The inherent design freedom of protein engineering and recombinant protein production enables specific tailoring of protein structure, function, and properties. Two areas of research where protein engineering has allowed for many advances in biomedical materials include the design of novel protein scaffolds for molecular recognition, as well as the use of recombinant proteins for production of next generation biomaterials. The main focus of my dissertation was to develop new biomedical materials using protein engineering. Chapters three and four discuss the engineering of repeat proteins as bio-recognition modules for biomedical sensing and imaging. Chapter three provides an overview of the most recent advances in engineering of repeat proteins in the aforementioned field. Chapter four discusses my contribution to this field. We have designed a de novo repeat protein scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Innate immunity receptors have been described as pattern recognition receptors in that they recognize "global features" of a family of pathogens versus one specific antigen. In mammals, two main protein families of such receptors are: extracellular Toll-like receptors (TLRs) and cytoplasmic Nucletide-binding domain- and Leucine-rich Repeat-containing proteins (NLRs). NLRs are defined by their tripartite domain architecture that contains a C-terminal LRR (Leucine Rich Repeat) domain, the nucleotide-binding oligomerization (NACHT) domain, and the N-terminal effector domain. It is proposed that pathogen sensing in NLRs occurs through ligand binding by the LRR domain. Thus, we hypothesized that LRRs would be suitable for the design of alternative binding scaffolds for use in molecular recognition. The NOD protein family plays a very important role in innate immunity, and consequently serves as a promising scaffold for design of novel recognition motifs. However, engineering of de novo proteins based on the NOD family LRR domain has proven challenging due to problems arising from protein solubility and stability. Consensus sequence design is a protein design tool used to create novel proteins that capture sequence-structure relationships and interactions present in nature in order to create a stable protein scaffold. We implement a consensus sequence design approach to develop proteins based on the LRR domain of NLRs. Using a multiple sequence alignment we analyzed all individual LRRs found in mammalian NLRs. This design resulted in a consensus sequence protein containing two internal repeats and separate N- and C- capping repeats named CLRR2. Using biophysical characterization methods of size exclusion chromatography, circular dichroism, and fluorescence, CLRR2 was found to be a stable, monomeric, and cysteine free scaffold. Additionally, CLRR2, without any affinity maturation, displayed micromolar binding affinity for muramyl dipeptide (MDP), a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand. Furthermore, CLRR2 demonstrated selective recognition to the biologically active stereoisomer of MDP. Results of this study indicate that LRRs are indeed a useful scaffold for development of specific and selective proteins for molecular recognition, creating much potential for future engineering of alternative protein scaffolds for biomedical applications. My second research interest focused on the development of proteins for novel biomaterials. In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues, as well as participates in important regulatory functions such as cell migration and proliferation and protein signalling. The aforementioned properties along with keratins' inherent capacity for self-assembly poise it as a promising scaffold for regenerative medicine and tissue engineering applications. However, due to the extraction process used to obtain natural keratin proteins from natural sources, protein damage and formation of by-products that alter network self-assembly and bioactivity often occur as a result of the extensive processing conditions required. Furthermore, natural keratins require exogenous chemistry in order to modify their properties, which greatly limits sequence tunability. Recombinant keratin proteins have the potential to overcome the limitations associated with the use of natural keratins while also maintaining their desired structural and chemical characteristics. Thus, we have used recombinant DNA technology for the production of human hair keratins, keratin 31 (K31) and keratin 81 (K81). The production of recombinant human hair keratins resulted in isolated proteins of the correct sequence and molecular weight determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry. Proteins with no unwanted sequence truncations, deletions, or mutations indicate recombinant DNA technology can be used to reliably generate full length keratin proteins. This allows for consistent starting materials with no observable impurities or undesired by-products, which combats a major challenge associated with natural keratins. Additionally, recombinant keratins must maintain the intrinsic propensity for self-assembly found in natural keratins. To test the propensity for self-assembly, we implemented size exclusion chromatography (SEC), dynamic light scattering (DLS), and transmission electron microscopy (TEM) to characterize K31, K81, and an equimolar mixture of K31 and K81. The results of the recombinant protein characterization reveal novel homo-polymerization of K31 and K81, not previously reported, and formation of characteristic keratin fibers for the K31 and K81 mixture. Therefore, recombinant K31 and K81 retain the intrinsic biological activity (i.e. self-assembly) of natural keratin proteins. We have also conducted a comparative study of recombinant and extracted heteropolymer K31/K81. Through solution characterization and TEM analysis it was found that use of the recombinant heteropolymer allows for increased purity of starting material while also maintaining self-assembly properties necessary for functional use in biomaterials design. However, under the processing condition implemented, extracted keratins demonstrated increased efficiency of assembly. Through each study we conclude that recombinant keratin proteins provide a promising solution to overcome the challenges associated with natural protein materials and present an exceptional design platform for generation of new biomaterials for regenerative medicine and tissue engineering. / Ph. D. / Protein engineering and synthetic protein production enables the creation of new proteins that can perform specific tasks. Many advances in biomedical materials and medical diagnostic tools stem from the use of synthetic proteins. The main focus of my dissertation was to develop new biomedical materials using protein engineering. In chapters three and four of the dissertation development of synthetic proteins for medical diagnostics is discussed. We have designed artificial protein sensors based on natural innate immunity proteins, which function in the body as the source for recognition of foreign pathogens, such as bacteria and viruses. Our goal was to create synthetic proteins with similar characteristics to the innate immunity receptors for the purpose of sensing bacteria and viruses in the form of a biosensors or medical diagnostic. Through our work we have developed an artificial protein scaffold that can selectively interact with a relevant biological target. This research provides the ground work for future development of proteins that can sense a wide variety of important pathogens and subsequently be manufactured into diagnostic devices. Our research involving protein design for biomaterials is the focus of chapters five and six of the dissertation. Keratin is a ubiquitous protein found in the human body. It functions as a structural protein and helps create the complex network that makes up skin, hair, and epidermal appendages. We have created synthetic keratin proteins in an effort to fabricate biomaterials that can be used for regenerative medicine and tissue engineering applications. Our strategy allows for development of proteins that can be designed to have characteristics not afforded to naturally occurring keratin proteins, and thus presents the opportunity to make materials with unique properties and characteristics that may make them more successful in our intended applications of tissue engineering. From our work we have shown that synthetic production of these proteins is possible and that the synthetically produced proteins retain the essential structural and functional properties associated with natural keratin proteins. Thus, this work highlights the potential for use of synthetic proteins for production of biomaterials with new and important features that cannot be obtained through use of natural proteins.

