Hsp70 molecular chaperones and Parkinson's disease

Because over expression of Hsp70 molecular chaperones suppresses the toxicity of aberrantly folded proteins that occur in Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis, and various polyQ‐diseases (Huntington's disease and ataxias), Hsp70 is garnering attention as a possible therapeutic agent for these various diseases. Here, I review progress in this fascinating field of molecular chaperones and neurodegeneration and describe our current understanding of the mechanisms by which Hsp70 protects cells from the PD‐related protein called alpha‐synuclein (α‐syn). © 2009 Wiley Periodicals, Inc. Biopolymers 93: 218–228, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformational stability of the full‐atom hexameric model of the ClpB chaperone from Escherichia coli

The Escherichia coli heat shock protein ClpB, a member of the Hsp100 family, plays a crucial role in cellular thermotolerance. In co‐operation with the Hsp70 chaperone system, it is able to solubilize proteins aggregated by heat shock conditions and refold them into the native state in an ATP‐dependent way. It was established that the mechanism of ClpB action depends on the formation of a ring‐shaped hexameric structure and the translocation of a protein substrate through an axial channel. The structural aspects of this process are not fully known. By means of homology modeling and protein–protein docking, we obtained a model of the hexameric arrangement of the full‐length ClpB protein complexed with ATP. A molecular dynamics simulation of this model was performed to assess its flexibility and conformational stability. The high mobility of the “linker” M‐domain, essential for the renaturing activity of ClpB, was demonstrated, and the size and shape of central channel were analyzed. In this model, we propose the coordinates for a loop between b4 and B6 structural elements, not defined in previous structural research, which faces the inside of the channel and may therefore play a role in substrate translocation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 47–60, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structure and function of the molecular chaperone Hsp104 from yeast

The molecular chaperone Hsp104 plays a central role in the clearance of aggregates after heat shock and the propagation of yeast prions. Hsp104's disaggregation activity and prion propagation have been linked to its ability to resolubilize or remodel protein aggregates. However, Hsp104 has also the capacity to catalyze protein aggregation of some substrates at specific conditions. Hence, it is a molecular chaperone with two opposing activities with respect to protein aggregation. In yeast models of Huntington's disease, Hsp104 is required for the aggregation and toxicity of polyglutamine (polyQ), but the expression of Hsp104 in cellular and animal models of Huntington's and Parkinson's disease protects against polyQ and α‐synuclein toxicity. Therefore, elucidating the molecular determinants and mechanisms underlying the ability of Hsp104 to switch between these two activities is of critical importance for understanding its function and could provide insight into novel strategies aimed at preventing or reversing the formation of toxic protein aggregation in systemic and neurodegenerative protein misfolding diseases. Here, we present an overview of the current molecular models and hypotheses that have been proposed to explain the role of Hsp104 in modulating protein aggregation and prion propagation. The experimental approaches and the evidences presented so far in relation to these models are examined. Our primary objective is to offer a critical review that will inspire the use of novel techniques and the design of new experiments to proceed towards a qualitative and quantitative understanding of the molecular mechanisms underlying the multifunctional properties of Hsp104 in vivo. © 2009 Wiley Periodicals, Inc. Biopolymers 93:252–276, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Plasticity of the Hsp90 chaperone machine in divergent eukaryotic organisms

Hsp90 is critical for the regulation and activation of numerous client proteins critical for diverse functions such as cell growth, differentiation, and reproduction. Cytosolic Hsp90 function is dependent on a battery of co-chaperone proteins that regulate the ATPase activity of Hsp90 function or direct Hsp90 to interact with specific client proteins. Little is known about how Hsp90 complexes vary between different organisms and how this affects the scope of clients that are activated by Hsp90. This study determined whether ten distinct Hsp90 co-chaperones were encoded by genes in 19 disparate eukaryotic organisms. Surprisingly, none of the co-chaperones were present in all organisms. The co-chaperone Hop/Sti1 was most widely dispersed (18 out of 19 species), while orthologs of Cdc37, which is critical for the stability and activation of diverse protein kinases in yeast and mammals, were identified in only nine out of 19 species examined. The organism with the smallest proteome, Encephalitozoon cuniculi, contained only three of these co-chaperones, suggesting a correlation between client diversity and the complexity of the Hsp90 co-chaperone machine. Our results suggest co-chaperones are critical for cytosolic Hsp90 function in vivo, but that the composition of Hsp90 complexes varies depending on the specialized protein folding requirements of divergent species.     

