Secondary Structure Preferences Research Articles

The presence of non-native helical structure in the unfolding of a beta-sheet protein MPT63.

MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a β-sandwich protein containing 11 β strands and a very short stretch of 310 helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete β-sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV-CD and FTIR spectra clearly suggest that the low-pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete β-sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non-native helical propensity of the protein structure.

Mechanism Underlying Conformational Effects of a Disease-Associated Hydrophobic-to-Hydrophobic Substitution on an Intrinsically Disordered Region

Disease-associated Single Nucleotide Polymorphisms (SNPs) are common in the disordered regions of proteins (21.7 % of SNPs are found in the disordered region). Most mutational studies of IDP's consider loss or gain of a charged residue. In this study, we explore the local and global effect of a charge-neutral mutation between two hydrophobic residues, with known effects on function: the Val66Met SNP in the 100-residue disordered prodomain of Brain Derived Neurotropic Factor (BDNF). Val66Met is the most frequently found SNP in the BDNF disordered domain, and is associated with various neurological and psychiatric disorders such as bipolar disorder and Alzheimer's disease. Previously, NMR studies demonstrated that the prodomain is disordered with different secondary structure preferences for Val and Met at 275K (Anastasia et al 2013). We attempt to elucidate this phenomenon at the microscopic level using large-scale, fully atomistic temperature replica exchange calculations of both the Val and Met forms of the BDNF prodomain. MD simulations indicate high propensity of secondary structure in regions similar to those identified in NMR studies. Interestingly, we observe reversed temperature dependence of the secondary structure around the SNP between Val and Met from 300K to 420K. With increasing temperature, Val66 is less likely to assume helical secondary structure, while Met66 is more likely, which is consistent with previous observations that entropy of the valine side-chain is significantly reduced upon helix formation. This cost likely offsets the entropic gain of nearby water molecules upon helix formation, associated with the hydrophobic effect and common to Val66 and Met66 helices. At room temperature, we also observe an increase in the radius of gyration of the Met66 prodomain relative to the Val66 prodomain, which can be reliably attributed to differential hydrogen bonding preferences of residues near in sequence to the SNP, affecting their likelihood of forming beta-bridges with distant residues. This is consistent with an experimentally observed higher hydrodynamic radius for the Met66 proregion than for Val66 proregion. These results indicate the seemingly subtle substitution may exert its effects by critically adjusting entropic cost of local secondary-structure elements, which, in turn, affects the conformational ensemble via differential long-range beta bridging patterns. Furthermore, although the SNP is neutral, it alters the exposure of charged residues around the SNP, which can potentially affect the binding characteristics of the prodomain to its highly charged receptor, SORCS2.

Temperature Dependent Pair-Wise Amino Acid Structural Propensity in ccβ and Implications for α to β Secondary Structural Conversion

Numerous proteins undergo an alpha-helix to beta-sheet conversion, which can then lead to the formation of toxic amyloid fibrils. The resulting fibrous protein aggregation is associated with the pathology of many debilitating illnesses and neurodegenerative disorders, such as type II diabetes, Alzheimer's disease, and Parkinson's disease. Many factors determine the preference that an amino acid has to assume an α-helix versus β-strand secondary structure. The large size of these proteins presents difficulties in investigating the molecular details of the dynamics of the structural transformation. We investigate the small, engineered protein ccβ that undergoes similar structural transformations. We use Replica Exchange Molecular Dynamics computer simulations to study the amino acids that make up the system. Based upon the importance of amino acid pairs, we examine the propensities for the various pairs of amino acids in ccβ to form specific secondary structures as a function of temperature. We find that there is an interplay between the inherent secondary structure preferences of amino acids with various molecular forces necessary to stabilize the helical trimer. Three specific pairs of amino acids in the ccβ primary sequence are especially important in the temperature-dependent structural conversion, and these pairs are located at positions along the sequence that facilitate the formation of the coiled-coil trimer. We show these effects vary with temperature, leading to the temperature dependent conversion of ccβ from helical to beta conformation.

Secondary Structure and Neighbor Preferences of Amino Acids

Secondary Structure and Neighbor Preferences of Amino Acids

Comparison of hydration behavior and conformational preferences of the Trp-cage mini-protein in different rigid-body water models.