Thesis (Ph. D.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains vi, 163 p. : ill. (some col.) Vita. Includes abstract. Includes bibliographical references.

A significant advance in understanding skeletal muscle adaptation to physical training has been the observation that nutrients and exercise work in synergy to enhance muscle protein synthesis. Physical activity triggers an adaptive response to which nutrition provides the necessary building blocks for an optimal response. The aim of the present work was to contribute to the understanding of the molecular events induced by exercise or nutrients (creatine and amino acids) to create the adaptive environment and to induce the cellular adaptation and growth, respectively. From a methodological point of view, two experimental models were used: muscle biopsies taken from the vastus lateralis of human volunteers and myotubes cultured from C2C12 cells. The transcription of a series of genes involved in muscle remodelling (MAFbx, MHCIIA, PGC-1á, PCNA and IL-6) was increased immediately after the completion of a resistance exercise session performed in the fasted state. The phosphorylation state of p38 and ERK1/2 was also increased, whereas the Akt/PKB pathway was negatively regulated. This contrasted with the high phosphorylation state observed on p70s6k and 4E-BP1 when subjects received a large amount of amino acids during the recovery period. Our results suggest that the MAPK pathway can be triggered by contractile activity alone, whereas the Akt/PKB pathway requires nutrients to be activated. Certain amino acids regulate the phosphorylation state of mTOR and its downstream targets, as demonstrated by one of our in vitro studies. However, that modulation did not lead to a systematic modification in the rate of protein synthesis. Amino acids were also able to influence the expression of muscle-specific genes, highlighting their importance in the control of muscle protein synthesis. Protein anabolism was largely enhanced and cell differentiation was accelerated by creatine in our in vitro model. We have identified the p38 and Akt/PKB pathways as mediators of these effects. Nevertheless, we were unable to confirm the existence of similar events in human skeletal muscle in vivo.

