Teses / dissertações sobre o tema "Structure en coiled-coil"

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2009.
Page 224 blank.
Includes bibliographical references.
The alpha-helical coiled coil is a protein sequence and structural motif that consists of two or more helices in a parallel or antiparallel orientation supercoiling around a central axis. Coiled coils have been observed in a wide range of protein families, and many studies have focused on their sequence and structural diversity over the past half-century. In particular, the observation that coiled coils can be involved in determining protein-protein interactions and protein architectures has prompted the developments of methods to predict the structure of a coiled-coil complex from sequence information alone. In this thesis, I discuss the development of a structurally annotated database of coiled-coil sequence useful for training statistics-based methods of coiled-coil structure prediction. This database was used to retrain and stringently cross-validate the Multicoil method of predicting coiled-coil oligomerization state. In addition, I describe recent work using implicit and explicit structure models to predict dimeric coiled-coil orientation and alignment. Improvements to existing models, insight into coiled-coil structure determinants, and the future of coiled-coil prediction are also discussed.
by Karl N. Gutwin.
Ph.D.

In the course of experiments designed to identify and characterize structural proteins of the nuclear matrix, one antibody was generated which recognized an extraction-resistant cytoplasmic protein. This antibody was used as the starting point in the cloning and molecular characterization of a novel protein of the inter-membrane space of the mitochondrion which has been named mitofilin. Mitofilin is expressed in all human cell types, and murine homologues also exist. Mitofilin associates only with mitochondria and not with other membrane-bounded organelles such as Golgi or endoplasmic reticulum. This observation has been confirmed both by biochemical fractionation and multi-label fluorescence microscopy. Recombinant mitofilin, purified to homogeneity by affinity chromatography and preparative electrophoresis, was used to raise second-generation antibodies. Results of Western blot and immunofluorescence microscopy experiments, identical to those obtained using the original monoclonal antibody, verify the cloning and biochemical characterization. The mitofilin polypeptide contains several regions which are predicted to interact by forming coiled coils; a mitochondrial targeting signal; and a hydrophobic, membrane-spanning domain. During the course of this work, a sequence match was found with a cDNA reported by Icho, et al (1994) for a mRNA preferentially expressed in heart muscle, which they have called HMP. Evidence is presented which contradicts those authors' contention that HMP is a kinesin-like motor protein. In the course of these investigations, methods were developed to detect and quantitate the expression of solubilization-resistant proteins of the nuclear matrix and the nuclear matrix-intermediate filament scaffold. This was accomplished by combining SDS-PAGE, high sensitivity chemiluminescent Western blots, and scanning densitometry. Sensitivity in the picogram range was obtained, and reproducibility was assessed. For semi-quantitative measurements of protein expression in tissue samples, cell number was normalized by measurement of lamin B, the major protein of the nuclear envelope. Results of screening several cell and tissue types for the expression of mitofilin and for the nuclear matrix proteins NuMA, the nucleoporin tpr, and lamin B are presented. These preliminary data suggest a potential connection of over-expression of NuMA, tpr, and mitofilin with ovarian carcinoma. In addition, quantitative analysis of mitofilin expression in a variety of human cell types was done using purified recombinant protein antigen as the standard. The presence of coiled coil domains in these and other proteins associated with cellular sub-structures gave rise to the third area of investigation described here. Experimental observations of the nuclear matrix-intermediate filament scaffold (NMIF), a tissue-wide structure greatly enriched in coiled coil proteins, led to the following hypothesis: that the differentiated cell and tissue architecture which characterizes Metazoa has evolved through the propagation and selective expression of genes encoding a wide variety of coiled coil proteins, and the integration of the gene products into a tissue-wide matrix based on coiled coil interactions. This hypothesis was explored by computer searches of sequence data files. The GenBank phylogenetic sequence files were examined with a heptad repeat analysis program to assess the occurrence of coiled coil proteins. how heptad repeat domains are organized within these proteins, and what structural/functional categories they comprised. Of 102,007 proteins analyzed, 5.95% (6074) contained coiled coil domains: 1.26% (1289) contained "extended" (> 75 amino acid) domains. While the frequency of proteins containing coiled coils was surprisingly constant among all biota, extended coiled coil proteins were 4-fold more frequent in the animal kingdom, and may reflect early events in the divergence of plants and animals. Structure/function categories of extended coils also revealed phylogenetic differences. In pathogens and parasites, many extended coiled coil proteins are external and bind host proteins. In animals, the majority of extended coiled coil proteins were identified as constituents of two categories: 1) myosins and motors, or 2) components of the NMIF. This scaffold, produced by sequential extraction of epithelial monolayers in situ, contains only 1-2% of the cell mass while accurately retaining morphological features of living epithelium. The NMIF incorporates many proteins with extensive, interrupted coiled coil forming domains. The increased occurrence of this type of protein in Metazoa compared to plants or protists supports the hypothesis that a tissue-wide matrix of coiled coil interactions underlies metazoan differentiated cell and tissue structure.

