Fluorescence Correlation Spectroscopic Study of Serpin Depolymerization by Computationally Designed Peptides

Members of the serine proteinase inhibitor (serpin) family play important roles in the inflammatory and coagulation cascades. Interaction of a serpin with its target proteinase induces a large conformational change, resulting in insertion of its reactive center loop (RCL) into the main body of the protein as a new strand within beta-sheet A. Intermolecular insertion of the RCL of one serpin molecule into the beta-sheet A of another leads to polymerization, a widespread phenomenon associated with a general class of diseases known as serpinopathies. Small peptides are known to modulate the polymerization process by binding within beta-sheet A. Here, we use fluorescence correlation spectroscopy (FCS) to probe the mechanism of peptide modulation of alpha(1)-antitrypsin (alpha(1)-AT) polymerization and depolymerization, and employ a statistical computationally-assisted design strategy (SCADS) to identify new tetrapeptides that modulate polymerization. Our results demonstrate that peptide-induced depolymerization takes place via a heterogeneous, multi-step process that begins with internal fragmentation of the polymer chain. One of the designed tetrapeptides is the most potent antitrypsin depolymerizer yet found

The Development of a Community-Based Drug Intervention for Filipino Drug Users

This article documents the development of a community-based drug intervention for low- to mild-risk drug users who surrendered as part of the Philippine government\u27s anti-drug campaign. It highlights the importance of developing evidence-informed drug recovery interventions that are appropriate to the Asian culture and to developing economies. Interviews and consultations with users and community stakeholders reveal the need for an intervention that would improve the drug recovery skills and life skills of users. Evidence-based interventions were adapted using McKleroy and colleagues’ (2006) Map of Adaptation Process (MAP) framework. The resulting intervention reflected the country\u27s collectivist culture, relational values, propensity for indirect and non-verbal communication, and interdependent self-construal. The use of small groups, interactive and creative methodologies, and the incorporation of music and prayer also recognised the importance of these in the Philippine culture

Calculation of the Free Energy and Cooperativity of Protein Folding

Calculation of the free energy of protein folding and delineation of its pre-organization are of foremost importance for understanding, predicting and designing biological macromolecules. Here, we introduce an energy smoothing variant of parallel tempering replica exchange Monte Carlo (REMS) that allows for efficient configurational sampling of flexible solutes under the conditions of molecular hydration. Its usage to calculate the thermal stability of a model globular protein, Trp cage TC5b, achieves excellent agreement with experimental measurements. We find that the stability of TC5b is attained through the coupled formation of local and non-local interactions. Remarkably, many of these structures persist at high temperature, concomitant with the origin of native-like configurations and mesostates in an otherwise macroscopically disordered unfolded state. Graph manifold learning reveals that the conversion of these mesostates to the native state is structurally heterogeneous, and that the cooperativity of their formation is encoded largely by the unfolded state ensemble. In all, these studies establish the extent of thermodynamic and structural pre-organization of folding of this model globular protein, and achieve the calculation of macromolecular stability ab initio, as required for ab initio structure prediction, genome annotation, and drug design

A Kinetic Model of Trp-Cage Folding from Multiple Biased Molecular Dynamics Simulations

Trp-cage is a designed 20-residue polypeptide that, in spite of its size, shares several features with larger globular proteins. Although the system has been intensively investigated experimentally and theoretically, its folding mechanism is not yet fully understood. Indeed, some experiments suggest a two-state behavior, while others point to the presence of intermediates. In this work we show that the results of a bias-exchange metadynamics simulation can be used for constructing a detailed thermodynamic and kinetic model of the system. The model, although constructed from a biased simulation, has a quality similar to those extracted from the analysis of long unbiased molecular dynamics trajectories. This is demonstrated by a careful benchmark of the approach on a smaller system, the solvated Ace-Ala3-Nme peptide. For the Trp-cage folding, the model predicts that the relaxation time of 3100 ns observed experimentally is due to the presence of a compact molten globule-like conformation. This state has an occupancy of only 3% at 300 K, but acts as a kinetic trap. Instead, non-compact structures relax to the folded state on the sub-microsecond timescale. The model also predicts the presence of a state at of 4.4 Å from the NMR structure in which the Trp strongly interacts with Pro12. This state can explain the abnormal temperature dependence of the and chemical shifts. The structures of the two most stable misfolded intermediates are in agreement with NMR experiments on the unfolded protein. Our work shows that, using biased molecular dynamics trajectories, it is possible to construct a model describing in detail the Trp-cage folding kinetics and thermodynamics in agreement with experimental data

