NMR Investigation of Structures of G-Protein Coupled Receptor Folding Intermediates

Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot et al., Biochemistry 29(1990), 4031) is postulated to proceed in 2 steps: Partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin et al., Biophys. J. 96(2009), 3187), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123 and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior

Structural and functional characterization of human ERp44: a closer look at a member of PDI family regulating protein quality control in the early secretory pathway

The Endoplasmic Reticulum (ER) is the site of folding and assembly of secretory proteins. Fidelity of protein-based intracellular communication is guaranteed by protein quality control mechanisms located at the Early Secretory Compartment (ESC), which restricts forward transport to native proteins. ERp44 plays a key role in the Thiol-Mediated Retention (TMR) of variety of client proteins (Ero1, SUMF1, Adiponectin, IgM) thus regulating their transport and localization. Little is known about the molecular mechanisms of ERp44-TMR and how it is regulated in living cells. Hence, the overall aim of this work is to investigate the structure-function relationship of ERp44. In collaboration with Wang's group the crystal structure of ERp44 was determined. The structure of ERp44 most likely represents a non-reactive conformation of the protein. Indeed, Cys29 is shielded from the bulk solvent by C-terminal tail and almost inaccessible for the formation of intermolecular disulfide bonds with client proteins. Based on the obtained structural data and functional studies, a panel of mutants of ERp44 has been characterized in order to understand how C-terminal tail rearrangements expose substrate binding site, thus modulating substrates binding/ release in view of its role in TMR. Moreover, the pH gradient between ESC organelles was investigated as major determinant of C-terminal tail rearrangements. Given the known pH differences in the ESC, the data support the hypothesis of a role of the pH variation in governing ERp44-TMR activity in vivo. A deeper knowledge of the structure/function relationship of ERp44 will shed light on the protein quality control mechanisms thus providing essential knowledge of ESC processing diseases and in biotechnology, improving the production of man-made therapeutic proteins