Now that the functioning of microtubules and the actin cytoskeleton has been worked out in enormous detail, the next important task is defining the structure of intermediate filaments that are far behind the other two major skeletal networks due to their inherent resistance to most structural techniques. The evolution of novel structural approaches for flexible proteins is making this possible now. In my thesis I will aim to elucidate the structure and assembly principles of lamin A nuclear intermediate filament protein. To study lamin A, I principally employed chemical cross-linking that allows the capturing of full-length protein structures in solution. I combined this with mass spectrometry approaches to identify cross-linked residues at the various stages of lamin A assembly that were additionally tracked with SILAC labelling and rotary metal shadowing TEM. Unlike previous cross-linking studies on intermediate filaments I use a zero-length self-excluding cross-linking agent EDC that is better tailored for investigation of the polar interactions between multiple unstructured or otherwise flexible charged sequences of lamins. Using this composite approach I interrogated lamin A dimeric and tetrameric assemblies. I elucidated hinge-like properties of the L12 and found indications that L1 and the region containing coil 2A and L2 and the beginning of coil 2B possess properties of linker-like flexibility and of predicted linear α-helical bundle and could act as molecular springs or compression buffers for the nuclear intermediate filaments. Further I confirm the role of the N-terminal unstructured region in lamin A assembly and for the first time show similar role for the C-terminal unstructured region flanking the rod domain of lamin A. Collected data strongly supports the model where both positively charged unstructured regions participate in extensive interaction with acidic rod termini and act as molecular bridges between these in the head-to-tail interface, confirming the uniformity of this principle between cytoplasmic and nuclear intermediate filaments. Formation of these bridges requires conformational change likely happening due to proline residues in the mitotic phosphorylation sites. Finally I suggest a mechanism of regulation of the order of assembly unique to the nuclear intermediate filament where C-terminal unstructured region blocks lateral interactions until it is tethered to the head-to-tail interface. Collected data on the dynamic behaviour of the C-terminal unstructured region and its ability to tether lamin A Ig domain may have far reaching implications for filament assembly and regulation of binding of hundreds of lamin A partner proteins presenting an important step in our understanding of relationship between lamin A structure and function and how altering the former could lead to disease.

Protein folding studies have provided important insights about the key role of non-covalent interactions in protein structure and conformational stability. Some of these interactions include salt bridges, cation-π, and anion-Ï€ interactions. Understanding these interactions is crucial to developing methods for predicting protein secondary, tertiary, quaternary structure from primary sequence and understanding protein-protein interactions and protein-ligand interactions. Several studies have described how the interaction between two amino acid side chains have a substantial effect on protein structure and conformational stability. This is under the assumption that the interaction between the two amino acids is independent of surrounding interactions. We are interested in understanding how salt bridges, cation-π, and anion-π interactions affect each other when they are in close proximity. Chapter 1 is a brief introduction on noncovalent interactions and noncovalent interaction cooperativity. Chapter 2 describes the progress we have made measuring the cooperativity between noncovalent interactions involving cations, anions and aromatic amino acids in a coiled-coil alpha helix model protein. Chapter 3 describes cooperativity between cation, anion, and nonaromatic hydrophobic amino acids in the context of a coiled-coil alpha helix. In chapter 4 we describe a strong anion-π interaction in a reverse turn that stabilizes a beta sheet model protein. In chapter 5 we measure the interaction between a cysteine linked maleimide and two lysines in a helix and show that it is a general strategy to stabilize helical structure.