The secondary structure conformational properties and hydration shell metrics of the Trp-cage mini-protein are examined in the folded and unfolded ensembles in mTIP3P, TIP4P, and TIP4P-Ew water models with the CHARMM22 force-field using molecular dynamics simulations at 250 K. Upon changing the water model, the conformational order metrics of the peptide show significant differences in the unfolded rather than in the folded ensemble. The unfolding temperatures for Trp-cage are observed to be around 460, 470, and 430 K in mTIP3P, TIP4P, and TIP4P-Ew, respectively. Upon comparing the results with a previous study on a 16-residue β-hairpin fragment of the 2GB1 protein, the same set of conformational order metrics are found to be insufficient in describing the free energy landscape of peptides having a distinct native secondary structure. However, the hydration shell properties of the peptide have been found to be independent of the sequence of the peptide and it changes in conformation upon unfolding. Our calculations reveal that for a particular water model, the secondary structure preferences in the unfolded ensembles of the two peptides are qualitatively different. The unfolded structures of Trp-cage prefer extended and compact structures in TIP4P-Ew and mTIP3P water, respectively, whereas the β-hairpin peptide prefers extended unfolded structures in mTIP3P. The conformational preferences of the unfolded peptide in a given water model have been found to depend on the peptide sequence, where the binding energies of the water molecules around the polar residues in the unfolded conformations show sensitivity to the multipole moments of the water models. The significance of an accurate description of peptide-solvent interactions in the parametrization of biomolecular force-fields, to obtain an accurate description of conformational preferences, in particular in the unfolded ensembles of proteins, is highlighted.

The dipeptide conformations of all twenty amino acid types in the context of biosynthesis.

There have been many studies of dipeptide structure at a high level of accuracy using quantum chemical methods. Such calculations are resource-consuming (in terms of memory, CPU and other computational imperatives) which is the reason why most previous studies were restricted to the two simplest amino-acid residue types, glycine and alanine. We improve on this by extending the scope of residue types to include all 20 naturally occurring residue types. Our results reveal differences in secondary structure preferences for the all residue types. There are in most cases very deep energy troughs corresponding either to the polyproline II (collagen) helix and the α-helix or both. The β-strand was not strongly favoured energetically although the extent of this depression in the energy surface is, while not “deeper” (energetically), has a wider extent than the other two types of secondary structure. There is currently great interest in the question of cotranslational folding, the extent to which the nascent polypeptide begins to fold prior to emerging from the ribosome exit tunnel. Accordingly, while most previous quantum studies of dipeptides were carried out in the (simulated) gas or aqueous phase, we wished to consider the first step in polypeptide biosynthesis on the ribosome where neither gas nor aqueous conditions apply. We used a dielectric constant that would be compatible with the water-poor macromolecular (ribosome) environment.

Ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB.

Molecular mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Average errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a physically motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reproduction of NMR χ1 scalar coupling measurements for proteins in solution. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.

Free Energy Landscapes of Alanine Oligopeptides in Rigid-Body and Hybrid Water Models.

Replica exchange molecular dynamics is used to study the effect of different rigid-body (mTIP3P, TIP4P, SPC/E) and hybrid (H1.56, H3.00) water models on the conformational free energy landscape of the alanine oligopeptides (acAnme and acA5nme), in conjunction with the CHARMM22 force field. The free energy landscape is mapped out as a function of the Ramachandran angles. In addition, various secondary structure metrics, solvation shell properties, and the number of peptide-solvent hydrogen bonds are monitored. Alanine dipeptide is found to have similar free energy landscapes in different solvent models, an insensitivity which may be due to the absence of possibilities for forming i-(i + 4) or i-(i + 3) intrapeptide hydrogen bonds. The pentapeptide, acA5nme, where there are three intrapeptide backbone hydrogen bonds, shows a conformational free energy landscape with a much greater degree of sensitivity to the choice of solvent model, though the three rigid-body water models differ only quantitatively. The pentapeptide prefers nonhelical, non-native PPII and β-sheet populations as the solvent is changed from SPC/E to the less tetrahedral liquid (H1.56) to an LJ-like liquid (H3.00). The pentapeptide conformational order metrics indicate a preference for open, solvent-exposed, non-native structures in hybrid solvent models at all temperatures of study. The possible correlations between the properties of solvent models and secondary structure preferences of alanine oligopeptides are discussed, and the competition between intrapeptide, peptide-solvent, and solvent-solvent hydrogen bonding is shown to be crucial in the relative free energies of different conformers.