구형성과 뒤틀림각에 기반한 단백질 구조 방법론 개발

학위논문 (박사)-- 서울대학교 대학원 : 협동과정 생물정보학전공, 2013. 2. 손현석.The structure of protein has intimate relationship with the function of protein. The structure of protein is experimentally determined through X-ray crystallography and NMR methods. However, X-ray crystallography is hard to obtain mobile protein structure and crystallization often causes practical problems. NMR structure is impossible in the observation of membranous or large proteins. Thus, theoretical methods for the determination of protein structures are highly concerned to circumvent practical problems. Homology, threading and ab initio modeling are the three typical approaches in protein structure modeling. ab initio modeling is often called as protein folding problem. The natural stable state of protein structure is believed to be the minimal energy state. The critical problem of protein folding research is the impossibility of the exhaustive search of possible conformations. Globularity of the protein structure was assessed in the pursuit of the universal structural constraint while approximated measurement name Gb-index was developed. Strong perfect globe-like character and the relationship between small size and the loss of globular structure was found among 7131 proteins which implies that living organisms have mechanisms to aid folding into the globular structure to reduce irreversible aggregation. This also implies the possible mechanisms of diseases caused by protein aggregation, including some forms of trinucleotide repeat expansion-mediated diseases. Torsion angle constraint mimics natural process of conformational change of proteins which lacks significant movement along covalent bonds and change in bond angles. This torsion angle system was applied to structure alignment to prove the validity as a structural representation. It was more effective to accurately anticipate homology among 1891 pairs of proteins of 62 different proteases and among 1770 pairs of 60 proteins of kinases and proteases with the string of φ and ψ dihedral angle array than famous 3D structural alignment tool TM-align. Secondary structure database and structure alignment web server was constructed from PDB and SCOP entries based on the simple classification scheme according to the backbone torsion angles. The database introduced here offers functions of secondary database searching, secondary structure calculation, and pair-wise protein structure comparison. Visualization during the process of the protein folding simulation is quite interesting regarding the fast apprehension of the states while previous algorithms such as molecular dynamics offers very few options of interference. Computational application named ProtTorter which visualizes three-dimensional conformation, calculates the potential energy, and supplies the user interface for backbone torsion angle manipulation was developed. Using this application, simple folding algorithm was newly investigated. Cotranslational and torsional folding path was utilized in the context of Levinthal paradox. The validity of the folding method was investigated using the test sets of small peptides. Positive result for the possibility of this method was obtained as the stable negative energy minimal structures and fast convergence. Application of torsional system of which validity was proved in the structure alignment assays and globular constraints which might infer solvent interactions by minimizing solvent accessible surface area might be worth for further studies based on the folding algorithm using ProtTorter application.1 Introduction 1 1.1 Background of Protein Research 1 1.1.1 The Function and Structure of Protein 2 1.1.2 Protein Secondary Structure 3 1.1.3 Torsion Angle 4 1.1.4 Hydrophobic Effect 5 1.2 Experimental Structure Determination Methods 6 1.2.1 X-ray Crystallography 6 1.2.2 NMR Spectroscopy 6 1.2.3 Limitations of Experimental Methods 7 1.3 Protein Structure Prediction Methods 8 1.3.1 Homology or Comparative Modeling Method 9 1.3.2 Threading Method 10 1.3.3 ab initio Method 12 1.3.3.1 Molecular Dynamics Simulation Method 13 1.3.3.2 Levinthal Paradox 15 1.3.3.3 Lattice Model 15 1.3.3.4 Monte Carlo Method 17 1.3.4 Competition of Protein Structure Prediction Methods: CASP 19 1.4 Studies and Concerns of the Protein Folding Research 20 2 Analysis of Globular Nature of Proteins 24 2.1 Introduction 24 2.2 Materials and Methods 26 2.2.1 Data Sets 26 2.2.2 Globularity Measurement 27 2.3 Results and Discussion 28 2.4 Conclusion 32 3 Validity of Protein Structure Alignment Based on Backbone Torsion Angles 39 3.1 Introduction 39 3.2 Materials and Methods 43 3.2.1 Definition of φ and ψ Angles 43 3.2.2 Ramachandran Plot RMSD (RamRMSD) 44 3.2.3 Statistical Similarity Measurement with Weight Imposition 45 3.2.4 Alignment Algorithm 46 3.2.5 Parameter Settings for Alignments and Clustering 47 3.2.6 Performance-evaluating Quantities 48 3.2.7 Test Set Preparation 49 3.3 Results and Discussion 50 3.3.1 Sequence and Structure Trees of Different Groups of Proteases 50 3.3.2 Comparison of Backbone Torsion Angle-based Method and TM-align 52 3.3.3 Clustring Trees and Accuracy Analysis with Delineation Set of 30 Kinases and 30 Proteases 55 3.3.4 Computational Time and Complexity 58 3.4 Conclusion 59 4 Secondary Structure Information Repository from Backbone Torsion Angle 67 4.1 Introduction 67 4.2 Materials and Methods 72 4.3 Results 72 4.3.1 User Interface and Architecture 72 4.3.2 Computational Mechanisms 75 4.4 Discussion 79 5 Computational Application for Protein Folding Modeling Based on Backbone Torsion Angle and for Protein Structure Viewing 86 5.1 Introduction 86 5.2 Materials and Methods 90 5.2.1 Computational Framework 90 5.2.2 Model Energy Calculation 90 5.3 Results 93 5.3.1 User Interface 93 5.3.2 Protein Structure File Import 96 5.3.3 Protein Structure File Export 96 5.3.4 Parsing and Initialization of Structure File 96 5.3.5 Structural Representation 98 5.3.6 Modifying Graphical Representation of Structure 99 5.3.7 Protein Model Building 101 5.3.8 Model Modification 103 5.3.9 Model Energy Calculation 104 5.3.10 Local Energy Minima Calculation and Cotranslational Folding 107 5.4 Discussion 107 6 Protein Folding of Cotranslational Initial Structure with Torsional Levinthal Path 114 6.1 Introduction 114 6.2 Materials and Methods 120 6.2.1 Dataset 120 6.2.2 Cotranslational Folding of Initial Structure 121 6.2.3 Iterative Optimization of Initial Structure Following Torsional Folding Path 122 6.3 Results and Discussion 123 6.4 Conclusion 128 7 Summary 137Docto

Profiling interactions of proximal nascent chains reveals a general co-translational mechanism of protein complex assembly