Background Maintenance of lean muscle mass and related strength is associated with lower risk for numerous chronic diseases of aging in women. Objective Our aim was to evaluate whether the association between dietary protein and lean mass differs by physical activity level, amino acid composition, and body mass index categories. Design We performed a cross-sectional analysis of a prospective cohort. Participants/setting Participants were postmenopausal women from the Womens Health Initiative with body composition measurements by dual-energy x-ray absorptiometry (n=8,298). Main outcome measures Our study measured percent lean mass, percent fat mass, and lean body mass index. Statistical analyses performed Linear regression models adjusted for scanner serial number, age, calibrated energy intake, race/ethnicity, neighborhood socioeconomic status, and recreational physical activity were used to determine the relationship between protein intake and body composition measures. Likelihood ratio tests and stratified analysis were used to investigate physical activity and body mass index as potential effect modifiers. Results Biomarker-calibrated protein intake was positively associated with percent lean mass; women in the highest protein quintile had 6.3 percentage points higher lean mass than the lowest quintile (P<0.001). This difference rose to 8.5 percentage points for physically active women in the highest protein quintile (P-interaction=0.023). Percent fat mass and lean body mass index were both inversely related to protein intake (both P<0.001). Physical activity further reduced percent fat mass (P-interaction=0.022) and lean body mass index (P-interaction=0.011). Leucine intake was associated with lean mass, as were branched chain amino acids combined (both P<0.001), but not independent of total protein. All associations were observed for normal-weight, overweight, and obese women. Conclusions Protein consumption up to 2.02 g/kg body weight daily is positively associated with lean mass in postmenopausal women. Importantly, those that also engage in physical activity have the highest lean mass across body mass index categories.

Introduction : Un hypométabolisme du glucose au cerveau peut apparaître des décennies avant le diagnostic de maladie d’Alzheimer. Pourtant, le métabolisme des cétones, le carburant alternatif principal au glucose, n’est pas altéré, autant chez les personnes plus âgées que chez les personnes atteintes de la maladie d’Alzheimer. Les méthodes connues (diète cétogène, jeûne, huile de triglycérides de moyennes chaînes) pour augmenter la production de cétones comportent leurs inconvénients. L’objectif de ces projets était de vérifier l’efficacité de produits commerciaux vendus à cet effet et d’évaluer d’autres molécules qui pourraient être utilisées pour leur propriété cétogène. Ce sont des suppléments alimentaires pouvant servir de substrat pour la cétogenèse (butyrate, monoglycéride d’octanoate (O-MAG), leucine) ou de facilitateur du transport des acides gras (carnitine). Méthodes : Pour ce faire, les participants devaient se présenter au laboratoire pour plusieurs demi-journées d’étude d’une durée de 4 heures. Lors de ces journées, un des suppléments leur était servi avec un déjeuner et des prises de sang étaient effectuées toutes les 30 minutes. Résultats : Le produit commercial contenant des MCT, le butyrate, l’O-MAG et la leucine ont permis d’augmenter respectivement les concentrations maximales en cétones plasmatiques de 568, 226, 67 et 70 µmol/L (p≤0,05) par rapport au contrôle. De plus, la concentration plasmatique en octanoate était corrélée significativement avec la concentration sanguine d’acétoacétate (r=0,85 ; p <0,0001) et de β-hydroxybutyrate (r=0,58 ; p=0,01). L’aire sous la courbe par gramme de produit ingéré montre que le butyrate était le supplément cétogène le plus efficace et était plus cétogène que le produit commercial, l’O-MAG et la leucine (p=0,009). Discussion : Le butyrate était 3 fois plus cétogène que tous les autres suppléments testés. Par contre, son ingestion a causé quelques effets secondaires à certains participants. La leucine a été le seul supplément à ne créer aucun inconfort chez les participants. Il faudrait donc étudier la possibilité de combiner plusieurs molécules dans un supplément chimiquement pensé pour être le plus efficace possible et pour être intéressant lors d’une utilisation quotidienne.

