than CGRP. The authors ascribe the overall tendency toward collapse to contacts of the N-loops with nonlocal residues, as previously described in Vaiana et al. (6). Under their solution conditions, CGRP carries only one more charge than amylin. By modulating charge interactions via solution pH, ionic strength, or charge mutants, the authors provide solid evidence that long-range charge repulsion underlies the observed net increase in CGRP size over amylin. These results underscore the critical role of charge repulsion effects on IDP structure (7,8) and even fibril assembly pathways of partially unfolded proteins (9). The authors then go on to compare the dynamical properties of both chains in aqueous solution by separating the observed quenching rates into their static reaction and dynamiccollision-rate components. As summarized in their Table 1, the rates of forming end-to-end contacts for CGRP are surprisingly insensitive to its overall size. One might presume that the chain dynamics simply slows down in proportion to CGRP compaction, which makes it more difficult to move between conformations. By quantifying the nearly twofold difference in intrachain dynamics of CGRP and amylin at identical overall size, the authors provide a compelling argument that compaction alone is insufficient to account for the size-induced changes in chain dynamics. Instead, the authors argue that the differences in peptide sequence result in distinct intrachain and chain-solvent interactions that are underlying the observed differences in chain dynamics of these two peptides. The above results provide a detailed image of both structural and dynamic features of these two peptides. The
http://dx.doi.org/10.1016/j.bpj.2015.07.024
New & Notable
authors also present qualitative arguments how the sensitivity of the CGRP structure to net charge (via pH) correlates with pH changes associated with of migraine attacks. Most importantly, though, this article emphasizes that a detailed analysis of chain dynamics in addition to the structural characterization of IDPs is essential for describing their physical state. REFERENCES 1. Xue, B., A. K. Dunker, and V. N. Uversky. 2012. Orderly order in protein intrinsic disor-
853 der distribution: disorder in 3500 proteomes from viruses and the three domains of life. J. Biomol. Struct. Dyn. 30:137–149. 2. Habchi, J., P. Tompa, ., V. N. Uversky. 2014. Introducing protein intrinsic disorder. Chem. Rev. 114:6561–6588. 3. Sizemore, S. M., S. M. Cope, ., S. M. Vaiana. 2015. Slow internal dynamics and charge expansion in the disordered protein CGRP: a comparison with amylin. Biophys. J. 109:1038–1048. 4. Lapidus, L. J., W. A. Eaton, and J. Hofrichter. 2000. Measuring the rate of intramolecular contact formation in polypeptides. Proc. Natl. Acad. Sci. USA. 97:7220–7225. 5. Rubinstein, M., and R. H. Colby. 2003. Polymer Physics. Oxford University Press, Oxford, UK.
6. Vaiana, S. M., R. B. Best, ., J. Hofrichter. 2009. Evidence for a partially structured state of the amylin monomer. Biophys. J. 97:2948– 2957. 7. Mu¨ller-Spa¨th, S., A. Soranno, ., B. Schuler. 2010. Charge interactions can dominate the dimensions of intrinsically disordered proteins. Proc. Natl. Acad. Sci. USA. 107:14609–14614. 8. Das, R. K., and R. V. Pappu. 2013. Conformations of intrinsically disordered proteins are influenced by linear sequence distributions of oppositely charged residues. Proc. Natl. Acad. Sci. USA. 110:13392–13397. 9. Hill, S. E., T. Miti, ., M. Muschol. 2011. Spatial extent of charge repulsion regulates assembly pathways for lysozyme amyloid fibrils. PLoS One. 6:e18171.
Biophysical Journal 109(5) 852–853
852
New and Notable What’s in a Sequence? Distinct Structures and Dynamics of Two Disordered Calcitonin Family Peptides Martin Muschol1,* 1 Department of Physics, University of South Florida, Tampa, Florida
Over the past 15 years, the study of intrinsically disordered proteins (IDPs) or protein regions (IDP regions) has undergone an explosion in research activity. Initially considered a curiosity, at best, intrinsic disorder proteins are recognized now as representing a large fraction of the proteome of eukaryotes (1). With the classical structure-function paradigm being of little use, IDPs continue to pose experimental and theoretical challenges of identifying the underlying principles that bestow this new class of proteins with their biological functions. This challenge has spawned a slew of creative experimental and theoretical approaches of how to characterize and categorize the structure(s) and dynamics of IDPs, and how to correlate those with their biological performance (for a recent review, see Habchi et al. (2)). In this issue of the Biophysical Journal, Sizemore et al. (3) apply one of those novel approaches, tryptophancysteine quenching (TCQ). Developed in the Eaton and Hofrichter lab at the National Institutes of Health, Bethesda, MD (Lapidus et al. (4)), TCQ monitors the quenching dynamics of the tryptophan triplet state by a cysteine (or cystine) located at the opposite end of a peptide. TCQ is as conceptually straightforward as it is elegant and offers multiple experimental advantages. It utilizes trypto-
Submitted June 19, 2015, and accepted for publication July 15, 2015. *Correspondence: [email protected] Editor: Elizabeth Rhoades. Ó 2015 by the Biophysical Society 0006-3495/15/09/0852/2
