Browsing by Subject "Protein"
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Item Open Access A Covalent Modification Technique for Protein-Ligand Binding Analysis Using Mass Spectrometry-Based Proteomics Platforms(2009) West, Graham MeldahlCurrently there is a dearth of analytical techniques for studying protein-ligand interactions on the proteomic scale. Existing techniques, which rely on various calorimetry or spectroscopy methods, are limited in their application to the proteomic scale due to their need for large amounts of pure protein. Recently, several mass spectrometry-based methods have been developed to study protein-ligand interactions. These mass spectrometry-based methods overcome some of the limitations of existing techniques by enabling the analysis of unpurified protein samples. However, the existing mass spectrometry-based methodologies for the analysis of protein-ligand binding interactions are not directly compatible with current mass spectrometry-based proteomics platforms.
Described here is the development and application of a new technique designed to detect and quantify protein-ligand binding interactions with mass spectrometry-based proteomic platforms. This technique, termed SPROX (Stability of Proteins from Rates of Oxidation), uses an irreversible covalent oxidation labeling reaction to monitor the global unfolding reactions of proteins to measure protein thermodynamic stability. Two variations of the SPROX technique are established here, including one variation that utilizes chemical denaturant to induce protein unfolding and a second variation that utilizes temperature to denature proteins. The SPROX methodology is tested on five proteins including ubiquitin, ribonuclease A, bovine carbonic anhydrase II, cyclophilin A, and calmodulin. Results obtained on these model systems are used to determine the method's ability to measure the thermodynamic parameters associated with each protein's folding/unfolding reaction. Results obtained on calmodulin and cyclophilin A are used to determine the method's ability to quantify the dissociation constants of protein-ligand complexes.
The primary motivation for the development of the SPROX protocols in this work was to create a protein-ligand binding assay that could be interfaced with conventional mass spectrometry-based platforms. Two specific SPROX protocols, including a label-free approach and an oxygen-16/18 labeling approach, are developed and demonstrated using the thermal SPROX technique to analyze ligand binding in a model four-protein component mixture consisting of ubiquitin, ribonuclease A, bovine carbonic anhydrase, and cyclophilin A. The thermal SPROX technique's ability to detect cyclosporin A binding to cyclophilin A in the context of the model mixture is shown using both labeling approaches.
An application using the SPROX technique combined with a multi-dimensional protein identification technology (MudPIT)-based proteomics platform is also described. In this application, which utilized an isobaric mass tagging strategy, 325 proteins in a yeast cell lysate are simultaneously assayed for CsA-binding. This study was also used to investigate the protein targets of an already well-studied immunosuppressive drug, cyclosporin A. Two of the ten protein targets identified in this work are known to interact with CsA, one through a direct binding event and one through an indirect binding event. The eight newly discovered protein targets of CsA suggest a molecular basis for post-transplant diabetes mellitus, which is a side effect of CsA in humans.
Item Open Access A randomized trial of supplemental parenteral nutrition in underweight and overweight critically ill patients: the TOP-UP pilot trial.(Crit Care, 2017-06-09) Wischmeyer, Paul E; Hasselmann, Michel; Kummerlen, Christine; Kozar, Rosemary; Kutsogiannis, Demetrios James; Karvellas, Constantine J; Besecker, Beth; Evans, David K; Preiser, Jean-Charles; Gramlich, Leah; Jeejeebhoy, Khursheed; Dhaliwal, Rupinder; Jiang, Xuran; Day, Andrew G; Heyland, Daren KBACKGROUND: Nutrition guidelines recommendations differ on the use of parenteral nutrition (PN), and existing clinical trial data are inconclusive. Our recent observational data show that amounts of energy/protein received early in the intensive care unit (ICU) affect patient mortality, particularly for inadequate nutrition intake in patients with body mass indices (BMIs) of <25 or >35. Thus, we hypothesized increased nutrition delivery via supplemental PN (SPN) + enteral nutrition (EN) to underweight and obese ICU patients would improve 60-day survival and quality of life (QoL) versus usual care (EN alone). METHODS: In this multicenter, randomized, controlled pilot trial completed in 11 centers across four countries, adult ICU patients with acute respiratory failure expected to require mechanical ventilation for >72 hours and with a BMI of <25 or ≥35 were randomized to receive EN alone or SPN + EN to reach 100% of their prescribed nutrition goal for 7 days after randomization. The primary aim of this pilot trial was to achieve a 30% improvement in nutrition delivery. RESULTS: In total, 125 patients were enrolled. Over the first 7 post-randomization ICU days, patients in the SPN + EN arm had a 26% increase in delivered calories and protein, whereas patients in the EN-alone arm had a 22% increase (both p < 0.001). Surgical ICU patients received poorer EN nutrition delivery and had a significantly greater increase in calorie and protein delivery when receiving SPN versus medical ICU patients. SPN proved feasible to deliver with our prescribed protocol. In this pilot trial, no significant outcome differences were observed between groups, including no difference in infection risk. Potential, although statistically insignificant, trends of reduced hospital mortality and improved discharge functional outcomes and QoL outcomes in the SPN + EN group versus the EN-alone group were observed. CONCLUSIONS: Provision of SPN + EN significantly increased calorie/protein delivery over the first week of ICU residence versus EN alone. This was achieved with no increased infection risk. Given feasibility and consistent encouraging trends in hospital mortality, QoL, and functional endpoints, a full-scale trial of SPN powered to assess these clinical outcome endpoints in high-nutritional-risk ICU patients is indicated-potentially focusing on the more poorly EN-fed surgical ICU setting. TRIAL REGISTRATION: NCT01206166.Item Open Access Analysis and Error Correction in Structures of Macromolecular Interiors and Interfaces(2009) Headd, Jeffrey JohnAs of late 2009, the Protein Data Bank (PDB) has grown to contain over 70,000 models. This recent increase in the amount of structural data allows for more extensive explication of the governing principles of macromolecular folding and association to complement traditional studies focused on a single molecule or complex. PDB-wide characterization of structural features yields insights that are useful in prediction and validation of the 3D structure of macromolecules and their complexes. Here, these insights lead to a deeper understanding of protein--protein interfaces, full-atom critical assessment of increasingly more accurate structure predictions, a better defined library of RNA backbone conformers for validation and building 3D models, and knowledge-based automatic correction of errors in protein sidechain rotamers.
