Probing Transient Partial Unfolding in Proteins by Native-State Proteolysis

Chiwook Park 한국구조생물학회 2014 Biodesign Vol.2 No.4

Even under native conditions, proteins can have various non-native conformations, which are in equilibrium with the native functional conformations. Though the populations of the non-native conformation are usually small under native conditions, knowledge of the energy and the structure of the non-native forms is critical in understanding how proteins acquire and lose their structures. Native-state proteolysis is an experimental approach to selectively investigate a dominant partially unfolded form using proteolysis as a structural probe. From analyzing the kinetics of proteolysis of a protein under native conditions, we determine the energetics of unfolding to the partially unfolded form. The effect of urea on the energetics of partial unfolding reports the degree of unfolding in the partially unfolded form. Also, by assessing the effect of a point mutation on the energetics of partial unfolding, we elucidate the structure of the partially unfolded form. Partially unfolded forms probed by native-state proteolysis provide valuable information on the mechanisms of protein folding and protein degradation. In this review, we survey the principle and the applications of native-state proteolysis and also examine the pros and cons of the method in comparison with other experimental approaches.

Highly sensitive and selective in vitro diagnostics based on DNA probes and aptamers

조헌호,이성환,반창일 한국구조생물학회 2015 Biodesign Vol.3 No.1

Medical diagnosis is very important and essential for maintaining a healthy life. In vitro diagnostics have recently been afocus of scientists and researchers because they have a number of merits over other diagnostic methods. In particular,nucleic acid-based diagnostic methods are powerful and promising techniques. In this review, various types of nucleicacid-based in vitro diagnostics are introduced. These methods can be categorized into three groups according to theiranalytical approaches. In addition, aptamer-based diagnostic methods are covered in greater detail because aptamers arepromising materials for diverse areas, not only as alternatives to antibodies but also as the core components of analyticalequipment. It is expected that in vitro diagnostics based on DNA probes and aptamers will become a valuable platformencompassing all types of diseases.

Two component signaling systems in Mycobacterium tuberculosis

조하언,강범식 한국구조생물학회 2015 Biodesign Vol.3 No.1

Mycobacterium tuberculosis is one of fearful pathogens and has the ability to persist within its host. Its successful survivalis due to alterations in gene expression in response to environmental changes by two component systems (TCS), whichconsist of sensor histidine kinases (HK) and their cognate response regulators (RR). M. tuberculosis has twelve TCSs andfive orphan RRs. A typical TCS involves sensing of internal or external signals by a HK, leading to its autophosphorylation,followed by phosphoryl transfer to the cognate RR, which functions as a transcriptional activator. To understand thefunction and the mechanism of M. tuberculosis TCSs, the components of HKs and RRs are subjected on the structuralstudies and the results could be useful for new antituberculosis drug development. Here, structural features of HKs andRRs currently revealed from M. tuberculosis are summarized. Those include GAF and PAS domains for senor domains andATP binding domains from HKs, and the receiver and effector domains from RRs.

Current advances in the development of G-protein-coupled receptor structure and their future application in drug design

김국래,강영금,조현수 한국구조생물학회 2014 Biodesign Vol.2 No.1

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that play important roles in signal transductionfrom the extracellular compartment to the cytoplasm. GPCR malfunction has been implicated in various diseases such ascancer, allergy, neuronal disease, rheumatism, and obesity. Because of the importance of GPCRs, GPCR-targeting drugsoccupy approximately 40% of the total drug market. Recently, three-dimensional structures of many GPCRs have beensuccessfully ascertained on the basis of various developed methods that improve protein stabilization, crystallization, anddiffraction. Some GPCR structures have elucidated the underlying activation mechanism by allowing comparison betweenthe receptor’s inactive and active states. β-adrenergic receptor bound with G-protein trimeric complex (Gαβγ) structure hasalso been determined. To date, XFEL technology is the latest technology used to determine GPCR structures. In this review,we describe GPCR structures, structure-based activation mechanisms and principles, and the application of XFEL indetermining GPCR structures and development of GPCR-based drugs.

Oxidative stress, mitochondrial dysfunction and endoplasmic reticulum stress

박용수 한국구조생물학회 2014 Biodesign Vol.2 No.1

Diabetes mellitus is a chronic complex metabolic condition that affects carbohydrate, lipid and protein metabolism andmay impair numerous organs and functions of the organism. Hyperglycemia is only the most obvious biochemical markerof diabetes, and the main contributor to the development of diabetes complications is the cumulative effect of chronichyperglycemia. Increasing evidence suggests that oxidative stress may be the key mediators of the deleterious effectsof hyperglycemia. Mitochondria play a central role in the generation of reactive oxygen species and cell apoptosis. Anumber of conditions including nutrient excess that interfere with proper endoplasmic reticulum (ER) function may leadto accumulation of unfolded proteins, which then trigger apoptotic as well as adaptive downstream signaling pathways. Many studies have also provided ample evidences that mitochondrial dysfunction and ER stress are most importantpathogenic causes for the development of diabetes and its complications. Regulation mechanisms of how mitochondriaplay in the metabolism of glucose and fatty acids, the primary fuels used by cells to produce ATP, have been the subject oftremendous interests. Nonetheless, much remains to be investigated such as tissue-specific fuel selection and its relationwith the pathogenesis of diabetes and complications. Cellular homeostasis depends upon the functional relationshipbetween mitochondria and the ER. Propagation of calcium signaling from ER to mitochondria is involved in both ATPproduction and cell death. On the other hand, the ER requires ATP to function properly, which may make it the best sitefor sensing metabolic stress. In this article, oxidative stress, mitochondrial dysfunction and ER stress, especially their realtimeinteraction in diabetes and complication development will be reviewed.

