The MdfA is a 410 amino acid-long integral membrane protein, which belongs to the Major Facilitator superfamily of multidrug transporters. It is predicted to consist of 12 transmembrane helices. MdfA uses the energy of the transmembrane proton gradient to pump a variety of toxic compounds out of E. coli cells. No high resolution structure of MdfA is available. The goals of this research project were to develop a practical method for purification of MdfA, to evaluate the feasibility of structure determination by Nuclear Magnetic Resonance (NMR) and X-ray crystallography, and to develop an activity assay for purified MdfA. To this end, MdfA, with a hexa-histidine tag attached to facilitate protein purification, was successfully expressed and incorporated into the cell membrane using an E. coli expression system. MdfA was extracted from the cell membrane with the detergents 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), n-dodecyl-B-D-maltoside (DDM), and 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG) and purified by affinity chromatography on nickel-nitrilotriacetic acid agarose. Pure protein was found to be monodisperse in DHPC, DDM and LMPG micelles. To achieve simple amino acid selective isotope labeling for high-resolution NMR studies, MdfA was expressed in a cell-free translation system. To determine if the purified protein was properly folded, 19F NMR experiments were carried out on 5-fluoro-tryptophan-labeled MdfA while titrating the MdfA substrates ethidium bromide and chloramphenicol into the fluoro-tryptophan-labeled MdfA sample. An activity assay was developed for MdfA incorporated into liposomes using the fluorescent dye 9-amino-6-chloro-2-methoxyacridine (ACMA) to detect proton translocation coupled to substrate transport. Results from both the 19F NMR and the transport activity assay indicated that the purified MdfA was properly folded and functional. NMR experiments with pure MdfA yielded spectra of insufficient quality for high-resolution structure determination but did indicate that structural studies of MdfA by NMR are feasible. Crystallization trials yielded crystals that are likely to contain protein and will serve as a starting point for further optimization of crystallization conditions for X-ray structure determination.

The MdfA is a 410 amino acid-long integral membrane protein, which belongs to the Major Facilitator superfamily of multidrug transporters. It is predicted to consist of 12 transmembrane helices. MdfA uses the energy of the transmembrane proton gradient to pump a variety of toxic compounds out of E. coli cells. No high resolution structure of MdfA is available. The goals of this research project were to develop a practical method for purification of MdfA, to evaluate the feasibility of structure determination by Nuclear Magnetic Resonance (NMR) and X-ray crystallography, and to develop an activity assay for purified MdfA. To this end, MdfA, with a hexa-histidine tag attached to facilitate protein purification, was successfully expressed and incorporated into the cell membrane using an E. coli expression system. MdfA was extracted from the cell membrane with the detergents 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), n-dodecyl-B-D-maltoside (DDM), and 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG) and purified by affinity chromatography on nickel-nitrilotriacetic acid agarose. Pure protein was found to be monodisperse in DHPC, DDM and LMPG micelles. To achieve simple amino acid selective isotope labeling for high-resolution NMR studies, MdfA was expressed in a cell-free translation system. To determine if the purified protein was properly folded, 19F NMR experiments were carried out on 5-fluoro-tryptophan-labeled MdfA while titrating the MdfA substrates ethidium bromide and chloramphenicol into the fluoro-tryptophan-labeled MdfA sample. An activity assay was developed for MdfA incorporated into liposomes using the fluorescent dye 9-amino-6-chloro-2-methoxyacridine (ACMA) to detect proton translocation coupled to substrate transport. Results from both the 19F NMR and the transport activity assay indicated that the purified MdfA was properly folded and functional. NMR experiments with pure MdfA yielded spectra of insufficient quality for high-resolution structure determination but did indicate that structural studies of MdfA by NMR are feasible. Crystallization trials yielded crystals that are likely to contain protein and will serve as a starting point for further optimization of crystallization conditions for X-ray structure determination.