My study of protein--protein interfaces identifies amino acid pairing preferences in a set of 146 transient interfaces. Using a geometric interface surface definition devoid of arbitrary cutoffs common to previous studies of interface composition, I calculate inter- and intrachain amino acid pairing preferences. As expected, salt-bridges and hydrophobic patches are prevalent, but likelihood correction of observed pairing frequencies reveals some surprising pairing preferences, such as Cys-His interchain pairs and Met-Met intrachain pairs. To complement my statistical observations, I introduce a 2D visualization of the 3D interface surface that can display a variety of interface characteristics, including residue type, atomic distance and backbone/sidechain composition.
My study of protein interiors finds that 3D structure prediction from sequence (as part of the CASP experiment) is very close to full-atom accuracy. Validation of structure prediction should therefore consider all atom positions instead of the traditional Calpha-only evaluation. I introduce six new full-model quality criteria to assess the accuracy of CASP predictions, which demonstrate that groups who use structural knowledge culled from the PDB to inform their prediction protocols produce the most accurate results.
My study of RNA backbone introduces a set of rotamer-like "suite" conformers. Initially hand-identified by the Richardson laboratory, these 7D conformers represent backbone segments that are found to be genuine and favorable. X-ray crystallographers can use backbone conformers for model building in often poor backbone density and in validation after refinement. Increasing amounts of high quality RNA data allow for improved conformer identification, but also complicate hand-curation. I demonstrate that affinity propagation successfully differentiates between two related but distinct suite conformers, and is a useful tool for automated conformer clustering.
My study of protein sidechain rotamers in X-ray structures identifies a class of systematic errors that results in sidechains misfit by approximately 180 degrees. I introduce Autofix, a method for automated detection and correction of such errors. Autofix corrects over 40% of errors for Leu, Thr, and Val residues, and a significant number of Arg residues. On average, Autofix made four corrections per PDB file in 945 X-ray structures. Autofix will be implemented into MolProbity and PHENIX for easy integration into X-ray crystallography workflows.
Item Open Access Bayesian Structural Phylogenetics(2013) Challis, ChristopherThis thesis concerns the use of protein structure to improve phylogenetic inference. There has been growing interest in phylogenetics as the number of available DNA and protein sequences continues to grow rapidly and demand from other scientific fields increases. It is now well understood that phylogenies should be inferred jointly with alignment through use of stochastic evolutionary models. It has not been possible, however, to incorporate protein structure in this framework. Protein structure is more strongly conserved than sequence over long distances, so an important source of information, particularly for alignment, has been left out of analyses.
I present a stochastic process model for the joint evolution of protein primary and tertiary structure, suitable for use in alignment and estimation of phylogeny. Indels arise from a classic Links model and mutations follow a standard substitution matrix, while backbone atoms diffuse in three-dimensional space according to an Ornstein-Uhlenbeck process. The model allows for simultaneous estimation of evolutionary distances, indel rates, structural drift rates, and alignments, while fully accounting for uncertainty. The inclusion of structural information enables pairwise evolutionary distance estimation on time scales not previously attainable with sequence evolution models. Ideally inference should not be performed in a pairwise fashion between proteins, but in a fully Bayesian setting simultaneously estimating the phylogenetic tree, alignment, and model parameters. I extend the initial pairwise model to this framework and explore model variants which improve agreement between sequence and structure information. The model also allows for estimation of heterogeneous rates of structural evolution throughout the tree, identifying groups of proteins structurally evolving at different speeds. In order to explore the posterior over topologies by Markov chain Monte Carlo sampling, I also introduce novel topology + alignment proposals which greatly improve mixing of the underlying Markov chain. I show that the inclusion of structural information reduces both alignment and topology uncertainty. The software is available as plugin to the package StatAlign.