Jun and Fos are components of the AP1 family of transcription factors that bind to the promoters of a diverse multitude of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryonic development and cancer. The specific protein-DNA interactions are driven by the binding of basic zipper (bZIP) domains of Jun and Fos to TPA response element (TRE) and cAMP response element (CRE) within the promoters of target genes. Here, using a diverse array of biophysical techniques, including in particular isothermal titration calorimetry in conjunction with molecular modeling and semi-empirical analysis, I characterize AP1-DNA interactions in thermodynamic and structural terms. My data show that the binding of bZIP domains of Jun-Fos heterodimer to TRE and CRE are under enthalpic control accompanied by entropic penalty at physiological temperatures. This is in agreement with the notion that protein-DNA interactions are largely driven by electrostatic interactions and intermolecular hydrogen bonding. A larger than expected heat capacity change suggests that the basic regions within the bZIP domains are unstructured in the absence of DNA and interact in a coupled folding and binding manner. Further analysis demonstrates that Jun-Fos heterodimer can tolerate single nucleotide variants of the TRE consensus sequence and binds in the biologically relevant micromolar to submicromolar range. Of particular interest is the observation that the Jun-Fos heterodimer binds to specific variants in a preferred orientation. 3D atomic models reveal that such preference in orientation results from asymmetric binding and may in part be attributable to chemically distinct but structurally equivalent residues within the basic regions of Jun and Fos. I further demonstrate that binding of the biologically relevant Jun-Jun homodimer to TRE and CRE occurs with favorable enthalpic contributions accompanied by entropic penalty at physiological temperatures in a manner akin to the binding of Jun-Fos heterodimer. However, anomalously large negative heat capacity changes provoke a model whereby Jun loads onto DNA as unfolded monomers coupled with subsequent folding and homodimerization upon association. The data also reveal that the heterodimerization of leucine zippers is modulated by the basic regions and these regions may undergo at least partial folding upon heterodimerization. Large negative heat capacity changes accompanying the heterodimerization of leucine zippers are consistent with the view that leucine zippers do not retain a-helical conformation in isolation and the formation of the native coiled coil a-helical dimer is attained through a coupled folding-dimerization mechanism. Taken together, this dissertation marks the first comprehensive thermodynamic analysis of an otherwise well-studied and vitally important transcription factor. My studies shed new light on the forces driving the AP1-DNA interaction in thermodynamic and structural terms. The implications of these novel findings on the development of novel therapies for the treatment of disease with greater efficacy coupled with low toxicity cannot be overemphasized.

Is Supplemental Leucine Intake Associated with Enhanced Post Exercise Muscle Protein Synthesis and Attenuated Muscle Protein Breakdown? Knight AD, Benardot D, Thompson W, and Henes ST Introduction: The role of individual amino acids on protein synthesis and their impact on physical performance is of high importance to athletes and to those studying the science of sports nutrition. Leucine, one of three branched-chain amino acids, is a frequently researched amino acid because of its potential stimulatory effect on muscle protein synthesis (MPS) following exercise in humans. Purpose: Although there have been many studies conducted on leucine’s muscle stimulatory effect, questions remain as to the efficacy and feasibility of leucine as an MPS catalyst. Contributing to these questions are the widely varied dosing and timing strategies that different researchers have employed. It is the purpose of this thesis, therefore, to assess the differences in study protocols and shed light on the potential effectiveness on leucine as a MPS stimulator. Central to this issue is whether supplemental leucine intake is associated with enhanced post exercise MPS and, if so, what associated factors, including timing and level of intake, are most likely to influence this effect. Methods: A comprehensive review of the literature on leucine and its effect on MPS was performed. Studies were organized into similar topics, with an assessment and summary of effect produced for each topic area. A general conclusion was made that was based on the summary of each topic area. Results: Leucine is involved in protein metabolism regulation through its role in stimulating the mammalian target of rapamycin (mTOR) signaling cascade and by indicating energy and amino acid availability. It functions to initiate MPS and decrease muscle protein breakdown by downregulating the ubiquitin-proteasome system, lysosomal activity, and/or increasing circulating insulin. Conclusions: Supplementation with the amino acid leucine effectively enhances MPS and attenuates muscle protein degradation in humans following bouts of physical exertion. Leucine intake in amounts greater than that found in ~20g whole protein saturates MPS and increases leucine oxidation. For this reason, an upper limit of leucine intake should be established. While leucine successfully increases MPS, it remains unclear whether this translates to enhanced physical performance, an area that requires more studies to be conducted.