Structural and functional annotation of sequences of putative proteins encoded in the newly sequenced genomes pose an important challenge. While much progress has been made towards high throughput experimental techniques for structure determination and functional assignment to proteins, most of the current genome-wide annotation systems rely on computational methods to derive cues on structure and function based on relationship with related proteins of known structure and/or function. Evolutionary pressure on proteins, forces the retention of sequence features that are important for structure and function. Thus, if it can be established that two proteins have descended from a common ancestor, then it can be inferred that the structural fold and biological function of the two proteins would be similar. Homology based information transfer from one protein to another has played a central role in the understanding of evolution of protein structures, functions and interactions. Many algorithmic improvements have been developed over the past two decades to recognize homologues of a protein from sequence-based searches alone, but there are still a large number of proteins without any functional annotation. The sensitivity of the available methods can be further enhanced by indirect comparisons with the help of intermediately-related sequences which link related families. However, sequence-based homology searches in the current protein sequence space are often restricted to the family members, due to the paucity of natural intermediate sequences that can act as linkers in detecting remote homologues. Thus a major goal of this thesis is to develop computational methods to fill up the sparse regions in the protein sequence space with computationally designed protein-like sequences and thereby create a continuum of protein sequences, which could aid in detecting remote homologues. Such designed sequences are further assessed for their effectiveness in detection of distant evolutionary relationships and functional annotation of proteins with unknown structure and function. Another important aspect in structural bioinformatics is to gain a good understanding of protein sequence - structure - function paradigm. Functional annotations by comparisons of protein sequences can be further strengthened with the addition of structural information; however, instances of functional divergence and convergence may lead to functional mis-annotations. Therefore, a systematic analysis is performed on the fold–function associations using binding site information and their inter-relationships using binding site similarity networks. Chapter 1 provides a background on proteins, their evolution, classification and structural and functional features. This chapter also describes various methods for detection of remote similarities and the role of protein sequence design methods in detection of distant relatives for protein annotation. Pitfalls in prediction of protein function from sequence and structure are also discussed followed by an outline of the thesis. Chapter 2 addresses the problem of paucity of available protein sequences that can act as linkers between distantly related proteins/families and help in detection of distant evolutionary relationships. Previous efforts in protein sequence design for remote homology detection and design of sequences corresponding to specific protein families are discussed. This chapter describes a novel methodology to computationally design intermediately-related protein sequences between two related families and thus fill-in the gaps in the sequence space between the related families. Protein families as defined in SCOP database are represented as position specific scoring matrices (PSSMs) and these profiles of related protein families within a fold are aligned using AlignHUSH -a profile-profile alignment method. Guided by this alignment, the frequency distribution of the amino acids in the two families are combined and for each aligned position a residue is selected based on the combined probability to occur in the alignment positions of two families. Each computationally designed sequence is then subjected to RPS-BLAST searches against an all profile pool representing all protein families. Artificial sequences that detect both the parent profiles with no hits corresponding to other folds qualify as ‘designed intermediate sequences’. Various scoring schemes and divergence levels for the design of protein-like sequences are investigated such that these designed sequences intersperse between two related families, thereby creating a continuum in sequence space. The method is then applied on a large scale for all folds with two or more families and resulted in the design of 3,611,010 intermediately-related sequences for 27,882 profile-profile alignments corresponding to 374 folds. Such designed sequences are generic in nature and can be augmented in any sequence database of natural protein sequences. Such enriched databases can then be queried using any sequence-based remote homology detection method to detect distant relatives. The next chapter (Chapter 3) explores the ability of these designed intermediate sequences to act as