Finally, I also examine limits on statistical inference of phylogeny through sequence information models. These limits arise due to the `cutoff phenomenon,' a term from probability which describes processes which remain far from their equilibrium distribution for some period of time before swiftly transitioning to stationarity. Evolutionary sequence models all exhibit a cutoff; I show how to find the cutoff for specific models and sequences and relate the cutoff explicitly to increased uncertainty in inference of evolutionary distances. I give theoretical results for symmetric models, and demonstrate with simulations that these results apply to more realistic and widespread models as well. This analysis also highlights several drawbacks to common default priors for phylogenetic analysis, I and suggest a more useful class of priors.
Item Open Access Chemical and Physical Analysis of Melanin in Complex Biological Matrices(2014) Glass, Keely ElizabethMelanin is a ubiquitous biological pigment found in bacteria, fungi, plants, and animals. It has a diverse range of ecological and biochemical functions including display, evasion, photoprotection, detoxification, and metal scavenging. Two forms of melanin produced from different molecular precursors are present in nature - eumelanin (dark brown-black in color) and pheomelanin (orange-red in color). Both eumelanin and pheomelanin are complex highly cross-linked biopolymers that are found intertwined with proteins, lipids, and metal ions in nature.
Recent reports have used morphological evidence to suggest the presence of melanin in the fossil record. These studies have been met with criticism due to their lack of chemical evidence to support melanin identification. This dissertation describes chemical approaches to unambiguously verify the presence of melanin in the fossil record and characterize the ancient pigment. It also explores the limitations for the survival of melanin in the fossil record and the possibility that melanin acts as a protective matrix to preserve other biomolecules that are embedded in the pigment.
Melanin has unique chemical signatures that are commonly used to characterize and compare the pigment of modern organisms. We applied these chemical approaches to the study of fossil pigmentation. Analysis of the black pigmentation of two > 160 million year old (Mya) Jurassic cephalopod ink sacs provided the first conclusive evidence for eumelanin in the fossil record. The preserved fossil eumelanin was then compared to modern cephalopod eumelanin from Sepia officinalis. Using these chemical approaches we found that fossil eumelanin was chemically and morphologically identical to S. officinalis eumelanin.
Although there is mounting chemical evidence for the presence and preservation of melanin in the fossil record, there is very little data constraining its long-term survival. We applied the analytical approaches designed to study fossil melanins and techniques used to study fossil sediments to compare the fossil inks from three deposits of similar age and lithology, but different maturation histories. Specifically, two ~ 180 Mya fossil ink sacs from a site that has entered the oil window in Holzmaden, Germany were compared to the previously analyzed fossil inks from two less mature sites in southern England. The chemistry of eumelanin was shown to alter at the onset of the oil window regardless of the age of the specimen. The decrease in surviving melanin was accompanied by an increase in the relative abundance of organic macromolecular material (kerogen), but no consistent change in melanin morphology.
Finally, the role of melanin as a matrix that enhances the preservation of other biomolecules in the fossil record was considered. Proteins, commonly associated with melanin in modern organisms, were discovered in the aforementioned fossil ink sacs during full-scale chemical analysis. The amino acid profile of the protein in each fossil specimen was determined with an amino acid analyzer and compared to the amino acid profile the protein in modern S. officinalis. Statistical analysis of the amino acid distributions indicated that there is no significant difference between the amino acid profile of modern and fossil melanins. In order to verify the ancient origin of the amino acids in the fossil ink sacs, the ratio of D/L amino acid isomers was determined. While the proteins of living organisms consist of only L-amino acids, post-mortem the amino acids slowly convert from L to D form until they reach equilibrium (D/L = 1). This process is called racemization. The amino acids in the fossil ink sacs were racemized, which suggests their ancient origin. This marks the oldest determination of protein in a fossil system and provides evidence that the longevity of proteins may be enhanced when associated with melanin.
Item Open Access Development and Application of a Mass Spectrometry-Based Assay for the High Throughput Analysis of Protein-Ligand Binding(2009) Hopper, Erin D.Many of the biological roles of proteins are modulated through protein-ligand interactions, making proteins important targets for drug therapies and diagnostic imaging probes. The discovery of novel ligands for a protein of interest often relies on the use of high throughput screening (HTS) technologies designed to detect protein-ligand binding. The basis of one such technology is a recently reported mass spectrometry-based assay termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is a technique that uses H/D exchange and MALDI-mass spectrometry for the measurement of protein stabilities and protein-ligand binding affinities. The single-point SUPREX assay is an abbreviated form of SUPREX that is capable of detecting protein-ligand interactions in a high throughput manner by exploiting the change in protein stability that occurs upon ligand binding.
This work is focused on the development and application of high throughput SUPREX protocols for the detection of protein-ligand binding. The first step in this process was to explore the scope of SUPREX for the analysis of non-two-state proteins to determine whether this large subset of proteins would be amenable to SUPREX analyses. Studies conducted on two model proteins, Bcl-xL and alanine:glyoxylate aminotransferase, indicate that SUPREX can be used to detect and quantify the strength of protein-ligand binding interactions in non-two-state proteins.