Systematic 3D Screening of Amino Acid Mutations in Pharmacogenes

The identification of genetic factors that influence drug responses often focuses on genes whose variants are predicted to alter pharmacokinetic and/or pharmacodynamic parameters—leading to an increased risk of drug toxicity or therapeutic failure. In this study, we selected 48 genes identified as “Very Important Pharmacogenes (VIPs)” by the PharmGKB database, and developed a fivefeature Structural Disturbance Score (SDS) for their amino acid variants. “SDS Pharmacogenes” is a score that categorizes distinguishable characteristic profiles that annotate VIP variants as functional rather than neutral mutations. Unlike most existing conservation-based measures, SDS Pharmacogenes can be used to evaluate unknown variants of currently 45/48 VIPs and predict the degree to which each one will have strong impacts towards pharmacogenomics, potentially aiding optimization of drug therapy. SDS Pharmacogenes is built upon a systematic screening for structural disturbance of amino acid mutations within the 45/48 VIPs in the context of their 3-dimensional (3D) protein structures. Our variant evaluation pipeline focuses on the changes in inter-residue bonding, protein stability, protein flexibility, drug binding capability, protein-protein interactions, and amino acid dissimilarity, in addition to the localization of the variants and the amino acid secondary structure preference. While expertise in 3D-protein analysis is beneficial, our implementation does not require that an individual with experience in protein structures be engaged in the personalized genome evaluation, nor expect the users must have bioinformatic backgrounds. In addition, the analysis pipeline is systematic and scalable, thus expected to keep pace with the rapid accumulation of pharmacogenomic data. The SDS Pharmacogenes web application is publicly available at http://sdsp. cig.biology.gatech.edu. Keywords: 3D protein structures, amino acid mutations, PharmGKB, SDS pharmacogenes, structural analysis, structural disruption index (SDI), structural disturbance score (SDS), very important pharmacogenes (VIP).

Molecular Dynamics Simulations of 441 Two-Residue Peptides in Aqueous Solution: Conformational Preferences and Neighboring Residue Effects with the Amber ff99SB-ildn-NMR Force Field.

Understanding the intrinsic conformational preferences of amino acids and the extent to which they are modulated by neighboring residues is a key issue for developing predictive models of protein folding and stability. Here we present the results of 441 independent explicit-solvent MD simulations of all possible two-residue peptides that contain the 20 standard amino acids with histidine modeled in both its neutral and protonated states. (3)J(HNHα) coupling constants and δ(Hα) chemical shifts calculated from the MD simulations correlate quite well with recently published experimental measurements for a corresponding set of two-residue peptides. Neighboring residue effects (NREs) on the average (3)J(HNHα) and δ(Hα) values of adjacent residues are also reasonably well reproduced, with the large NREs exerted experimentally by aromatic residues, in particular, being accurately captured. NREs on the secondary structure preferences of adjacent amino acids have been computed and compared with corresponding effects observed in a coil library and the average β-turn preferences of all amino acid types have been determined. Finally, the intrinsic conformational preferences of histidine, and its NREs on the conformational preferences of adjacent residues, are both shown to be strongly affected by the protonation state of the imidazole ring.

RNA Bind-n-Seq: Measuring the Binding Affinity Landscape of RNA-Binding Proteins.

RNA-binding proteins (RBPs) coordinate post-transcriptional control of gene expression, often through sequence-specific recognition of primary transcripts or mature messenger RNAs. Hundreds of RBPs are encoded in the human genome, most with undefined or incompletely defined biological roles. Understanding the function of these factors will require the identification of each RBP's distinct RNA binding specificity. RNA Bind-n-Seq (RBNS) is a high-throughput, cost-effective in vitro method capable of resolving sequence and secondary structure preferences of RBPs. Dissociation constants can also be inferred from RBNS data when provided with additional experimental information. Here, we describe the experimental procedures to perform RBNS and discuss important parameters of the method and ways that the experiment can be tailored to the specific RBP under study. Additionally, we present the conceptual framework and execution of the freely available RBNS computational pipeline and describe the outputs of the pipeline. Different approaches to quantify binding specificity, quality control metrics, and estimation of binding constants are also covered.