linkers of two related families and aid in detection of remote homologues. To assess the applicability of these designed sequences two types of databases have been generated, namely a CONTROL database containing protein sequences from natural sequence databases and an AUGMENTED database in which designed sequences are included in the database of natural sequences. Detailed assessments of the utility of such designed sequences using traditional sequence-based searches in the AUGMENTED database showed an enhanced detection of remote homologues for almost 74% of the folds. For over 3,000 queries, it is demonstrated that designed sequences are positioned as suitable linkers, which mediate connections between distantly related proteins. Using examples from known distant evolutionary relationships, we demonstrate that homology searches in augmented databases show an increase of up to 22% in the number of /correct evolutionary relationships "discovered". Such connections are reported with high sensitivities and very low false positive rates. Interestingly, they fill-in void and sparse regions in sequence space and relate distant proteins not only through multiple routes but also through SCOP-NrichD database, SUPFAM+ database, SUPERFAMILY database, protein domain library queried by pDomTHREADER and HHsearch against HMM library of SCOP families. This approach detected evolutionary relationships for almost 20% of all the families with no known structure or function. Detailed report of predictions for 614 DUFs, their fold and species distribution are provided in this chapter. These predictions are then enriched with GO terms and enzyme information wherever available. A detailed discussion is provided for few of the interesting assignments: DUF1636, DUF1572 and DUF2092 which are functionally annotated as thioredoxin-like 2Fe-2S ferredoxin, putative metalloenzyme and lipoprotein localization factors respectively. These 614 novel structure-function relationships of which 193 are supported by consensus between at least two of the five methods, can be accessed from http://proline.biochem.iisc.ernet.in/RHD_DUFS/. Protein functions can be appreciated better in the light of evolutionary information from their structures. Chapter 6 describes a database of evolutionary relationships identified between Pfam families. The grouping of Pfam families is important to obtain a better understanding on evolutionary relationships and in obtaining clues to functions of proteins in families of yet unknown function. Many structural genomics initiative projects have made considerable efforts in solving structures and bridging the growing gap between protein sequences and their structures. The results of such experiments suggest that often the newly solved structure using X-ray crystallography or NMR methods has structural similarity to a protein with already known structure. These relationships often remain undetected due to unavailability of structural information. Therefore, SUPFAM+ database aims to detect such distant relationships between Pfam families by mapping the Pfam families and SCOP domain families. The work presented in this chapter describes the generation of SUPFAM+ database using a sensitive AlignHUSH method to uncover hidden relationships. Firstly, Pfam families are queried against a profile database of SCOP families to derived Pfam-SCOP associations, and then Pfam families are queried against Pfam database to derive Pfam-Pfam relationships. Pfam families that remain without a mapping to a SCOP family are mapped indirectly to a SCOP family by identifying relationships between such Pfam families and other Pfam families that are already mapped to a SCOP family. The criteria are kept stringent for these mappings to minimize the rate of false positives. In case of a Pfam family mapping to two or more SCOP superfamilies, a decision tree is implemented to assign the Pfam family to a single SCOP superfamily. Using these direct and indirect evolutionary relationships present in the SCOP database, associations between Pfam families are derived. Therefore, relationship between two Pfam families that do not have significant sequence similarity can be identified if both are related to same SCOP superfamily. Almost 36% of the Pfam families could be mapped to SCOP families through direct or indirect association. These Pfam-SCOP associations are grouped into 1,646 different superfamilies and cataloguing changes that occur in the binding sites between two functions, which are analysed in this study to trace possible routes between different functions in evolutionarily related enzymes. The main conclusions of the entire thesis are summarized in Chapter 8, contributing in the area of remote homology detection from sequence information alone and understanding the ‘sequence-structure-function’ paradigm from a binding site perspective. The chapter illustrates the importance of the work presented here in the post-genomic era. The development of the algorithm for the design of ‘intermediately-related sequences’ that could serve as effective linkers in remote homology detection, its subsequent large scale assessment and amenability to be augmented into any protein sequence