Knowledge of how a polypeptide folds from a space-filling random coil into a biologically-functional, three-dimensional structure has been the essence of the protein folding problem. Though mechanistic details of DNA transcription and RNA translation are well understood, a specific code by which the primary structure dictates the acquisition of secondary, tertiary, and quarternary structure remains unknown. However, the demonstrated reversibility of in vitroprotein folding allows for a thermodynamic analysis of the folding reaction. By probing both the equilibrium and kinetics of protein folding, a protein folding mechanism can be postulated. Over the past 40 years, folding mechanisms have been determined for many proteins; however, a generalized folding code is far from clear. Furthermore, most protein folding studies have focused on monomeric proteins even though a majority of biological processes function via the association of multiple subunits. Consequently, a complete understanding of the acquisition of quarternary protein structure is essential for applying the basic principles of protein folding to biology. The studies presented in this dissertation examined the folding and assembly of two very different multimeric proteins. Underlying both of these investigations is the need for a combined analysis of a repertoire of approaches to dissect the folding mechanism for multimeric proteins. Chapter II elucidates the detailed folding energy landscape of HIV-1 protease, a dimeric protein containing β-barrel subunits. The folding of this viral enzyme exhibited a sequential three-step pathway, involving the rate-limiting formation of a monomeric intermediate. The energetics determined from this analysis and their applications to HIV-1 function are discussed. In contrast, Chapter III illustrates the association of a coiled coil component of L. terrestriserythrocruorin. This extracellular hemoglobin consists of a complex scaffold of linker chains with a central ring of interdigitating coiled coils. Allostery is maintained by twelve dodecameric hemoglobin subunits that dock upon this scaffold. Modest association was observed for this coiled coil, and the implications of this fragment to linker assembly are addressed. These studies depict the complexity of multimeric folding reactions. Chapter II demonstrates that a detailed energy landscape of a dimeric protein can be determined by combining traditional equilibrium and kinetic approaches with information from a global analysis of kinetics and a monomer construct. Chapter III indicates that fragmentation of large complexes can show the contributions of separate domains to hierarchical organization. As a whole, this dissertation highlights the importance of pursuing mulitmeric protein folding studies and the implications of these folding mechanisms to biological function.

Les remorines (REMs) sont des protéines multifonctionnelles qui jouent des rôles essentiels dans l'immunité des plantes, le développement et la symbiose en s'associant à la membrane plasmique et en séquestrant des lipides spécifiques dans des nanodomaines membranaires fonctionnels. Ces protéines sont classées dans une famille multigénique avec six groupes caractérisés par des compositions distinctes de domaines de protéines. Tous les membres de la famille des REMs partagent une ancre membranaire C-terminale (REM-CA), un domaine d'homo-oligomérisation, et une région N-terminale intrinsèquement désordonnée (IDR) de longueur variable. De manière unique, les REMs contournent la voie sécrétoire pour cibler la membrane et se localisent dans distincts nanodomaines en fonction de leur groupe phylogénétique. Dans cette étude, nous avons combiné la spectroscopie de Résonance Magnétique Nucléaire (RMN), les calculs de structure de protéines et des simulations avancées de dynamique moléculaire (MD) pour révéler les propriétés de structuration et de dynamiques des REMs. Nous avons découvert que les REMs forment des dimères stables pré-structurés en coiled-coil dans le cytosol, qui agissent comme des unités modulables pour cibler des nanodomaines. Ces dimères présentent, avant l'association avec la membrane, une charge positive de la surface en forme de code-barres, dépendante des REMs. En outre, les REM-CA montrent