Evolution and function of diverse Hsp90 homologs and cochaperone proteins

Members of the Hsp90 molecular chaperone family are found in the cytosol, ER, mitochondria and chloroplasts of eukaryotic cells, as well as in bacteria. These diverse family members cooperate with other proteins, such as the molecular chaperone Hsp70, to mediate protein folding, activation and assembly into multiprotein complexes. All examined Hsp90 homologs exhibit similar ATPase rates and undergo similar conformational changes. One of the key differences is that cytosolic Hsp90 interacts with a large number of cochaperones that regulate the ATPase activity of Hsp90 or have other functions, such as targeting clients to Hsp90. Diverse Hsp90 homologs appear to chaperone different types of client proteins. This difference may reflect either the pool of clients requiring Hsp90 function or the requirement for cochaperones to target clients to Hsp90. This review discusses known functions, similarities and differences between Hsp90 family members and how cochaperones are known to affect these functions. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Interplay between protein homeostasis networks in protein aggregation and proteotoxicity

The misfolding and aggregation of disease proteins is characteristic of numerous neurodegenerative diseases. Particular neuronal populations are more vulnerable to proteotoxicity while others are more apt to tolerate the misfolding and aggregation of disease proteins. Thus, the cellular environment must play a significant role in determining whether disease proteins are converted into toxic or benign forms. The endomembrane network of eukaryotes divides the cell into different subcellular compartments that possess distinct sets of molecular chaperones and protein interaction networks. Chaperones act as agonists and antagonists of disease protein aggregation to prevent the accumulation of toxic intermediates in the aggregation pathway. Interacting partners can also modulate the conformation and localization of disease proteins and thereby influence proteotoxicity. Thus, interplay between these protein homeostasis network components can modulate the self‐association of disease proteins and determine whether they elicit a toxic or benign outcome. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 229–236, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Farnesylation of Ydj1 is required for in vivo interaction with Hsp90 client proteins

Ydj1 of Saccharomyces cerevisiae is an abundant cytosolic Hsp40, or J-type, molecular chaperone. Ydj1 cooperates with Hsp70 of the Ssa family in the translocation of preproteins to the ER and mitochondria and in the maturation of Hsp90 client proteins. The substrate-binding domain of Ydj1 directly interacts with steroid receptors and is required for the activity of diverse Hsp90-dependent client proteins. However, the effect of Ydj1 alteration on client interaction was unknown. We analyzed the in vivo interaction of Ydj1 with the protein kinase Ste11 and the glucocorticoid receptor. Amino acid alterations in the proposed client-binding domain or zinc-binding domain had minor effects on the physical interaction of Ydj1 with both clients. However, alteration of the carboxy-terminal farnesylation signal disrupted the functional and physical interaction of Ydj1 and Hsp90 with both clients. Similar effects were observed upon deletion of RAM1, which encodes one of the subunits of yeast farnesyltransferase. Our results indicate that farnesylation is a major factor contributing to the specific requirement for Ydj1 in promoting proper regulation and activation of diverse Hsp90 clients.     