database and exploration by any sequence-based search method is highlighted. Databases in the NrichD resource are made available in the public domain along with a portal to design artificial sequence for or between protein families. This thesis also provides useful and meaningful predictions for protein families with yet unknown structure and function using NrichD database as well as four other state-of-the-art sequence-based remote homology detection methods. A different aspect addressed in this thesis provides a fundamental understanding of the relationships between protein structure and functions. Evolutionary relationships between functional families are identified using the inherent structural information for these families and fold-function relationships are studied from a perspective of similarities in their binding sites. Such studies help in the area of functional annotation, polypharmacology and protein engineering. Chapter 2 addresses the problem of paucity of available protein sequences that can act as linkers between distantly related proteins/families and help in detection of distant evolutionary relationships. Previous efforts in protein sequence design for remote homology detection and design of sequences corresponding to specific protein families are discussed. This chapter describes a novel methodology to computationally design intermediately-related protein sequences between two related families and thus fill-in the gaps in the sequence space between the related families. Protein families as defined in SCOP database are represented as position specific scoring matrices (PSSMs) and these profiles of related protein families within a fold are aligned using AlignHUSH -a profile-profile alignment method. Guided by this alignment, the frequency distribution of the amino acids in the two families are combined and for each aligned position a residue is selected based on the combined probability to occur in the alignment positions of two families. Each computationally designed sequence is then subjected to RPS-BLAST searches against an all profile pool representing all protein families. Artificial sequences that detect both the parent profiles with no hits corresponding to other folds qualify as ‘designed intermediate sequences’. Various scoring schemes and divergence levels for the design of protein-like sequences are investigated such that these designed sequences intersperse between two related families, thereby creating a continuum in sequence space. The method is then applied on a large scale for all folds with two or more families and resulted in the design of 3,611,010 intermediately-related sequences for 27,882 profile-profile alignments corresponding to 374 folds. Such designed sequences are generic in nature and can be augmented in any sequence database of natural protein sequences. Such enriched databases can then be queried using any sequence-based remote homology detection method to detect distant relatives. The next chapter (Chapter 3) explores the ability of these designed intermediate sequences to act as linkers of two related families and aid in detection of remote homologues. To assess the applicability of these designed sequences two types of databases have been generated, namely a CONTROL database containing protein sequences from natural sequence databases and an AUGMENTED database in which designed sequences are included in the database of natural sequences. Detailed assessments of the utility of such designed sequences using traditional sequence-based searches in the AUGMENTED database showed an enhanced detection of remote homologues for almost 74% of the folds. For over 3,000 queries, it is demonstrated that designed sequences are positioned as suitable linkers, which mediate connections between distantly related proteins. Using examples from known distant evolutionary relationships, we demonstrate that homology searches in augmented databases show an increase of up to 22% in the number of /correct evolutionary relationships "discovered". Such connections are reported with high sensitivities and very low false positive rates. Interestingly, they fill-in void and sparse regions in sequence space and relate distant proteins not only through multiple routes but also through SCOP-NrichD database, SUPFAM+ database, SUPERFAMILY database, protein domain library queried by pDomTHREADER and HHsearch against HMM library of SCOP families. This approach detected evolutionary relationships for almost 20% of all the families with no known structure or function. Detailed report of predictions for 614 DUFs, their fold and species distribution are provided in this chapter. These predictions are then enriched with GO terms and enzyme information wherever available. A detailed discussion is provided for few of the interesting assignments: DUF1636, DUF1572 and DUF2092 which are functionally annotated as thioredoxin-like 2Fe-2S ferredoxin, putative metalloenzyme and lipoprotein localization factors respectively. These 614 novel structure-function relationships of which 193 are supported by consensus between at least two of the five methods, can be accessed from http://proline.biochem.iisc.ernet.in/RHD_DUFS/. Protein