Methods for measuring exchangeable protons in glycosaminoglycans.

Recent NMR studies of the exchangeable protons of GAGs in aqueous solution, including those of the amide, sulfamate, and hydroxyl moieties, have demonstrated potential for the detection of intramolecular hydrogen bonds, providing insights into secondary structure preferences. GAG amide protons are observable by NMR over wide pH and temperature ranges; however, specific solution conditions are required to reduce the exchange rate of the sulfamate and hydroxyl protons and allow their detection by NMR. Building on the vast body of knowledge on detection of hydrogen bonds in peptides and proteins, a variety of methods can be used to identify hydrogen bonds in GAGs including temperature coefficient measurements, evaluation of chemical shift differences between oligo- and monosaccharides, and relative exchange rates measured through line shape analysis and EXSY spectra. Emerging strategies to allow direct detection of hydrogen bonds through heteronuclear couplings offer promise for the future. Molecular dynamic simulations are important in this effort both to predict and confirm hydrogen bond donors and acceptors.

Investigation of membrane penetration depth and interactions of the amino-terminal domain of huntingtin: refined analysis by tryptophan fluorescence measurement.

The membrane-association properties of the amino-terminal domain of huntingtin are accompanied by subcellular redistribution of the protein in cellular compartments. In this study we used tryptophan substitution of amino-acid residues at different positions of the huntingtin 1-17 domain (Htt17) to precisely determine, for the first time, the depth of penetration of the peptides within the lipid bilayer. Initially, secondary structure preferences and membrane association properties were quantitatively determined for several membrane lipid compositions; they were found to be closely related to those of the natural peptide, indicating that changes in the sequence had little effect on these characteristics of the domain. The tryptophan-substituted peptides became inserted into the membranes' interfacial region, with average tryptophan positions between 7.5 and 11Å from the bilayer center, in agreement with in-plane orientation of the peptide. Participation of the very-amino terminus of the peptide in the membrane-association process was demonstrated. The results not only revealed the occurrence of association intermediates when the huntingtin 1-17 anchoring sequence became inserted into the membrane but also suggest the formation of aggregates and/or oligomers during membrane association. When inserted, the F11W site was of crucial importance in lipid anchoring and stabilization of the whole peptide, whereas the terminal residues are located close to the membrane surface. The carboxy-terminal tryptophan (F17W), which also constitutes the site of the polyglutamine extension in the natural domain, was found closest to the aqueous environment, accompanied with the highest aqueous quenching constants. These results were used to propose a refined model of lipid interactions of the huntingtin 1-17 domain.

Secondary structure preferences of mn (2+) binding sites in bacterial proteins.

3D structures of proteins with coordinated Mn2+ ions from bacteria with low, average, and high genomic GC-content have been analyzed (149 PDB files were used). Major Mn2+ binders are aspartic acid (6.82% of Asp residues), histidine (14.76% of His residues), and glutamic acid (3.51% of Glu residues). We found out that the motif of secondary structure “beta strand-major binder-random coil” is overrepresented around all the three major Mn2+ binders. That motif may be followed by either alpha helix or beta strand. Beta strands near Mn2+ binding residues should be stable because they are enriched by such beta formers as valine and isoleucine, as well as by specific combinations of hydrophobic and hydrophilic amino acid residues characteristic to beta sheet. In the group of proteins from GC-rich bacteria glutamic acid residues situated in alpha helices frequently coordinate Mn2+ ions, probably, because of the decrease of Lys usage under the influence of mutational GC-pressure. On the other hand, the percentage of Mn2+ sites with at least one amino acid in the “beta strand-major binder-random coil” motif of secondary structure (77.88%) does not depend on genomic GC-content.