The throughput and efficiency of a high throughput SUPREX protocol (i.e., single-point SUPREX) was also evaluated in this work. As part of this evaluation, cyclophilin A, a protein target of diagnostic and therapeutic significance, was screened against the 880-member Prestwick Chemical Library to identify novel ligands that might be useful as therapeutics or imaging agents for lung cancer. This screening not only established the analytical parameters of the assay, but it revealed a limitation of the technique: the efficiency of the assay is highly dependent on the precision of each mass measurement, which generally decreases as protein size increases.
To overcome this limitation and improve the efficiency and generality of the assay, a new SUPREX protocol was developed that incorporated a protease digestion step into the single-point SUPREX protocol. This new protocol was tested on two model proteins, cyclophilin A and alanine:glyoxylate aminotransferase, and was found to result in a significant improvement in the efficiency of the SUPREX assay in HTS applications. This body of work resulted in advancements in the use of SUPREX for high throughput applications and laid the groundwork for future HTS campaigns on target proteins of medical significance.
Item Open Access Fundamental Mechanisms in the Extreme UV Resistance of Adenovirus(2009) Eischeid, AnneThe adenoviruses are nonenveloped double stranded DNA viruses, which cause enteric dysentary and respiratory infection. Adenovirus has become a focus of the water treatment community because of its apparent resistance to ultraviolet disinfection; it is the basis for stringent new EPA regulations regarding all viruses in both surface and ground waters. Most of the work done so far, however, has involved the use of monochromatic (254 nm) low pressure (LP) UV sources and subsequent assay of viral infectivity in cell culture models. LP UV lamps primarily damage DNA, while polychromatic UV sources may damage other parts of the virus as well. Recent research has shown that these newer, polychromatic UV sources--such as medium pressure (MP) UV--are more effective than monochromatic LP UV for disinfection of adenovirus. The objectives of this work were to study adenoviral response to UV using both LP and MP UV as well as using both standard cell culture infectivity assays and more direct methods of assessment based on molecular biology. These include quantitative long PCR for assessment of DNA damage and SDS-PAGE for assessment of protein damage; transmission electron microscopy was used to examine the structure of UV treated viral particles. This work was only the second significant study to show the response of adenoviruses to medium pressure UV and the first to thoroughly examine the response of adenoviruses to both LP and MP UV using cell culture-independent methods. Results confirm that adenovirus is sensitive to MP UV when assayed in cell culture; they show that LP and MP UV are equally effective at inducing damage to the adenoviral genome and that MP UV is more effective than LP UV at damaging the viral proteins. This work helps deepen our understanding of UV disinfection of adenovirus.
Item Open Access Improved Function With Enhanced Protein Intake per Meal: A Pilot Study of Weight Reduction in Frail, Obese Older Adults.(J Gerontol A Biol Sci Med Sci, 2016-10) Porter Starr, Kathryn N; Pieper, Carl F; Orenduff, Melissa C; McDonald, Shelley R; McClure, Luisa B; Zhou, Run; Payne, Martha E; Bales, Connie WBACKGROUND: Obesity is a significant cause of functional limitations in older adults; yet, concerns that weight reduction could diminish muscle along with fat mass have impeded progress toward an intervention. Meal-based enhancement of protein intake could protect function and/or lean mass but has not been studied during geriatric obesity reduction. METHODS: In this 6-month randomized controlled trial, 67 obese (body mass index ≥30kg/m(2)) older (≥60 years) adults with a Short Physical Performance Battery score of 4-10 were randomly assigned to a traditional (Control) weight loss regimen or one with higher protein intake (>30g) at each meal (Protein). All participants were prescribed a hypo-caloric diet, and weighed and provided dietary guidance weekly. Physical function (Short Physical Performance Battery) and lean mass (BOD POD), along with secondary measures, were assessed at 0, 3, and 6 months. RESULTS: At the 6-month endpoint, there was significant (p < .001) weight loss in both the Control (-7.5±6.2kg) and Protein (-8.7±7.4kg) groups. Both groups also improved function but the increase in the Protein (+2.4±1.7 units; p < .001) was greater than in the Control (+0.9±1.7 units; p < .01) group (p = .02). CONCLUSION: Obese, functionally limited older adults undergoing a 6-month weight loss intervention with a meal-based enhancement of protein quantity and quality lost similar amounts of weight but had greater functional improvements relative to the Control group. If confirmed, this dietary approach could have important implications for improving the functional status of this vulnerable population (ClinicalTrials.gov identifier: NCT01715753).Item Unknown Multiphase, Multicomponent Systems: Divalent Ionic Surfactant Phases and Single-Particle Engineering of Protein and Polymer Glasses(2011) Rickard, DeborahThis thesis presents an analysis of the material properties and phase behavior of divalent ionic surfactant salts, and protein and polymer glasses. There has been extensive interest in understanding the phase behavior of divalent ionic surfactants due to the many applications of ionic surfactants in which they come into contact with divalent ions, such as detergency, oil recovery, and surfactant separation processes. One goal of determining the phase boundaries was to explore the option of incorporating a hydrophobic molecule into the solid phase through the micelle-to-crystal bilayer transition, either for drug delivery applications (with a biologically compatible surfactant) or for the purpose of studying the hydrophobic molecule itself. The liquid micellar and solid crystal phases of the alkaline earth metal dodecyl sulfates were investigated using calorimetry, visual inspection, solubilization of a fluorescent probe, and x-ray diffraction. The Krafft temperature and dissolution enthalpy were determined for each surfactant, and partial composition-temperature phase diagrams of magnesium dodecyl sulfate-water, calcium dodecyl sulfate-water, as well as sodium dodecyl sulfate with MgCl2 and CaCl2 are presented. As a proof of concept, fluorescence microscopy images showed that it is, in fact, possible to incorporate a small hydrophobic molecule, diphenylhexatriene, into the solid phase.