functions can be appreciated better in the light of evolutionary information from their structures. Chapter 6 describes a database of evolutionary relationships identified between Pfam families. The grouping of Pfam families is important to obtain a better understanding on evolutionary relationships and in obtaining clues to functions of proteins in families of yet unknown function. Many structural genomics initiative projects have made considerable efforts in solving structures and bridging the growing gap between protein sequences and their structures. The results of such experiments suggest that often the newly solved structure using X-ray crystallography or NMR methods has structural similarity to a protein with already known structure. These relationships often remain undetected due to unavailability of structural information. Therefore, SUPFAM+ database aims to detect such distant relationships between Pfam families by mapping the Pfam families and SCOP domain families. The work presented in this chapter describes the generation of SUPFAM+ database using a sensitive AlignHUSH method to uncover hidden relationships. Firstly, Pfam families are queried against a profile database of SCOP families to derived Pfam-SCOP associations, and then Pfam families are queried against Pfam database to derive Pfam-Pfam relationships. Pfam families that remain without a mapping to a SCOP family are mapped indirectly to a SCOP family by identifying relationships between such Pfam families and other Pfam families that are already mapped to a SCOP family. The criteria are kept stringent for these mappings to minimize the rate of false positives. In case of a Pfam family mapping to two or more SCOP superfamilies, a decision tree is implemented to assign the Pfam family to a single SCOP superfamily. Using these direct and indirect evolutionary relationships present in the SCOP database, associations between Pfam families are derived. Therefore, relationship between two Pfam families that do not have significant sequence similarity can be identified if both are related to same SCOP superfamily. Almost 36% of the Pfam families could be mapped to SCOP families through direct or indirect association. These Pfam-SCOP associations are grouped into 1,646 different superfamilies and cataloguing changes that occur in the binding sites between two functions, which are analysed in this study to trace possible routes between different functions in evolutionarily related enzymes. The main conclusions of the entire thesis are summarized in Chapter 8, contributing in the area of remote homology detection from sequence information alone and understanding the ‘sequence-structure-function’ paradigm from a binding site perspective. The chapter illustrates the importance of the work presented here in the post-genomic era. The development of the algorithm for the design of ‘intermediately-related sequences’ that could serve as effective linkers in remote homology detection, its subsequent large scale assessment and amenability to be augmented into any protein sequence database and exploration by any sequence-based search method is highlighted. Databases in the NrichD resource are made available in the public domain along with a portal to design artificial sequence for or between protein families. This thesis also provides useful and meaningful predictions for protein families with yet unknown structure and function using NrichD database as well as four other state-of-the-art sequence-based remote homology detection methods. A different aspect addressed in this thesis provides a fundamental understanding of the relationships between protein structure and functions. Evolutionary relationships between functional families are identified using the inherent structural information for these families and fold-function relationships are studied from a perspective of similarities in their binding sites. Such studies help in the area of functional annotation, polypharmacology and protein engineering.

A cell is a highly complex, ordered, and above all, a robust system. It copes with in-trennel and external uncertainties like heterogeneous stimuli, errors in processing and execution, and changes within and outside the cell. Maintenance of such a system critically depends on a large body of signalling networks and associated regulatory mechanisms. Of the recurrent manoeuvres in cell signalling, protein phosphorylation is the most prominent, and is used as a switch to transmit information and effect-ate various outcomes. It is estimated that 30% of the entire proteome of a typical eukaryotic cell is phosphorylated at one time or another, almost exclusively at the hydroxyl groups of one or more Seer(S)/Thru(T)/Tyr(Y) residues. This phosphorylation is accomplished through the transfer of g-phosphate of ATP in the presence of cations by a superfamily of enzymes called protein kinases, or STY kinases. In accordance with widespread phosphorylation events, STY kinases form a large and diverse superfamily, constituting 2% of the proteins encoded in an eukaryotic genome and about 500 proteins in the human proteome. Distantly related STY kinases share less than 20% sequence identity, phosphorylate