Stimuli-Responsivity of Secondary Structures of Glycopolypeptides Derived from Poly(l-glutamate-co-allylglycine)

Copolypeptides containing L-glutamate and various amounts of either D-/DL-/L-allylglycine or D-/DL-/L-(3-(β-D-glucopyranosyl)thio)propylglycine defect units were studied by circular dichroism (CD) and infrared (FT-IR) spectroscopy according to their secondary structures in dependence of pH and temperature. All samples adopt random coil conformation at high pH and α-helix at low pH without evidence for β-sheet formation. Folding into the α-helix structure is strongly affected by the number and configuration of allylglycine defects (which intrinsically stabilize β-sheets). Helix folding is facilitated upon the attachment of D-glucopyranose to the L- (but not the D-) allylglycine units, which is attributed to a different secondary structure preference of the L-(3-(β-D-glucopyranosyl)thio)propylglycine (L: random coil; D: β-sheet) and a majority rule effect.

Structure and Internal Dynamics of Calcitonin Family Peptides: Implications for Amyloid Formation

The calcitonin peptide (Ct) family comprises the intrinsically disordered proteins amylin (IAPP), calcitonin gene-related peptide (CGRP), calcitonin, and adrenomedullins. These are genetically and structurally related hormone peptides that are able to bind to each other's receptors, though with varying degrees of affinity. Some of these peptides form amyloid fibers, while others do not. They contain highly conserved sequence elements that have been experimentally shown to affect the secondary structural preferences of these peptides. The effect of such conserved elements on tertiary structure has not been experimentally explored to the same extent. Detecting tertiary structural properties of IDPs is considerably more challenging due to fast reconfigurations of the backbone over a wide range of possible conformations. High resolution time-resolved techniques are needed. We use a nanosecond laser spectroscopy technique to measure transient tertiary contact formation. This technique reveals information on the structure and internal dynamics of IDPs. We compare members of the Ct family which differ in hydrophobicity and net charge, and study the effect of proline mutations on contact formation rates. We find that functionally required, conserved sequence elements play an important role in determining the structure, internal dynamics and aggregation properties of these peptides.

Sensitivity of local hydration behaviour and conformational preferences of peptides to choice of water model

Hydration of the 16-residue β-hairpin fragment of the protein in the folded and unfolded ensembles is studied with mTIP3P and TIP4P solvent models using the CHARMM22 protein force-field. mTIP3P is a three-site water model which is used for parameterization of the CHARMM force-field and is known to exhibit liquid-state anomalies of water at temperatures about 80 K lower than the experimental temperature. TIP4P is a four-site water model which gives a better description of the experimental phase diagram and liquid-state anomalies of water. At a temperature of 250 K, where the folded ensemble of the peptide is stable and the unfolded ensemble is metastable, secondary structure metrics are much more sensitive to the choice of solvent model in the unfolded, rather than folded, ensemble. In particular, mean values as well as variation in the positional root mean square displacements (RMSD) and configurational entropy are greater in mTIP3P compared to the TIP4P solvent. The peptide structure is relatively more compact in the TIP4P solvent, which supports unfolded as well as hydrophobic core states. In terms of average local order and binding energy of the water surrounding the peptide, strong deviations from bulk behaviour are restricted to the first hydration shell and differences between the folded and unfolded ensembles in the two solvents are small. The strong coupling between the solvent and the peptide is demonstrated, however, by the dependence of the unfolding temperature on the water model (400 K in mTIP3P and 465 K in TIP4P) and the qualitatively different temperature dependence of the hydration layer occupancy signalling the unfolding transition in the two solvents. A residue-wise decomposition of different contributions to the configurational energy indicates that the TIP4P solvent shows far greater variation in the interaction with charged sidegroups of amino acid residues than the mTIP3P solvent. The implications of sequence-dependent sensitivity of peptide secondary structures to the choice of water models for simulating folding-unfolding equilibria and free energy landscapes are discussed.

Improved Generalized Born Solvent Model Parameters for Protein Simulations.