des variations en structures et dynamiques au sein de la famille, fournissant une plateforme sélective pour l’association avec les phospholipides lors du contact avec la membrane. L'IDR N-terminale forme un ensemble structural flexible en forme de « fuzzy coat » autour du coeur coiled-coil. Les ancres C-terminales créent une avidité à travers des interactions électrostatiques multivalentes entre les groupes des lipides anioniques et la surface chargée positivement du dimère, indiquant un mécanisme synergique entre REM-CA et le domaine coiled-coil pour ségréguer les nanodomaines lipides-protéines. La RMN du solide et les simulations MD à gros grain de REMs sur la membrane lipidique ont également révélé le comportement distinct des REM-CA lorsqu'ils sont associés aux lipides de la membrane. Nous observons des différences dans les profils d'association des REM-CA et des coiled-coils chargés à la membrane, en fonction des charges de surface du dimère et des lipides présents dans la membrane. La stabilité du coiled-coil et l'intensité de l'association à la membrane sont modulées par les groupement des tête chargées des lipides présents à la surface de la membrane. Ces découvertes améliorent notre compréhension des mécanismes moléculaires sous-jacents au rôle des REMs dans l'organisation de la membrane des plantes, des localisations séléctives des REMs dans les nanodomaines des membranes et des facteurs structurales contribuant aux différentes fonctions des remorines. Cette recherche propose une base pour de futures études visant à élucider les comportements complexes des REMs associés aux membranes et les ajustement des structures lors des mécanismes de signalisation et de défense cellulaires
Remorins are multifunctional proteins that play vital roles in plant immunity, development, and symbiosis by associating with the plasma membrane and sequestering specific lipids into functional membrane nanodomains. These proteins are classified into a multigenic family with six groups characterized by distinct protein-domain compositions. All remorin family members share a C-terminal membrane anchor (REM-CA), a homo-oligomerization domain, and the N-terminal is an intrinsically disordered region (IDR) of variable length. Uniquely, REMs bypass the secretory pathway for membrane targeting and localize to different nanodomains based on their phylogenetic group. In this study, we combined Nuclear Magnetic Resonance (NMR) spectroscopy, protein structure calculations, and advanced molecular dynamics (MD) simulations to reveal the structural and dynamic properties of REMs. We discovered that remorins form stable pre-structured coiled-coil dimers in the cytosol, which act as tunable nanodomain-targeting units. These dimers feature a REM-dependent barcode-like positive surface charge before membrane association. Furthermore, the REM-CAs exhibit structural and dynamic variations across the family, providing a selective platform for phospholipid binding upon membrane contact. The N-terminal IDR forms a flexible fuzzy structural ensemble around the coiled-coil core. The C-terminal anchors create avidity through multivalent electrostatic interactions between anionic lipid headgroups and the positively charged dimer surface, supporting a synergistic mechanism between REM-CA and the coiled-coil domain to segregate lipid-protein nanodomains. Solid-state NMR and coarse-grained MD simulations further revealed the distinct behavior of REM-CAs when associated to the lipid membrane. We observe differences in membrane association profiles of the REM-CAs and of the charged coiled-coils dependent on the dimer surface charges and dependent on the lipids present in the membrane. Coiled-coil stability and the intensity of membrane association is tuned by the lipid headgroups on the membrane surface. The insights enhance our understanding of the molecular mechanisms underlying the role of remorins in membrane organization in plants, the distinct localizations of remorins in membrane nanodomains and the structural factors contributing to the different remorin functions. This research lays the groundwork for future studies to elucidate the complex behaviors of membrane-associated REMs and their structural tuning during cellular signaling and defense mechanisms