specific substrates, bind to dis-tint interaction partners, localise in different cellular compartments and are regulated by different mechanisms. Despite flexibly accommodating these specific attributes, all STY kinases share a conserved 3-dimensional fold and retain the catalytic function. Moreover, all STY kinases can be manipulated by the signalling machinery to be in the “on” (functionally active) or “off” (inactive) state, thereby adding another layer of regulatory control. Such versatility of the STY kinase domain in harbouring specific substrate recognition motifs, binding interfaces, domain architectures and functional states makes it one of the most influential players in cell signalling and a desirable drug target. Despite decades of studies, a comprehensive understanding of the kinase domain, and the features that dictate its catalytic activity and specificity is lacking. This is reflected by the fact that whereas kinases specifically bind and phosphorylate their cognate substrates, most drugs targeted at them are non-specific and beget cross-reactivity. This gap in understanding potentially ensues from an awry outlook of STY kinases from the viewpoint of sequences and structures alone. It is now well established that the function and regulation of a protein molecule, along with its stability and evolution, is closely related to its dynamics. In this premise, this thesis explores the mechanistic and dynamics underpinnings of STY kinases, and interprets them in the light of their multitude of functional responsibilities and specificities In Chapter 1 of the thesis, we broadly discuss the complexity of cell signalling and the pivotal role of STY kinases in it. After a brief introduction to cell signalling in eukaryotes, several signal cascades mediated by different secondary messengers (camp, cGMP, DAG, IP3) are described. In these signal pathways, modularity is identified as a recurrent theme at all levels of hierarchy: within domain, within protein, within signalling pathway and across signalling pathways. One such modular regulation, protein phosphorylation, is discussed in detail and its catalytic enzyme STY kinase is introduced. An overview and historical perspective of the STY kinase superfamily is presented along with the review of literature pertaining to their sequence, structure and catalytic function characteristics. We note that in the active state, all STY kinases adopt a specific spatial conformation characterised by precise positioning of crucial structural motifs, while the inactive state is usually a case of some deviation from these structural constraints. Chapter 2 addresses a fundamental question in the protein dynamics and function paradigm. If mobility and dynamics of a protein is intimately coupled to its function, how does it manifest in STY kinases? Is there a discernible inter-relationship be-tween the mobility of an STY kinase and its functional competence? To answer these questions, we collated 55 crystal structures of 14 STY kinases from diverse groups and families, and subjected their kinase catalytic domains to Gaussian network model (GNM) based normal mode analysis (NMA). GNM models the kinase structure as a 3-dimensional mass-spring system in a coarse-grained fashion, with masses/nodes at Ca atom positions. Proximate Ca nodes, within a 7 A˚ distance cut-off, are con-nested by identical virtual springs, resulting in a simplified network of Ca-Ca bonded and non-bonded interactions modelled as harmonic potentials. Based purely on the topology of mechanical constraints imposed by the springs, GNM analytically deter-mines the isotropic vibrational normal modes available to the kinase structure. This method approximates the energy of the protein structure harmonically, and thus any micro-motion of the kinase can be theoretically described by a linear cSombination of the calculated normal modes. It is known from previous studies that the modes of low frequencies correspond to biologically feasible and meaningful motions like hinge movements, protein folding and catalysis. We note that the multiple crystal structures analysed in each of the 14 STY kinases are identical in sequence and gross structural fold, and vary only in local backbone conformations corresponding to the functional state of the kinase (active/inactive). Upon examining the fluctuations of kinases in the normal mode of the least frequency (or, global mode), we found systematically higher structural fluctuations in the inactive states when compared to the corresponding active states. This observation held true within individual kinases and across all the 14 kinases. Taken together, a more number of residues have higher fluctuations in the inactive states (n = 1095), than those with higher fluctuations in the active states (n = 525; Chi-square test, p value < 0.05). This skewed fluctuation distribution is in corroboration with higher B-factors and con-formational energies of the inactive state crystal structures. Moreover, high fluctuation is observed across the different inactive forms, except a small fraction of DFG-“in” in-active conformations. Interestingly, the regions of differential fluctuation localised to activation