The generalized Born (GB) model is one of the fastest implicit solvent models and it has become widely adopted for Molecular Dynamics (MD) simulations. This speed comes with tradeoffs, and many reports in the literature have pointed out weaknesses with GB models. Because the quality of a GB model is heavily affected by empirical parameters used in calculating solvation energy, in this work we have refit these parameters for GB-Neck, a recently developed GB model, in order to improve the accuracy of both the solvation energy and effective radii calculations. The data sets used for fitting are significantly larger than those used in the past. Comparing to other pairwise GB models like GB-OBC and the original GB-Neck, the new GB model (GB-Neck2) has better agreement to Poisson-Boltzmann (PB) in terms of reproducing solvation energies for a variety of systems ranging from peptides to proteins. Secondary structure preferences are also in much better agreement with those obtained from explicit solvent MD simulations. We also obtain near-quantitative reproduction of experimental structure and thermal stability profiles for several model peptides with varying secondary structure motifs. Extension to non-protein systems will be explored in the future.

Structural Ensembles of Intrinsically Disordered FG-Nucleoporins Depend on Force Field

Intrinsically disordered proteins (IDPs) fulfill important biological roles including cell signaling, cell cycle regulation, and rubber-like elasticity. IDPs pose a tremendous challenge both to traditional structural determination methods as well as their theoretical description via molecular dynamics (MD) simulations: it is difficult to obtain sufficient data to determine the ensembles of structurally heterogeneous systems. Furthermore, because established MD force fields have been developed primarily to study folded proteins, it is not clear how accurately these force fields are able to describe disordered states.Here, we performed microsecond-timescale MD simulations using four recently-developed force fields: Amber ff99SB∗-ILDN, Amber ff03w, CHARMM22∗, and CHARMM36. We studied a set of FG-nucleoporin peptides with sequences derived from yeast Nsp1p. FG-nucleoporins are IDPs responsible for the high selectivity of the nuclear pore complex (NPC). They form a mesh-like structure in the central region of the NPC that controls the passage of macromolecules into and out of the nucleus. FG-nucleoporins are a prototypic example of the biological role of protein disorder, and beyond their particular function are key model systems for disordered proteins.Overall trends, such as temperature-induced unfolding and differences in compactness between cohesive and extended coil domains of Nsp1p, are described reasonably well by all force fields. However, we find marked differences in the extent of hydrogen bonding and secondary structure preferences. The average chain dimensions with the CHARMM force fields are more than 20% larger than the Amber force fields. Taken together, our results strongly suggest that disordered states are particularly sensitive to force field choice. As a next step, we will therefore compare to experimental measurements of both chain dimensions and secondary structure to determine which force field provides the most accurate description.

Electrostatic Interactions Modulate the Structure and Dynamics of Calcitonin Gene-Related Peptide

Calcitonin gene-related peptide (CGRP) is an intrinsically disordered, 37 residue neuropeptide that acts as a potent vasodilator. It is a member of the calcitonin peptide (Ct) family, together with amylin, calcitonin and adrenomedullin. Understanding how sequence variations affect the conformation and dynamics of these genetically and functionally related IDPs is of considerable interest. While the secondary structural preferences of these peptides have been experimentally determined, the tertiary structural preferences are more challenging to detect due to fast reconfigurations of the backbone over a wide range of conformations, and to the very low solubility of certain Ct family peptides. High resolution time-resolved techniques are needed.We use a nanosecond-resolved spectroscopic technique based on tryptophan triplet quenching by cystine to detect tertiary contact formation in CGRP under varying solvent and temperature conditions. Using this technique we had previously found that conserved structural elements of amylin and CGRP induce compact states, characterized by short end-to-end distances(1,2). Here we compare contact formation rates of wild type and mutant CGRPs with different net charge. We find that electrostatic interactions modulate the degree of compaction of CGRP. The observed difference between CGRP and amylin can be rationalized in terms of electrostatic interactions. Interestingly, even with a net charge of +6, CGRP remains more compact in aqueous solvent than in denaturant with high ionic strength, indicating strong attractive intra-molecular interactions. Overall our data are consistent with a common mechanism driving compaction in CGRP and amylin, with electrostatic interactions modulating the degree of compaction in CGRP. We discuss our findings in relation to secondary structural preferences of these peptides and discuss their possible functional role.1 Vaiana S.M. et al. Biophys. J. 97 2009.2 Sara M. Sizemore et al.2012 Biophys. J, Supplement, Abstract.