Thesis (Ph. D.)--Indiana University, Dept. of Chemistry, 2009.
Title from PDF t.p. (viewed Feb. 10, 2010). Source: Dissertation Abstracts International, Volume: 70-05, Section: B, page: 2913. Adviser: Martha G. Oakley.

Les septines forment une famille de GTPases conservée chez les champignons et dans les cellules animales [Kinoshita et al., 2003]. Pendant la division cellulaire, elles se localisent aux sites de cytocinèse et sont essentielles pour ce processus dans la levure bourgeonnante, les embryons de drosophile et les cellules de mammifère en culture [Faty et al., 2002]. Dans les levures bourgeonnantes, les septines, composées de réseaux parallèles de 1laments [Byers et al., 1976], forment un anneau au cou mère-fille. Cet anneau est étroitement associé à la membrane plasmique et constitue un échafaudage pour le recrutement de la myosine II et d'autres facteurs cytocinétiques au futur site de clivage [Longtine et al., 2003]. De plus, l'anneau de septines contribue à la formation d'une barrière de diffusion latérale sur la membrane plasmique, qui aide à maintenir les facteurs de la polarité cellulaire dans le bourgeon [Barral et al., 2000; Takizawa et al., 2000]. Chez les métazoaires, les septines sont aussi requises pour la compartimentation du cortex cellulaire [Schmidt et al., 2004; Joo et al., 2005] et sont impliquées dans une myriade de processus cellulaires, y compris l'assemblage et l'orientation du corps polaire du fuseau [Kusch et al., 2002; Spiliotis et al., 2005], l'exocytose et le transport vésiculaire [Hsu et al., 1998;Beites et al., 1999], la migration cellulaire [Finger et al., 2003], et l'apoptose Larisch et al., 2000; Gottfried et al., 2004].[...]Dans ce mémoire, après avoir passé en revue les fonctions essentielles des septines chez la levure bourgeonnante Saccharomyces cerevisiae, je présente une étude dans laquelle j’ai démontré que UNC-59 et UNC-61 forment un complexe hétérotétramérique capable de s’associer en structures d’ordre supérieur et en filaments dans les cellules et que le complexe hétérotétramérique de septines est assemblé à partir d’hétérodimères UNC-59/UNC-61. De plus, au cours d’un projet visant à développer un outil moléculaire pour l’étude des fonctions des septines in vivo dans des cellules sauvages de Saccharomyces, j’ai mis au point une méthode d’isolement d’intrabodies à partir de la banque d’anticorps recombinants humains en phage display ETH-2 et démontré leur activité d’inhibition de la formation de l’anneau de septines au col du bourgeon dans les cellules de Saccharomyces cerevisiae. L’ensemble des résultats présentés met en lumière l'arrangement moléculaire des monomères dans le complexe de septines et suggère que les complexes de septines s’assemblent dans les filaments et les structures d'ordre supérieur au col du bourgeon chez Saccharomyces cerevisiae. Ainsi, nous proposons que les septines forment la quatrième composante du cytosquelette
Septins form a family of GTPases conserved in fungi and animal cells [Kinoshita etal., 2003]. During cell division, they localize at cytokinesis sites and are essential for this process in budding yeast, Drosophila embryos and cultured mammalian cells [Fatyet al., 2002].In budding yeasts, septins, composed of parallel networks of filaments [Byers et al.,1976], form a mother-daughter neck ring. This ring is closely associated with the plasma membrane and constitutes a scaFold for the recruitment of myosin II and other cytokinetic factors at the future cleavage site [Longtine et al., 2003]. In addition, the septin ring contributes to the formation of a lateral diffusion barrier on the plasma membrane, which helps maintain the factors of cell polarity in the bud [Barral et al.,2000; Takizawa et al., 2000].In metazoans, septins are also required for compartmentalization of the cellular cortex [Schmidt et al., 2004; Joo et al., 2005] and are involved in a myriad of cellular processes, including assembly and orientation of the polar body of the spindle [Kusch etal., 2002; Spiliotis et al., 2005], exocytosis and vesicular transport [Hsu et al., 1998;Beites et al., 1999], cell migration [Finger et al., 2003], and apoptosis [Larisch et al.,2000; Gottfried et al., 2004].[...]The set of results presented highlights the molecular arrangement of the monomersin the septin complex and suggests that the septin complexes assemble in filaments and higher order structures at the bud neck in Saccharomyces cerevisiae. Thus, wepropose that septins form the fourth component of the cytoskeleton

The structure-property relationships of an artificial protein hydrogel, which was constructed from a triblock protein (designated AC10A) that contained two associative leucine-zipper endblocks and a water-soluble random coil midblock, were investigated to provide guidelines for the rational design of new generations of artificial protein hydrogels. The leucine zipper A domain is composed of six heptad repeating units designated as abcdefg, where the a and d positions are occupied by hydrophobic residues, and the e and g positions are mainly occupied by glutamic acid residues. In contrast to hydrogels formed from synthetic hydrophobically modified polymers, the normalized plateau storage modulus G/nkT of the AC10A gel was below 13% at all concentrations examined. This indirect evidence that AC10A tends to form a substantial fraction of looped configurations was supported by a fluorescence quenching experiment: significant quenching occurred in labeled d-AC10A-a (d=tryptophan at the N-terminus, a=coumarin at the C terminus) chains mixed with a great excess of unlabelled AC10A chains in a solution. The strong tendency to form loops originates in large part from the compact size of the random coil midblock domain (mean RH, C10~20 A, determined from quasi-elastic light scattering of C10). Despite the small aggregation number of the leucine zipper domains (tetrameric aggregates, determined from multi-angle static light scattering of AC10 diblock), the average center-to-center distance between aggregates in a 7% w/v AC10A solution is roughly 3 times the radius of gyration and 1.5 times the average end-to-end distance of the C10 domain. To avoid the energy penalty for stretching the C10 domain to form bridges, the chains tend to form loops. The importance of loops explains the nonmonotonic effect of pH on modulus and the decrease in modulus with increasing ionic strength. It also led to the design concept of increasing the midblock length or charge density to increase storage modulus.

Dynamic properties of the AC10A hydrogel show correlation between network relaxation behavior and molecular exchange kinetics of the associative domain. The longest stress relaxation time changes from ca. 70 seconds at pH 8.0 to ca. 1000 seconds at pH 7.0, determined by creep measurements on 7% w/v gels. In a parallel manner, the characteristic time of the leucine zipper strand exchange varies from ca. 200 seconds at pH 8.0 to ca. 4500 seconds at pH 7.0, determined by fluorescence de-quenching after mixing a fluorescein-labeled leucine zipper solution (in which fluorescence was quenched) with a great excess of an unlabeled leucine zipper solution. Both time scales vary strongly with pH due to the associated change in charge on the e and g residues of the leucine zipper.