Whey protein supplementation, containing large amount of leucine, has been a traditional intervention to maintain net protein balance in the past decades. It has been recognized that leucine alone is able to stimulate protein synthesis by activating mTOR and its related downstream pathway without affecting protein degradation, whereas its metabolite β-hydroxy-β-methylbutyrate (HMB) is known to attenuate protein degradation when provided chronically. However, the mechanism of HMB’s benefit remains unclear. To address how HMB regulates protein synthesis and degradation signaling pathways, we compared one dose of whey protein (187.5mg/kg), HMB (400mg/kg) or leucine (1.4g/kg) by oral gavage. Blood was collected at 0, 45 and 90 min for blood glucose and plasma insulin analysis. Red and white gastrocnemius muscle was taken separately 90 min after gavage. Blood glucose was reduced by leucine at 45 and 90 min post gavage. Plasma insulin was enhanced by leucine at 45 min and then decreased at 90 min post gavage, whereas HMB decreased plasma insulin through 90 min post gavege. Western blot analysis showed that HMB phosphorylated Akt in red gastronemius, and enhanced phosphorylation of mTOR in both types of muscles. Leucine phosphorylated mTOR, p70s6k and 4E-BP1 in both red and white gastronemius. Regarding protein degradation signals, phosphorylation of FOXO3A was enhanced by HMB, but not in the other treatment groups. Whey protein had no effect on those cellular signaling. Our results indicate that both HMB and leucine may stimulate protein synthesis through the mTOR pathway in red and white gastrocnemius muscles by different degrees with leucine more effective than HMB. HMB may have a greater effect than leucine on limiting protein degradation by phosphorylating Akt and FOXO3A in red and white gastrocnemius muscles. A combination of HMB and leucine, as a new interventional strategy, is predicted to maximize protein accretion by increasing protein synthesis as well as inhibiting protein degradation. / text

Carbohydrate plus additional protein supplementation provided immediately after exercise has been found to increase the rate of muscle glycogen restoration compared to carbohydrate alone. To examine whether leucine, and/or β-hydroxy-β-methylbutyrate (HMB) to carbohydrate plus protein supplementation affects short-term recovery (45 min) of muscle glycogen, we compared plasma glucose and insulin, the muscle glycogen concentration, and the cellular signaling proteins controlling muscle glycogen synthesis 45 min after supplementation. Rats (n=35) underwent high-intensity resistance exercise followed by supplementation with carbohydrate (CHO: 1.2g/kg body weight), carbohydrate with whey protein (CP: 1.2g CHO + 375mg whey protein/kg body weight), carbohydrate with whey protein plus HMB (CPH: 1.2g CHO + 375mg whey protein + 400mg HMB/kg body weight), carbohydrate with whey protein, HMB plus leucine (CPHL: 1.2g CHO + 375mg whey protein + 400mg HMB + 444mg leucine/kg body weight) or exercise only (CON). Blood samples were collected immediately after exercise and 45 min after supplementations. Muscle samples of plantaris were excised immediately and 45 min post-exercise. Plasma glucose was increased by CHO and CPH supplementation and reduced by CPHL at 45 min post-exercise. Plasma insulin was elevated by CP and CPHL treatments compare to CHO. Muscle glycogen concentration was unaffected by all treatments and did not differ from CON. Phosphorylation of Akt/PKB, GSK3α/β, and GS at 45 min of recovery for all supplements was not significant difference from CON. Phosphorylation of mTOR was significantly increased by CPHL and CP supplementation compared to CON, CHO, and CPH. Phosphorylation of AS160 was markedly reduced by CPH supplementation compared to CON. These results suggest that supplementing with carbohydrate plus protein with or without leucine and its metabolite, HMB, to enhance muscle glycogen replenishment following exercise may not provide an advantage during the early phase of recovery (45 min). Furthermore, there is some indication that HMB may elicit insulin resistance, and this needs further evaluation. / text

Luman (LZIP/CREB3) is a basic leucine zipper (bZIP) transcription factor that has been linked to the endoplasmic reticulum (ER) stress response. In the event of ER stress, Luman is proteolytically cleaved, or ‘activated’, through regulated intramembrane proteolysis (RIP), resulting in an amino-terminal fragment that translocates to the nucleus to activate transcription of downstream unfolded protein response (UPR)-related genes. The general mode of activation of the key signal transducers of the UPR appears to be an alteration of their oligomeric states. Structural and functional similarities to these proteins suggest that Luman may be activated in a similar manner. In this thesis, we demonstrate through in vitro and in vivo studies that Luman can form homodimers in the cell. Through the use of mutagenesis, we show that Luman dimerization is mediated through the leucine zipper and we provide evidence that Luman dimerization is required for its transcription activation function. However, we found that Luman dimerization is not required for its activation by proteolytic cleavage.