The second, and main, part of this thesis expands on work done previously in the lab by using the micropipette technique to study two-phase microsystems. These microsystems consist of a liquid droplet suspended in a second, immiscible liquid medium, and can serve as direct single-particle studies of drug delivery systems that are formed using solvent extraction (e.g., protein encapsulated in a biodegradable polymer), and as model systems with which to study the materials and principles that govern particle formation. The assumptions of the Epstein-Plesset model, which predicts the rate of droplet dissolution, are examined in the context of the micropipette technique. A modification to the model is presented that accounts for the effect a solute has on the dissolution rate. The modification is based on the assumption that the droplet interface is in local thermodynamic equilibrium, and that the water activity in a solution droplet can be used to determine its dissolution (or dehydration) rate. The model successfully predicts the dissolution rates of NaCl solutions into octanol and butyl acetate up to the point of NaCl crystallization. The dehydration of protein solutions (lysozyme or bovine serum albumin) results in glassified microbeads with less than a monolayer of water coverage per protein molecule, which can be controlled by the water activity of the surrounding organic medium. The kinetics of dehydration match the prediction of the activity-based model, and it is shown how the micropipette technique can be used to study the effect of dissolution rate on final particle morphology. By using a stable protein with a simple geometry (lyosyzme), this technique was be used to determine the distance dependence of protein-protein interactions in the range of 2-25 Å, providing the first calculation of the hydration pressure decay length for globular proteins. The distance-dependence of the interaction potential at distances less than 9 Å was found to have a decay length of 1.7 Å, which is consistent with the known decay length of hydration pressure between other biological materials. Biodegradable polyesters, such as poly(lactide-co-glycolide) (PLGA), are some of the most common materials used for the encapsulation of therapeutics in microspheres for long-term drug release. Since they degrade by hydrolysis, release rates depend on water uptake, which can be affected by processing parameters and the material properties of the encapsulated drug. The micropipette technique allows observations not possible on any bulk preparation method. Single-particle observations of microsphere formation (organic solvent extraction into a surrounding aqueous phase) show that as solvent leaves the microsphere and the water concentration in the polymer matrix becomes supersaturated, water phase separates and inclusions initially grow quickly. Once the concentration in the polymer matrix equilibrates with the surrounding aqueous medium, the water inclusions continue to grow due to dissolved impurities, solvent, and/or water-soluble polymer fragments resulting from hydrolysis, all of which locally lower the water activity in the inclusion. Experiments are also presented in which glassified protein microbeads were suspended in PLGA solution prior to forming the single microspheres. This technique allowed the concentration of protein in a single microbead/inclusion to be determined as a function of time.
Item Open Access Multiscale Simulations of Biomolecules in Condensed Phase: from Solutions to Proteins(2010) Zeng, XianchengThe thesis contains two directions in the simulations of biomolecular systems. The first part (Chapter 2 - Chapter 4) mainly focuses on the simulations of electron transfer processes in condensed phase; the second part (Chapter 5 - Chapter 6) investigates the conformational sampling of polysaccharides and proteins. Electron transfer (ET) reaction is one of the most fundamental processes in chemistry and biology. Because of the quantum nature of the processes and the complicated roles of the solvent, calculating the accurate kinetic and dynamic properties of ET reactions is challenging but extremely useful. Based on the Marcus theory for thermal ET in weak coupling limit, we combined the rigorous ab initio quantum mechanical (QM) method and well-established molecular mechanical (MM) force field and developed an approach to directly calculate a key factor that affects the ET kinetics: the redox free energy. A novel reaction order parameter fractional number of electrons (FNE) was used to characterize the ET progress and to drive the QM/MMMD sampling of the nonadiabatic free energy surface. This method was used for two aqueous metal cations, iron and ruthenium in solution, and generated satisfactory results compared to experiments. In order to further reduce the computational cost, a QM/MM-minimum free energy path (MFEP) method is implemented and combined with the FNE in the calculation of redox free energies. The calculation results using QM/MM-MFEP+FNE generated identical results as the direct QM/MM-MD method for the two metal cations, demonstrating the consistency of the two different sampling strategy. Furthermore, this new