phan phosphorescence instead of requiring one to attach large extrinsic fluorophores. Furthermore, the experimentally observed lifetime yields two basic parameters of IDPs simultaneously. The static tryptophan-cysteine reaction rate kR is closely related to the end-to-end distribution of the peptide. This allows for comparisons with various theoretical polymer models such as the random Gaussian or the wormlike chain (5). In addition, TCQ provides information about chain dynamics via the diffusive rate kD by which the ends come into contact. Dynamic parameters are particularly important for understanding the biological functionality of IDPs, which relies on their intrinsic pliability. It will be interesting to see whether TCQ can be further extended to obtain information on both intra- and intermolecular interactions, the latter of which are of particular interest to the process of fibril formation. Here, the authors use this experimental approach to compare the mean sizes and internal chain dynamics for two closely related members of the calcitonin peptide family: calcitoningene-related peptide (CGRP) and amylin (islet amyloid polypeptide, IAPP). Both peptides are of immediate biomedical relevance. CGRP is a known trigger of migraine attacks while amylin forms the substrate for amyloid fibril growth in type-II diabetes. The sequences of both peptides have a length of 37 amino acids, have a 47% sequence homology, and share a loop structure near the N-terminus formed by a C2–C7 disulfide bond. The immediate question the authors pose is whether, despite these similarities, there are discernible differences in the solution structure and dynamics of these two closely related peptides. Their starting point is a comparison of the end-to-end separation of CGRP in a good solvent (6 M guanidine hydrochloride) to prior measurements of amylin under the same conditions (6). There, both peptides assume rather extended states, with CGRP margin-
ally more expanded than amylin. Upon transfer to aqueous solutions, both peptides collapse, with amylin being noticeably more compact than CGRP. The authors ascribe the overall tendency toward collapse to contacts of the N-loops with nonlocal residues, as previously described in Vaiana et al. (6). Under their solution conditions, CGRP carries only one more charge than amylin. By modulating charge interactions via solution pH, ionic strength, or charge mutants, the authors provide solid evidence that long-range charge repulsion underlies the observed net increase in CGRP size over amylin. These results underscore the critical role of charge repulsion effects on IDP structure (7,8) and even fibril assembly pathways of partially unfolded proteins (9). The authors then go on to compare the dynamical properties of both chains in aqueous solution by separating the observed quenching rates into their static reaction and dynamiccollision-rate components. As summarized in their Table 1, the rates of forming end-to-end contacts for CGRP are surprisingly insensitive to its overall size. One might presume that the chain dynamics simply slows down in proportion to CGRP compaction, which makes it more difficult to move between conformations. By quantifying the nearly twofold difference in intrachain dynamics of CGRP and amylin at identical overall size, the authors provide a compelling argument that compaction alone is insufficient to account for the size-induced changes in chain dynamics. Instead, the authors argue that the differences in peptide sequence result in distinct intrachain and chain-solvent interactions that are underlying the observed differences in chain dynamics of these two peptides. The above results provide a detailed image of both structural and dynamic features of these two peptides. The
http://dx.doi.org/10.1016/j.bpj.2015.07.024
New & Notable
authors also present qualitative arguments how the sensitivity of the CGRP structure to net charge (via pH) correlates with pH changes associated with of migraine attacks. Most importantly, though, this article emphasizes that a detailed analysis of chain dynamics in addition to the structural characterization of IDPs is essential for describing their physical state. REFERENCES 1. Xue, B., A. K. Dunker, and V. N. Uversky. 2012. Orderly order in protein intrinsic disor-
853 der distribution: disorder in 3500 proteomes from viruses and the three domains of life. J. Biomol. Struct. Dyn. 30:137–149. 2. Habchi, J., P. Tompa, ., V. N. Uversky. 2014. Introducing protein intrinsic disorder. Chem. Rev. 114:6561–6588. 3. Sizemore, S. M., S. M. Cope, ., S. M. Vaiana. 2015. Slow internal dynamics and charge expansion in the disordered protein CGRP: a comparison with amylin. Biophys. J. 109:1038–1048. 4. Lapidus, L. J., W. A. Eaton, and J. Hofrichter. 2000. Measuring the rate of intramolecular contact formation in polypeptides. Proc. Natl. Acad. Sci. USA. 97:7220–7225. 5. Rubinstein, M., and R. H. Colby. 2003. Polymer Physics. Oxford University Press, Oxford, UK.
6. Vaiana, S. M., R. B. Best, ., J. Hofrichter. 2009. Evidence for a partially structured state of the amylin monomer. Biophys. J. 97:2948– 2957. 7. Mu¨ller-Spa¨th, S., A. Soranno, ., B. Schuler. 2010. Charge interactions can dominate the dimensions of intrinsically disordered proteins. Proc. Natl. Acad. Sci. USA. 107:14609–14614. 8. Das, R. K., and R. V. Pappu. 2013. Conformations of intrinsically disordered proteins are influenced by linear sequence distributions of oppositely charged residues. Proc. Natl. Acad. Sci. USA. 110:13392–13397. 9. Hill, S. E., T. Miti, ., M. Muschol. 2011. Spatial extent of charge repulsion regulates assembly pathways for lysozyme amyloid fibrils. PLoS One. 6:e18171.
Biophysical Journal 109(5) 852–853