The observed structure-property relationships suggest that the rapid dissolution that occurs with AC10A hydrogels in open systems originates from the tendency of the protein to form loops, the small aggregation number of the associative domains, and the transient nature of association. For applications in which materials are surrounded by excess fluids, we demonstrated two molecular design approaches to avoid the rapid dissolution. One way to slow dissolution is to suppress loops by engineering a triblock protein with dissimilar associative endblocks, PC10A, such that P associates only with P and A associates only with A. A PC10A gel erodes 500 times more slowly and exhibits a 5-fold increase in modulus compared to an AC10A gel at the same concentration. Alternatively, hydrogel stability in open systems can be improved by engineering a cysteine residue into each leucine zipper domain to allow covalent bond formation following physical association of leucine zippers. Asymmetric placement of the cysteine residue in each leucine zipper domain suppresses locking-in loops and creates linked "multichains". The increased valency of the building units stabilizes the hydrogels in open systems, while the physical nature of their association retains the reversibility of gelation. The gel networks dissolve at pH 12.2, where the helicity of the leucine zipper domains is reduced by ca. 90%, and re-form upon acidification.

The ability to form robust artificial protein hydrogels in open systems opens the way to biomedical applications. Therefore, we examined their toxicity and incorporated an RGD cell-binding domain into the midblock backbone. Viability assays for mammalian 3T3 fibroblast cells cultured in the presence of the AC10A protein revealed no evidence of toxicity. Anchorage-dependent epithelial cells spread well on hydrogel films bearing an RGD cell-binding domain. In contrast, cells remained round on films without the cell-binding domain; significant apoptosis was induced. Encapsulated 3T3 fibroblast cells remained viable inside the hydrogel for at least 12 hours, suggesting that these materials have proper permeability for transferring oxygen, nutrients, and metabolic waste. The hydrogel containing the RGD domain was micropatterned on a PEG-modified glass surface and limited cell adhesion to the hydrogel region.

Hyperthermophilic proteins are of great interest in both the academic and industrial world in understanding how these proteins are capable of retaining their biological activity under such harsh environmental conditions. This thesis studies a tetrabrachion stalk domain from Staphylothermus marinus, know as Right Handed Coiled Coil (RHCC). This protein is of interest due to its extreme thermostability and its affinity for heavy metals. We aim to better understand the reason for the extreme thermal stability of the protein and to take advantage of the proteins affinity for heavy metals with a view to developing a novel approach to bioremediate Hg2+, a major environmental pollutant. Our results clearly indicated that the protein is more thermostable in alkaline conditions in comparison to acidic conditions. This observation can be explained by careful inspection of the high resolution structure. Our data also clearly show that RHCC is able to bind ionic mercury compounds such as mercury nitrate and dipotassium mercury iodide.

碩士
國立臺灣大學
化學研究所
104
Ion pairing interactions play important roles in protein structure stability. Stabilizing ion pairing interactions are formed between two oppositely charged residues such as Arg/Lys and Asp/Glu. To gain insight into ion pairing interactions and their potential roles in protein structure, we have designed a β-hairpin peptide system, allowing the measurement of the stability effects of individual charged residues and ion pairing interactions. Herein, we study the effect of side chain length on cross-strand diagonal ion pairing interactions. Peptides were synthesized by solid phase methods and purified by reverse phase HPLC. The sequence specific assignments were obtained based on TOCSY, ROESY, and DQF-COSY spectra. The β-hairpin structures were confirmed by chemical shift deviation, 3JHNα coupling constants, and NOE signals. The fraction folded and ΔGfold of the peptides were derived by comparing the chemical shifts with the folded and unfolded reference peptides. The stability of the peptides followed the trend : HPDAadDab ~ HPDAadDap > HPDGluDab ~ HPDGluDap > HPDAspDab ~ HPDAspDap. The results indicate that pairing the longer side chains provides the most stable β-hairpin. Coiled coil is a common motif found in nature. These motifs are ideal models for the study of protein secondary and teriary inteactions because the relationship between sequence and stability are well-understood. Incorporation of fluorinated amino acid residues into coiled coil peptides can stabilize coiled coils. This stabilization has been referred to as the fluoro-stabilization effect. To further understand the fluoro-stabilization effect in proteins, peptides based on the leucine zipper region of the yeast transcription factor GCN4 was designed and fluorinated amino acids were incorporated at the hydrophobic positions.