Grp94, the endoplasmic reticulum Hsp90, has a similar solution conformation to cytosolic Hsp90 in the absence of nucleotide

The molecular chaperone, Hsp90, is an essential eukaryotic protein that assists in the maturation and activation of client proteins. Hsp90 function depends upon the binding and hydrolysis of ATP, which causes large conformational rearrangements in the chaperone. Hsp90 is highly conserved from bacteria to eukaryotes, and similar nucleotide‐dependent conformations have been demonstrated for the bacterial, yeast, and human proteins. There are, however, important species‐specific differences in the ability of nucleotide to shift the conformation from one state to another. Although the role of nucleotide in conformation has been well studied for the cytosolic yeast and human proteins, the conformations found in the absence of nucleotide are less well understood. In contrast to cytosolic Hsp90, crystal structures of the endoplasmic reticulum homolog, Grp94, show the same conformation in the presence of both ADP and AMPPNP. This conformation differs from the yeast AMPPNP‐bound crystal state, suggesting that Grp94 may have a different conformational cycle. In this study, we use small angle X‐ray scattering and rigid body modeling to study the nucleotide free states of cytosolic yeast and human Hsp90s, as well as mouse Grp94. We show that all three proteins adopt an extended, chair‐like conformation distinct from the extended conformation observed for the bacterial Hsp90. For Grp94, we also show that nucleotide causes a small shift toward the crystal state, although the extended state persists as the major population. These results provide the first evidence that Grp94 shares a conformational state with other Hsp90 homologs.     

Stimulation of the weak ATPase activity of human hsp90 by a client protein.

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the folding and assembly of a limited set of "client" proteins, many of which are involved in signal transduction pathways. In vivo, it is found in complex with additional proteins, including the chaperones Hsp70, Hsp40, Hip and Hop (Hsp-interacting and Hsp-organising proteins, respectively), as well as high molecular mass immunophilins, such as FKBP59, and the small acidic protein p23. The role of these proteins in Hsp90-mediated assembly processes is poorly understood. It is known that ATP binding and hydrolysis are essential for Hsp90 function in vivo and in vitro.Here we show, for the first time, that human Hsp90 has ATPase activity in vitro. The ATPase activity is characterised using a sensitive assay based on a chemically modified form of the phosphate-binding protein from Escherichia coli. Human Hsp90 is a very weak ATPase, its activity is significantly lower than that of the yeast homologue, and it has a half-life of ATP hydrolysis of eight minutes at 37 degrees C. Using a physiological substrate of Hsp90, the ligand-binding domain of the glucocorticoid receptor, we show that this "client" protein can stimulate the ATPase activity up to 200-fold. This effect is highly specific and unfolded or partially folded proteins, which are known to bind to Hsp90, do not affect the ATPase activity. In addition, the peroxisome proliferator-activated receptor, which is related in both sequence and structure to the glucocorticoid receptor but which does not bind Hsp90, has no observable effect on the ATPase activity.We establish the effect of the co-chaperones Hop, FKBP59 and p23 on the basal ATPase activity as well as the client protein-stimulated ATPase activity of human Hsp90. In contrast with the yeast system, human Hop has little effect on the basal rate of ATP hydrolysis but significantly inhibits the client-protein stimulated rate. Similarly, FKBP59 has little effect on the basal rate but stimulates the client-protein stimulated rate further. In contrast, p23 inhibits both the basal and stimulated rates of ATP hydrolysis.Our results show that the ATPase activity of human Hsp90 is highly regulated by both client protein and co-chaperone binding. We suggest that the rate of ATP hydrolysis is critical to the mode of action of Hsp90, consistent with results that have shown that both over and under-active ATPase mutants of yeast Hsp90 have impaired function in vivo. We suggest that the tight regulation of the ATPase activity of Hsp90 is important and allows the client protein to remain bound to Hsp90 for sufficient time for activation to occur.     