The association of proteins into functional oligomeric complexes is crucial for nearly all cellular processes. Despite rapid progress in characterizing the structure of native assemblies, the underlying mechanisms that guide faithful complex formation in the crowded cellular environment are understood only superficially. To secure efficient complex biogenesis and limit the exposure of aggregation-prone intermediates, many proteins assemble co-translationally, via interaction of a fully synthetized and a nascent protein subunit (co-post assembly). Here, we explore the prevalence and the mechanistic principles of a putative co-translational assembly mechanism, which involves the direct interaction of nascent subunits emerging from proximal ribosomes (co-co assembly). To obtain direct evidence of this putative assembly mode, we apply a newly developed method based on Ribosome Profiling, named Disome Selective Profiling (DiSP), which allows to monitor the conversion of single ribosomes to nascent chain connected ribosome pairs across the proteome with high resolution. We use this approach to analyse co-co assembly in two human cell lines and demonstrate that it constitutes a general mechanism inside cells that is employed by hundreds of high confidence and thousands of low confidence candidates, comprising 11 to 32% of all complex subunits. Analysing the features of the co-co assembly proteome, we reveal that this mechanism guides formation of mostly homomeric complexes and typically relies on interaction of N-terminal nascent chain segments. We further identify five dimerization domains mediating the majority of co-co interactions, which are either partially or completely exposed at the onset of nascent chain dimerization, implying different folding and assembly mechanisms. The detectable fraction of each candidate’s nascent chains that co-co assemble is in median 40% and in some cases exceeds 90%, suggesting that this co-translational assembly path may be employed as the main route for complex formation. To gain deeper insights into the mechanistic basis of co-co assembly, we took a series of experimental approaches that distinguish between interactions of nascent chains emerging from the same or different polysomes (termed assembly in cis and in trans, respectively). These experiments could not support a model of assembly in trans. Conversely, we find indications supporting a cis assembly model for nuclear lamin C, one of our high confidence candidates. This mechanism provides a simple explanation for the remarkable specificity of lamin homodimer formation in vivo, where splice variants with largely overlapping sequences do not mix. We propose that assembly in cis more generally secures specific homomer formation of isoforms and structurally-related proteins which are highly prone to promiscuous interactions inside cells. In conclusion, this study provides a global annotation of nascent chain interactions across the human proteome and elucidates the basic principles of this widespread assembly pathway. Our findings raise a number of fundamental questions concerning the mechanisms ensuring high-fidelity protein biogenesis, including the implications of co-co assembly on polysome structure, the possible consequences of co-co assembly failure, the inter-dependence with co-translational folding and the synchronization and coordination with translation kinetics

Peptide Model of the Mutant Proinsulin Syndrome. I. Design and Clinical Correlation

The mutant proinsulin syndrome is a monogenic cause of diabetes mellitus due to toxic misfolding of insulin's biosynthetic precursor. Also designated mutant INS-gene induced diabetes of the young (MIDY), this syndrome defines molecular determinants of foldability in the endoplasmic reticulum (ER) of β-cells. Here, we describe a peptide model of a key proinsulin folding intermediate and variants containing representative clinical mutations; the latter perturb invariant core sites in native proinsulin (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser). The studies exploited a 49-residue single-chain synthetic precursor (designated DesDi), previously shown to optimize in vitro efficiency of disulfide pairing. Parent and variant peptides contain a single disulfide bridge (cystine B19-A20) to provide a model of proinsulin's first oxidative folding intermediate. The peptides were characterized by circular dichroism and redox stability in relation to effects of the mutations on (a) in vitro foldability of the corresponding insulin analogs and (b) ER stress induced in cell culture on expression of the corresponding variant proinsulins. Striking correlations were observed between peptide biophysical properties, degree of ER stress and age of diabetes onset (neonatal or adolescent). Our findings suggest that age of onset reflects the extent to which nascent structure is destabilized in proinsulin's putative folding nucleus. We envisage that such peptide models will enable high-resolution structural studies of key folding determinants and in turn permit molecular dissection of phenotype-genotype relationships in this monogenic diabetes syndrome. Our companion study (next article in this issue) employs two-dimensional heteronuclear NMR spectroscopy to define site-specific perturbations in the variant peptides