method was applied to the calculation of organic molecules and enhanced the computational efficiency 15-30 times than the direct QM/MM-MD method, while maintaining high accuracy. Finally, I successfully extended the QM/MM-MFEP+FNE method to a series of redox proteins, azurin and its mutants, and obtained very accurate redox free energy differences with relative error less than 0.1 eV. The new method demonstrated its excellent transferability, reliability and accuracy among various conditions from aqueous solutions to complex protein systems. Therefore, it shows great promises for applications of the studies on redox reactions in biochemistry. In the studies of force-induced conformational transitions of biomolecules, the large time-scale difference from experiments presents the challenge of obtaining convergent sampling for molecular dynamics simulations. To circumvent this fundamental problem, an approach combining the replica-exchange method and umbrella sampling (REM-US) is developed to simulate mechanical stretching of biomolecules under equilibrium conditions. Equilibrium properties of conformational transitions can be obtained directly from simulations without further assumptions. To test the performance, we carried out REM-US simulations of atomic force microscope (AFM) stretching and relaxing measurements on the polysaccharide pustulan, a (1→6)-β-D-glucan, which undergoes well-characterized rotameric transitions in the backbone bonds. With significantly enhanced sampling convergence and efficiency, the REMUS approach closely reproduced the equilibrium force-extension curves measured in AFM experiments. Consistent with the reversibility in the AFM measurements, the new approach generated identical force-extension curves in both stretching and relaxing simulations, an outcome not reported in previous studies, proving that equilibrium conditions were achieved in the simulations. In addition, simulations of nine different polysaccharides were performed and the conformational transitions were reexamined using the REM-US approach. The new approach demonstrated consistent and reliable performance among various systems. With fully converged samplings and minimized statistical errors, both the agreement and the deviations between the simulation results and the AFM data were clearly presented. REM-US may provide a robust approach to modeling of mechanical stretching on polysaccharides and even nucleic acids. However, the performance of the REM-US in protein systems, especially with explicit solvent model, is limited by the large system size and the complex interactions. Therefore, a Go-like model is employed to simulate the protein folding/unfolding processes controlled by AFM. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. The trajectories obtained in the simulation captured the critical conformational transitions and the rate-limiting nucleation event. Together with the AFM experiments, the coarse-grained simulations revealed the protein folding and unfolding pathways under the mechanical tension.
Item Open Access Novel Algorithms for Computational Protein Design, with Applications to Enzyme Redesign and Small-Molecule Inhibitor Design(2009) Georgiev, Ivelin StefanovComputational protein design aims at identifying protein mutations and conformations with desired target properties (such as increased protein stability, switch of substrate specificity, or novel function) from a vast combinatorial space of candidate solutions. The development of algorithms to efficiently and accurately solve problems in protein design has thus posed significant computational and modeling challenges. Despite the inherent hardness of protein design, a number of computational techniques have been previously developed and applied to a wide range of protein design problems. In many cases, however, the available computational protein design techniques are deficient both in computational power and modeling accuracy. Typical simplifying modeling assumptions for computational protein design are the rigidity of the protein backbone and the discretization of the protein side-chain conformations. Here, we present the derivation, proofs of correctness and complexity, implementation, and application of novel algorithms for computational protein design that, unlike previous approaches, have provably-accurate guarantees even when backbone or continuous side-chain flexibility are incorporated into the model. We also describe novel divide-and-conquer and dynamic programming algorithms for improved computational efficiency that are shown to result in speed-ups of up to several orders of magnitude as compared to previously-available techniques. Our novel algorithms are further incorporated as part of K*, a provably-accurate ensemble-based algorithm for protein-ligand binding prediction and protein design. The application of our suite of protein design algorithms to a variety of problems, including enzyme redesign and small-molecule inhibitor design, is described. Experimental validation, performed by our collaborators, of a set of our computational predictions confirms the feasibility and usefulness of our novel algorithms for computational protein design.