Regulation of the Hsp90 system

Hsp90 is a highly conserved and abundant chaperone. It participates in essential cellular activities by supporting the maturation process of its client proteins, many of which are protein kinases and steroid receptors. Client processing is achieved via extensive conformational changes within the dimeric chaperone. This requires an ATP hydrolysis activity that is controlled by auto-inhibitory mechanisms and several structurally diverse cofactors. Especially the client-specificity of Hsp90 depends on client-specific cofactors, which can adapt Hsp90's activities to the client requirements at different conditions and in different cell types. Additionally, post-translational modifications can influence almost every aspect of Hsp90's interactions and activities. In this review, we present these regulatory principles, discuss the factors that have an impact on Hsp90's function and elaborate the mechanisms that are responsible for regulating the Hsp90 machinery.     

5′‐N‐ethylcarboxamidoadenosine is not a paralog‐specific Hsp90 inhibitor

The molecular chaperone Hsp90 facilitates the folding and modulates activation of diverse substrate proteins. Unlike other heat shock proteins such as Hsp60 and Hsp70, Hsp90 plays critical regulatory roles by maintaining active states of kinases, many of which are overactive in cancer cells. Four Hsp90 paralogs are expressed in eukaryotic cells: Hsp90α/β (in the cytosol), Grp94 (in the endoplasmic reticulum), Trap1 (in mitochondria). Although numerous Hsp90 inhibitors are being tested in cancer clinical trials, little is known about why different Hsp90 inhibitors show specificity among Hsp90 paralogs. The paralog specificity of Hsp90 inhibitors is likely fundamental to inhibitor efficacy and side effects. In hopes of gaining insight into this issue we examined NECA (5′‐N‐ethylcarboxamidoadenosine), which has been claimed to be an example of a highly specific ligand that binds to one paralog, Grp94, but not cytosolic Hsp90. To our surprise we find that NECA inhibits many different Hsp90 proteins (Grp94, Hsp90α, Trap1, yeast Hsp82, bacterial HtpG). NMR experiments demonstrate that NECA can bind to the N‐terminal domains of Grp94 and Hsp82. We use ATPase competition experiments to quantify the inhibitory power of NECA for different Hsp90 proteins. This scale: Hsp82 > Hsp90α > HtpG ≈ Grp94 > Trap1, ranks Grp94 as less sensitive to NECA inhibition. Because NECA is primarily used as an adenosine receptor agonist, our results also suggest that cell biological experiments utilizing NECA may have confounding effects from cytosolic Hsp90 inhibition.     

Specific DNA G‐quadruplexes bind to ethanolamines

A significant number of G‐quadruplex‐forming sequences have been revealed in human genome by bioinformatic searches, implying that G‐quadruplexes may be involved in important biological processes and may be new chemotherapeutic targets. Therefore, it is important to discover the potential interactions of G‐quadruplexes with other molecules or groups. Here we describe a class of G‐quadruplexes, which can bind to ethanolamine groups that widely exist in biomolecules and drug molecules. The specific interaction of these G‐quadruplexes with ethanolamine groups was identified by high performance affinity chromatography (HPAC) using immobilized ethanolamine and diethanolamine as stationary phase reagents. The circular dichroism (CD) spectra and native polyacrylamide gel electrophoresis (PAGE) show that these ethanolamine binding quadruplexes adopt an intramolecularly parallel structure. The relationship of ethanolamine binding and G‐quadruplexe structure provides new clues for the G‐quadruplex‐related studies as well as for the molecular designs of therapeutic reagents. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 874–883, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Enzymatic activity and thermal stability of metallo proteins in hydrated ionic liquids

Hydrated choline dihydrogen phosphate (Hy[ch][dhp]) containing 30 wt% water was investigated as a novel protein solvent. The Hy[ch][dhp] dissolved some metallo proteins (cytochrome c, peroxidase, ascorbate oxidase, azurin, pseudoazurin and fructose dehydrogenase) without any modification. These proteins retained the surroundings of the active site after dissolution in Hy[ch][dhp]. Some metallo proteins were found to retain their activity in the Hy[ch][dhp]. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1093–1099, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The co-chaperone p23 arrests the Hsp90 ATPase cycle to trap client proteins