Understanding the folding mechanisms of small model proteins

Most proteins require appropriate folding in order to perform their respective functions, whereas misfolding can lead to pathological conditions. Thus, protein folding presents a complex problem which requires extensive study from both physical and biological perspectives. While significant progress has been made towards a solution to the protein folding problem, a quantitative and predictive understanding of how proteins fold is yet to be reached. This is partly due to the fact that protein molecules are complex, and that many (weak) interactions work together to define a protein\u27s native structure. To reduce the complexity of the problem, we have taken a bottom-up approach and focused on studying the folding dynamics and mechanism of small peptides that exhibit folding characteristics of large proteins. These studies not only yield insights into the early steps of protein folding, including the theoretical folding speed limit, but they also provide ideal models for computer simulations. The systems described in this thesis include simple protein secondary structural elements and miniproteins (i.e., trp-cage, GCN4-p1, and villin headpiece subdomain). Since these peptides fold on the sub-millisecond timescale, we have employed a laser-induced temperature jump infrared technique to measure their folding kinetics. Whenever necessary, we have also made use of sequence and structure perturbing methods to systematically assess the mechanistic role of various native-state properties, including protein length, hydrophobic and electrostatic interactions, and backbone-backbone hydrogen bonding, in the formation of the transition state ensemble. For example, our study of Trp-cage folding indicates that, contrary to molecular dynamics simulation, the formation of a solvent exposed salt-bridge is not required for achieving fast folding; instead, a well-placed aromatic interaction lowers the folding free energy barrier. Also, through investigation of the folding dynamics of a GCN4 coiled-coil variant, we show that the folding of such dihelical structural motifs is likely initiated by contacts throughout the sequence rather than those localized in a previously identified trigger sequence. Furthermore, using amide-to-ester backbone mutations, we are able to demonstrate that helix formation is not necessary for acquiring the transition state in the folding of the helical subdomain of villin headpiece, discrediting the backbone-centered view of protein folding

Understanding the folding mechanisms of small model proteins

Most proteins require appropriate folding in order to perform their respective functions, whereas misfolding can lead to pathological conditions. Thus, protein folding presents a complex problem which requires extensive study from both physical and biological perspectives. While significant progress has been made towards a solution to the protein folding problem, a quantitative and predictive understanding of how proteins fold is yet to be reached. This is partly due to the fact that protein molecules are complex, and that many (weak) interactions work together to define a protein\u27s native structure. To reduce the complexity of the problem, we have taken a bottom-up approach and focused on studying the folding dynamics and mechanism of small peptides that exhibit folding characteristics of large proteins. These studies not only yield insights into the early steps of protein folding, including the theoretical folding speed limit, but they also provide ideal models for computer simulations. The systems described in this thesis include simple protein secondary structural elements and miniproteins (i.e., trp-cage, GCN4-p1, and villin headpiece subdomain). Since these peptides fold on the sub-millisecond timescale, we have employed a laser-induced temperature jump infrared technique to measure their folding kinetics. Whenever necessary, we have also made use of sequence and structure perturbing methods to systematically assess the mechanistic role of various native-state properties, including protein length, hydrophobic and electrostatic interactions, and backbone-backbone hydrogen bonding, in the formation of the transition state ensemble. For example, our study of Trp-cage folding indicates that, contrary to molecular dynamics simulation, the formation of a solvent exposed salt-bridge is not required for achieving fast folding; instead, a well-placed aromatic interaction lowers the folding free energy barrier. Also, through investigation of the folding dynamics of a GCN4 coiled-coil variant, we show that the folding of such dihelical structural motifs is likely initiated by contacts throughout the sequence rather than those localized in a previously identified trigger sequence. Furthermore, using amide-to-ester backbone mutations, we are able to demonstrate that helix formation is not necessary for acquiring the transition state in the folding of the helical subdomain of villin headpiece, discrediting the backbone-centered view of protein folding

New Aspects on using Artificial Intelligence to Shape the Future of Entrepreneurs

One way for enterprises to be successful in today’s challenging market is to be agile and be flexible to handle market changes. Using a conceptual and operational framework for improving the enterprise and keeping their desired situation, is always required. In this paper, a service oriented decision support system-based framework is proposed. The framework seeks for service oriented architecture (SOA) governance and suggests the initial architecture of the enterprise to support agility and optimality. Also, for the stability purpose a structure including service platforms, analyzers and decision support systems are employed to analyze the enterprise and make better decisions