Item Open Access Personalized nutrition therapy in critical care: 10 expert recommendations.(Critical care (London, England), 2023-07) Wischmeyer, Paul E; Bear, Danielle E; Berger, Mette M; De Waele, Elisabeth; Gunst, Jan; McClave, Stephen A; Prado, Carla M; Puthucheary, Zudin; Ridley, Emma J; Van den Berghe, Greet; van Zanten, Arthur RHPersonalization of ICU nutrition is essential to future of critical care. Recommendations from American/European guidelines and practice suggestions incorporating recent literature are presented. Low-dose enteral nutrition (EN) or parenteral nutrition (PN) can be started within 48 h of admission. While EN is preferred route of delivery, new data highlight PN can be given safely without increased risk; thus, when early EN is not feasible, provision of isocaloric PN is effective and results in similar outcomes. Indirect calorimetry (IC) measurement of energy expenditure (EE) is recommended by both European/American guidelines after stabilization post-ICU admission. Below-measured EE (~ 70%) targets should be used during early phase and increased to match EE later in stay. Low-dose protein delivery can be used early (~ D1-2) (< 0.8 g/kg/d) and progressed to ≥ 1.2 g/kg/d as patients stabilize, with consideration of avoiding higher protein in unstable patients and in acute kidney injury not on CRRT. Intermittent-feeding schedules hold promise for further research. Clinicians must be aware of delivered energy/protein and what percentage of targets delivered nutrition represents. Computerized nutrition monitoring systems/platforms have become widely available. In patients at risk of micronutrient/vitamin losses (i.e., CRRT), evaluation of micronutrient levels should be considered post-ICU days 5-7 with repletion of deficiencies where indicated. In future, we hope use of muscle monitors such as ultrasound, CT scan, and/or BIA will be utilized to assess nutrition risk and monitor response to nutrition. Use of specialized anabolic nutrients such as HMB, creatine, and leucine to improve strength/muscle mass is promising in other populations and deserves future study. In post-ICU setting, continued use of IC measurement and other muscle measures should be considered to guide nutrition. Research on using rehabilitation interventions such as cardiopulmonary exercise testing (CPET) to guide post-ICU exercise/rehabilitation prescription and using anabolic agents such as testosterone/oxandrolone to promote post-ICU recovery is needed.Item Open Access POEGMAlation – A Next-Generation PEGylation Technology(2016) Qi, YizhiThe delivery of therapeutic peptides and proteins is often challenged by a short circulation half-life, necessitating frequent injections that limit efficacy, reduce patient compliance and increase treatment cost. The covalent conjugation of therapeutic peptides and proteins, and more recently oligonucleotide-based drugs, with the “stealth” polymer poly(ethylene glycol) (PEG), termed PEGylation, is one of the most commonly used approaches to increase the in vivo half-life and reduce the immunogenicity of these therapeutic biomolecules. However, after several decades of research and clinical use, the limitations of PEGylation have begun to emerge.
Conventional methods for synthesizing peptide/protein-polymer conjugates have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of control over site and stoichiometry of conjugation, which results in heterogeneous products with significantly compromised biological activity. Additionally, anti-PEG antibodies have been induced in patients treated with PEGylated drugs and have been shown to correlate with rapid clearance of these drugs. High levels of pre-existing anti-PEG antibodies have also been found in individuals naïve to PEGylated agents, which are associated with serious first-exposure allergic reactions.
To address the synthetic limitations of PEGylation, a general approach for the high-yield synthesis of site-specific (C-terminal) and stoichiometric (1:1) peptide/protein-polymer conjugates, named sortase-catalyzed polymer conjugation, was developed. Demonstrating proof-of-concept of the approach with green fluorescent protein (GFP) as a model protein, sortase A from Staphylococcus aureus was used to site-specifically attach an initiator solely at the C-terminus of GFP, followed by in situ growth of the PEG-based brush polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) from the protein macroinitiator by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeded with high specificity and near-complete (~ 95%) product conversion. Subsequent in situ ATRP in aqueous buffer produced 1:1 stoichiometric conjugates with > 90% yield, tunable MW, low dispersity, and no denaturation of the protein. The extraordinarily high yield compares favorably to order of magnitude losses typically seen in conventional PEGylation processes.
Next, the therapeutic potential of POEGMAlation, or the conjugation of POEGMA to a peptide or protein, was demonstrated by implementing the developed sortase-catalyzed polymer conjugation strategy with exendin-4 (exendin), a therapeutic peptide for treating type 2 diabetes, to synthesize exendin-C-POEGMA conjugates with a wide and tunable range of molecular weights (MWs) and low dispersity. A single subcutaneous injection of exendin-C-POEGMA conjugates lowered blood glucose for up to 120 h in a diabetic mouse model. Most intriguingly, we showed that appending PEG as oligomeric side-chains on the conjugated POEGMA and tuning the side-chain length completely eliminated the reactivity of exendin-C-POEGMA conjugates toward patient-derived anti-PEG antibodies without compromising in vivo efficacy. Clinically, the lack of anti-PEG antigenicity of POEGMA conjugates is expected to completely eliminate serious first-exposure allergic reactions and the accelerated blood clearance of POEGMA-drug conjugates due to pre-existing anti-PEG antibodies in patients.
Collectively, these results establish POEGMAlation as a next-generation PEGylation technology that is highly useful for improving the pharmacological performance of therapeutic biomolecules while providing a timely solution to the increasing levels of pre-existing anti-PEG antibodies in patients that are seriously hindering the safety and efficacy of traditional PEGylated drugs.
Item Open Access Protein-DNA Binding: Discovering Motifs and Distinguishing Direct from Indirect Interactions(2009) Gordan, Raluca MihaelaThe initiation of two major processes in the eukaryotic cell, gene transcription and DNA replication, is regulated largely through interactions between proteins or protein complexes and DNA. Although a lot is known about the interacting proteins and their role in regulating transcription and replication, the specific DNA binding motifs of many regulatory proteins and complexes are still to be determined. For this purpose, many computational tools for DNA motif discovery have been developed in the last two decades. These tools employ a variety of strategies, from exhaustive search to sampling techniques, with the hope of finding over-represented motifs in sets of co-regulated or co-bound sequences. Despite the variety of computational tools aimed at solving the problem of motif discovery, their ability to correctly detect known DNA motifs is still limited. The motifs are usually short and many times degenerate, which makes them difficult to distinguish from genomic background. We believe the most efficient strategy for improving the performance of motif discovery is not to use increasingly complex computational and statistical methods and models, but to incorporate more of the biology into the computational techniques, in a principled manner. To this end, we propose a novel motif discovery algorithm: PRIORITY. Based on a general Gibbs sampling framework, PRIORITY has a major advantage over other motif discovery tools: it can incorporate different types of biological information (e.g., nucleosome positioning information) to guide the search for DNA binding sites toward regions where these sites are more likely to occur (e.g., nucleosome-free regions).