The action of the molecular chaperone Hsp90 is essential for the activation and assembly of an increasing number of client proteins. This function of Hsp90 has been proposed to be governed by conformational changes driven by ATP binding and hydrolysis. Association of co-chaperones and client proteins regulate the ATPase activity of Hsp90. Here, we have examined the inhibition of the ATPase activity of human Hsp90beta by one such co-chaperone, human p23. We demonstrate that human p23 interacts with Hsp90 in both the absence and presence of nucleotide with a higher affinity in the presence of the ATP analogue AMP-PNP. This is consistent with an analysis of the effect of p23 on the steady-state kinetics that revealed a mixed mechanism of inhibition. Mass spectrometry of the intact Hsp90.p23 complex determined the stoichiometry of binding to be one p23 to each subunit of the Hsp90 dimer. p23 was also shown to interact with a monomeric, truncated fragment of Hsp90, lacking the C-terminal homodimerisation domain, indicating dimerisation of Hsp90 is not a prerequisite for association with p23. Complex formation between Hsp90 and p23 increased the apparent affinity of Hsp90 for AMP-PNP and completely inhibited the ATPase activity. We propose a model where the role of p23 is to lock individual subunits of Hsp90 in an ATP-dependent conformational state that has a high affinity for client proteins.     

Peptidic modulators of protein‐protein interactions: Progress and challenges in computational design

With the decline in productivity of drug‐development efforts, novel approaches to rational drug design are being introduced and developed. Naturally occurring and synthetic peptides are emerging as novel promising compounds that can specifically and efficiently modulate signaling pathways in vitro and in vivo. We describe sequence‐based approaches that use peptides to mimic proteins in order to inhibit the interaction of the mimicked protein with its partners. We then discuss a structure‐based approach, in which protein‐peptide complex structures are used to rationally design and optimize peptidic inhibitors. We survey flexible peptide docking techniques and discuss current challenges and future directions in the rational design of peptidic inhibitors. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 505–513, 2009. This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Exploring Mechanisms of Allosteric Regulation and Communication Switching in the Multiprotein Regulatory Complexes of the Hsp90 Chaperone with Cochaperones and Client Proteins: Atomistic Insights from Integrative Biophysical Modeling and Network Analysis of Conformational Landscapes

Understanding molecular principles underlying Hsp90 chaperone functions and modulation of client activity is fundamental to dissect activation mechanisms of many proteins. In this work, we performed a computational investigation of the Hsp90-Hsp70-Hop-CR client complex to examine allosteric regulatory mechanisms underlying dynamic chaperone interactions and principles of chaperone-dependent client recognition and remodeling. Conformational dynamics analysis using high-resolution coarse-grained simulations and ensemble-based local frustration analysis suggest that the Hsp90 chaperone could recognize and recruit the GR client by invoking reciprocal dynamic exchanges near the intermolecular interfaces with the client. Using mutational scanning of the intermolecular residues in the Hsp90-Hsp70-Hop-GR complex, we identified binding energy hotspots in the regulatory complex. Perturbation-based network analysis and dynamic fluctuations-based modeling of allosteric residue potentials are employed for a detailed analysis of allosteric interaction networks and identification of conformational communication switches. We found that allosteric interactions between the Hsp90, the client-bound Hsp70 and Hop cochaperone can define two allosteric residue clusters that control client recruitment in which the intrinsic Hsp70 allostery is exploited to mediate integration of the Hsp70-bound client into the Hsp90 chaperone system. The results suggest a model of dynamics-driven allostery that enables efficient client recruitment and loading through allosteric couplings between intermolecular interfaces and communication switch centers. This study showed that the Hsp90 interactions with client proteins may operate under dynamic-based allostery in which ensembles of preexisting conformational states and intrinsic allosteric pathways present in the Hsp90 and Hsp70 chaperones can be exploited for recognition and integration of substrate proteins.     