Prediction of Protein Structure and Interaction by GALAXY Protein Modeling Programs

신웅희,이규리,허림,이하섭,석차옥 한국구조생물학회 2014 Biodesign Vol.2 No.1

In this review, recently developed GALAXY protein modeling programs are introduced and advantages and disadvantagesof these programs for both program users and method developers are discussed. The GALAXY package consists of thetemplate-based modeling program GalaxyTBM, the loop/terminus modeling program GalaxyLoop, the model refinementprogram GalaxyRefine, the homo-oligomer prediction program GalaxyGemini, and the protein-ligand docking programGalaxyDock. These programs have been tested with some success in community-wide competition Critical Assessmentof Techniques for Protein Structure Prediction (CASP) experiments. For the development of these programs, modelingproblems have been posed as global optimization problems of designed energy functions. The free energy functionsof GALAXY have been carefully designed by combining physical chemistry principles and structure and sequenceinformation. Efficient conformational search methods such as conformational space annealing and triaxial loop closurehave been employed. Freely accessible web servers of the modeling programs are available at http://galaxy.seoklab.org,and some programs can be downloaded from http://galaxy.seoklab.org/softwares.

Side-chain Prediction and Computational Protein Design Problems

김덕수,유중현 한국구조생물학회 2014 Biodesign Vol.2 No.1

Protein is fundamental for life and thus protein design has been one of the hottest research issues since a couple ofdecades ago and computational approach has been developed with some significant design cases with experimentalverification. One of the fundamental building blocks of computational protein design is to predict side-chain conformationof backbone structure. This paper first reviews prior efforts of computationally predicting sidechains of a given backboneand then reviews computational protein design efforts based on side-chain prediction. The paper views computationalprotein design problem as three categories: Redesign, De novo design of Type I, and De novo design of Type II. Some wellknowncomputer programs and algorithms related with computational protein design are also reviewed.

Improvement of crystal quality for X-ray diffraction of the problematic redox protein complex, thioredoxin and thioredoxininteracting protein

황중원,김명희 한국구조생물학회 2014 Biodesign Vol.2 No.2

Thioredoxin-interacting protein (TXNIP) regulates many biological processes by interacting with thioredoxin (TRX) in aredox-dependent fashion. Thus, elucidation of the mechanism, by which these two proteins interact, is a key to understandredox-dependent cell signaling. Recently, the TXNIP-TRX complex structure and their interacting mechanism have beenpublished. Both TRX (containing 5 cysteine residues) and TXNIP (containing 11 cysteine residues) are highly redoxsensitiveproteins, and are therefore extremely difficult to handle in vitro. Here, we present details of how these problematicredox proteins can be expressed, purified, and crystallized to a suitable quality for X-ray diffraction. Both proteins wereexpressed as a soluble complex in the E. coli Rosetta-gami (DE3) system in which disulfide bonds can form owing to trxB/gor mutation. Moreover, catching TXNIP in an intermediate state, in which TRX was bound, was crucial to obtain stablecomplex proteins linking to crystallization.

Structure-based drug discovery using NMR spectroscopy

윤지혜,이원태 한국구조생물학회 2014 Biodesign Vol.2 No.2

The structural information of biomolecules is essential for novel drug discovery. Recently, several new high-throughputdrug-screening methods have been established. Nuclear magnetic resonance (NMR) spectroscopy is one of the mostpowerful techniques for drug development, providing critical information on molecular interactions for drug design insolution. Knowledge of the 3D structure and dynamic properties of drug target molecules is mandatory in performinginitial hit selection and lead optimization. For this purpose, NMR spectroscopy has been developed and widely used. Inparticular, NMR is ideal for the fragment-based drug discovery (FBDD) strategy, because molecular interactions betweentarget proteins and small ligands can be quickly identified at the atomic level. In this review, NMR techniques used forstructure-based drug design will be discussed together with practical examples.

Structural characteristics of human histidyltRNA synthetase

김영관,장지은,김성훈,전영호 한국구조생물학회 2014 Biodesign Vol.2 No.4

Aminoacyl-tRNA synthetase is an enzyme that recognizes its cognate amino acid and tRNA to catalyze the reaction offorming of aminoacyl-tRNAs. Histidyl-tRNA synthetase (HisRS) belongs to the class II family of amioacyl-tRNA synthetasesand is responsible for the incorporation of histidine into proteins. To elucidate the structure, full-length human HisRS(hHisRS) was over-expressed, purified and crystallized. We determined the crystal structure to 2.8 Å resolutions by themolecular replacement. The space group was P41212 with a = b = 97.66 Å, c = 254.40 Å. α=90.00°, β=90.00°, γ=90.00°. Overall structure of human HisRS is quite similar to that of other HisRSs except the insertion domain in the catalyticdomain of hHisRS (residues 214-290), which likely binds to tRNA. The N-terminal WHEP-TRS domain of hHisRS, whichplays an important role in non-canonical function of HisRS, do not appear in this structure. While the anticodon binding andinsertion domain showed structural diversity among the class II ARS sub-families, the catalytic domain showed substantialstructural conservation in class II ARSs. Structural comparison with bacterial HisRSs and human ARSs identified that novelinsertion domain and strictly conserved catalytic site residues for the interaction with ATP and histidine.