We use transcription factor (TF) binding data from yeast chromatin immunoprecipitation (ChIP-chip) experiments to test the performance of our motif discovery algorithm when incorporating three types of biological information: information about nucleosome positioning, information about DNA double-helical stability, and evolutionary conservation information. In each case, incorporating additional biological information has proven very useful in increasing the accuracy of motif finding, with the number of correctly identified motifs increasing with up to 52%. PRIORITY is not restricted to TF binding data. In this work, we also analyze origin recognition complex (ORC) binding data and show that PRIORITY can utilize DNA structural information to predict the binding specificity of the yeast ORC.
Despite the improvement obtained using additional biological information, the success of motif discovery algorithms in identifying known motifs is still limited, especially when applied to sequences bound in vivo (such as those of ChIP-chip) because the observed protein-DNA interactions are not necessarily direct. Some TFs associate with DNA only indirectly via protein partners, while others exhibit both direct and indirect binding. We propose a novel method to distinguish between direct and indirect TF-DNA interactions, integrating in vivo TF binding data, in vivo nucleosome occupancy data, and in vitro motifs from protein binding microarrays. When applied to yeast ChIP-chip data, our method reveals that only 48% of the ChIP-chip data sets can be readily explained by direct binding of the profiled TF, while 16% can be explained by indirect DNA binding. In the remaining 36%, we found that none of the motifs used in our analysis was able to explain the ChIP-chip data, either because the data was too noisy or because the set of motifs was incomplete. As more in vitro motifs become available, our method can be used to build a complete catalog of direct and indirect TF-DNA interactions.
Item Open Access Self-Assembled Protein-Based Biomaterials with Tailorable Physical Properties(2015) Goodwin, MorganSoft biomaterials are used in a variety of applications such as scaffolds for cell growth and coatings for implants or transplants. We aim to create a protein hydrogel that will self-assemble upon the mixing of two different protein constructs. This is accomplished using Streptavidin, a protein that tetramerizes, and SpyTag-SpyCatcher, a protein-peptide that spontaneously forms covalent bonds, as the crosslinking mechanisms. Further, using protein building blocks whose viscoelastic properties are known from single-molecule force spectroscopy (SMFS), we aim to create a hydrogel whose physical properties are tailorable by altering the building blocks incorporated in the constructs. This thesis focuses on using an atomic force microscope for force spectroscopy and imaging to analyze the formation of networks upon mixing various protein constructs. We find that small scale networks form using both Streptavidin and SpyTag-SpyCatcher as crosslinkers and that SpyCatcher can be used to detect molecular crosslinking and polyprotein polarization through SMFS.
Item Open Access Tailoring nutrition therapy to illness and recovery.(Crit Care, 2017-12-28) Wischmeyer, Paul EWithout doubt, in medicine as in life, one size does not fit all. We do not administer the same drug or dose to every patient at all times, so why then would we live under the illusion that we should give the same nutrition at all times in the continuum of critical illness? We have long lived under the assumption that critical illness and trauma lead to a consistent early increase in metabolic/caloric need, the so-called "hypermetabolism" of critical illness. What if this is incorrect? Recent data indicate that early underfeeding of calories (trophic feeding) may have benefits and may require consideration in well-nourished patients. However, we must confront the reality that currently ICU nutrition delivery worldwide is actually leading to "starvation" of our patients and is likely a major contributor to poor long-term quality of life outcomes. To begin to ascertain the actual calorie and protein delivery required for optimal ICU recovery, an understanding of "starvation" and recovery from starvation and lean body mass (LBM) loss is needed. To begin to answer this question, we must look to the landmark Minnesota Starvation Study from 1945. This trial defines much of the world's knowledge about starvation, and most importantly what is required for recovery from starvation and massive LBM loss as occurs in the ICU. Recent and historic data indicate that critical illness is characterized by early massive catabolism, LBM loss, and escalating hypermetabolism that can persist for months or years. Early enteral nutrition during the acute phase should attempt to correct micronutrient/vitamin deficiencies, deliver adequate protein, and moderate nonprotein calories in well-nourished patients, as in the acute phase they are capable of generating significant endogenous energy. Post resuscitation, increasing protein (1.5-2.0 g/kg/day) and calories are needed to attenuate LBM loss and promote recovery. Malnutrition screening is essential and parenteral nutrition can be safely added following resuscitation when enteral nutrition is failing based on pre-illness malnutrition and LBM status. Following the ICU stay, significant protein/calorie delivery for months or years is required to facilitate functional and LBM recovery, with high-protein oral supplements being essential to achieve adequate nutrition.