Structural studies of SNARE complex and its interaction with complexin by molecular dynamics simulation

Since neurotransmitter releasing into the synaptic space delivers electrical signals from presynaptic neural cell to the postsynaptic cell, neurotransmitter secretion must be much orchestrated. Crowded intracellular vesicles involving neurotransmitters present a question of the how secretory vesicles fuse onto the plasma membrane in a fast synchronized fashion. Complexin is one of the most experimentally studied proteins that regulate assembly of fusogenic four‐helix SNARE complex to synchronized neurotransmitter secretion. We used MD simulation to investigate the interaction of complexin with the neural SNARE complex in detail. Our results show that the SNARE complex interacts with the complexin central helix by forming salt bridges and hydrogen bonds. Complexin also can interact with the Q‐SNARE complex instead of synaptobrevin to decrease the Q‐SNARE flexibility. The complexin alpha‐accessory helix and the C‐terminal region of synaptobrevin can interact with the same region of syntaxin. Although the alpha‐accessory helix aids the tight binding of the central helix to the SNARE complex, its proximity with synaptobrevin causes the destabilization of syntaxin and Sn1 helices. This study suggests that the alpha‐accessory helix of complexin can be an inhibiting factor for membrane fusion by competing with synaptobrevin for binding to the Q‐SNARE complex. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 560–570, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Hsp90 Governs Echinocandin Resistance in the Pathogenic Yeast Candida albicans via Calcineurin

Candida albicans is the leading fungal pathogen of humans, causing life-threatening disease in immunocompromised individuals. Treatment of candidiasis is hampered by the limited number of antifungal drugs whose efficacy is compromised by host toxicity, fungistatic activity, and the emergence of drug resistance. We previously established that the molecular chaperone Hsp90, which regulates the form and function of diverse client proteins, potentiates resistance to the azoles in C. albicans and in the model yeast Saccharomyces cerevisiae. Genetic studies in S. cerevisiae revealed that Hsp90''s role in azole resistance is to enable crucial cellular responses to the membrane stress exerted by azoles via the client protein calcineurin. Here, we demonstrate that Hsp90 governs cellular circuitry required for resistance to the only new class of antifungals to reach the clinic in decades, the echinocandins, which inhibit biosynthesis of a critical component of the fungal cell wall. Pharmacological or genetic impairment of Hsp90 function reduced tolerance of C. albicans laboratory strains and resistance of clinical isolates to the echinocandins and created a fungicidal combination. Compromising calcineurin function phenocopied compromising Hsp90 function. We established that calcineurin is an Hsp90 client protein in C. albicans: reciprocal co-immunoprecipitation validated physical interaction; Hsp90 inhibition blocked calcineurin activation; and calcineurin levels were depleted upon genetic reduction of Hsp90. The downstream effector of calcineurin, Crz1, played a partial role in mediating calcineurin-dependent stress responses activated by echinocandins. Hsp90''s role in echinocandin resistance has therapeutic potential given that genetic compromise of C. albicans HSP90 expression enhanced the efficacy of an echinocandin in a murine model of disseminated candidiasis. Our results identify the first Hsp90 client protein in C. albicans, establish an entirely new role for Hsp90 in mediating resistance to echinocandins, and demonstrate that targeting Hsp90 provides a promising therapeutic strategy for the treatment of life-threatening fungal disease.     

Viral structural transition mechanisms revealed by multiscale molecular dynamics/order parameter extrapolation simulation

On the basis of an all‐atom multiscale analysis theory of nanosystem dynamics, a multiscale molecular dynamics/order parameter extrapolation (MD/OPX) approach has recently been developed. It accelerates MD for long‐time simulation of large bionanosystems and addresses rapid atomistic fluctuations and slowly varying coherent dynamics simultaneously. In this study, MD/OPX is optimized and implemented to simulate viral capsid structural transitions. Specifically, 200 ns MD/OPX simulation of the swollen state of cowpea chlorotic mottle virus capsid reveals that it undergoes significant energy‐driven shrinkage in vacuum, which is a symmetry‐breaking process involving local initiation and front propagation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 61–73, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com