loop, catalytic loop, aC-helix and aG-helix, which are implied in kinase function and regulation. Further investigation of 476 crystal structures of kinase com-plexes with other proteins revealed a remarkable correspondence of these regions of differential fluctuation to contact interfaces. We further verified that this differential fluctuation is not a trivial consequence of bound small molecules or mutations, but an inherent attribute of the kinase catalytic domains. In Chapter 3, we verified the accuracy of differential fluctuation observed between the active and inactive STY kinases, as perceived from GNM based NMA, using the more rigorous method of molecular dynamics (MD) simulations. GNM is minimal-is tic in that the STY kinase catalytic domain is coarse-grained and reduced to a 3-dimensional mechanical network of Ca atom nodes. Thus, the role of side chains and their biophysical character, intra-protein interactions, mutations and bound factors are grossly overlooked. In this premise, we conducted all-atom MD simulations using AMBER ff14SB force-field of 6 structural variants of cAMP-dependent protein kinase (PKA) for 1 ms each. We chose 2 crystal structures of active and inactive PKA (PDB IDs 3FJQ and 1SYK respectively) whose kinase domains shared high structural similarity (gRMSD = 2.6 A)˚. They were modified in silico to obtain 6 starting structures for MD simulations: phosphorylated kinase domain in active and inactive states, kinase do-main along with its C-terminal tail in active and inactive states, active kinase domain bound to ATP/2Mg2+, and unphosphorylated inactive kinase domain. In the absence of external domains, the inactive kinase domain conformation elicits higher mobility in terms of Ca RMSD and Ca RMSF than the active kinase domain. Of the 255 residues in PKA, remarkable 198 residues have higher Ca RMSF in the inactive state, with predominant contributions from ATP binding loop, catalytic loop and aG-helix. In the presence of C-terminal tail, the differential mobility of the kinase domain is exaggerated, with 241 out of 255 residues showing higher Ca RMSF in the inactive state. Upon close investigation, we found that in the presence of C-terminal tail, al-though the mobility of residues is generally suppressed in both the functional states, a few functional regions like activation loop and hinge residues experience higher Ca RMSF in the inactive state. This sheds light on the role of C-terminal tail in the dynam-ics of the activation loop, potentially operating through the hinge residues. Absence of phosphorylation in the inactive kinase domain increases the mobility of residues in general, except of those in the aG-helix. When bound to ATP/2Mg2+, active ki-nase domain (active-holo) showed higher mobility than the active-apo and inactive structures, contrary to the previous results and studies. Intrigued, we examined the simulation closely and found a transition of the active-holo structure to another con-formation, named s2, at 450 ns. Upon analysis of the trajectory before the transition, the active-holo form was indeed found to be more stable and less mobile than the inac-tive state(s). Thus, all the inactive variants are found to be consistently more agile and mobile than their active counterparts, in agreement with the results obtained using NMA. Chapter 4 discusses the transition of the active-holo simulation to a new state, named s2, characterises its structural features and explores the possibility of its func-tional relevance. In the previous chapter, while attempting to verify the presence of differential mobility between various active and inactive forms of PKA through MD simulations, we chanced upon the transition of an active PKA state bound to ATP/2Mg2+ (active-holo) to s2 conformation. The s2 state has a Ca RMSD of up to 4.1 A˚ from the initial starting conformations, mainly contributed by the ATP binding loop, abs-helix, act-helix and age-helix, which are implicated in catalysis and substrate recognition. Once formed, s2 was stable and did not revert back to the active-hole starting structure or any other conformation. We calculated all-vs.-all Ca RMSDs of the conformations sampled during the simulation and identified 3 time periods: 0 - 200 ns of initial conformations similar to the starting structure, 201 - 500 ns of transition, and 501 - 1000 ns of s2 conformations. Principle component analysis (PCA) of the Ca spatial positions during the entire trajectory also categorically exposed two energy wells corresponding to the initial and s2 conformations in the first and second PCs (variance = 56%). Upon systematically comparing the conformers sampled in MD with every known kinase structure, no structure hit with Ca RMSD 2 A˚ was found for conformers sampled after 500 ns, deeming s2 as a novel and hitherto unknown conformation. Investigation of persistent intra-protein interactions unique to the s2 state revealed two stabilising interactions: a salt bridge between K73 and E106 in the b-sheet behind the ATP binding cleft and a network of hydrophobic interactions anchoring act-helix to the age-helix. Aside from these defining interactions, s2 is also characterised by a higher density of intra-protein hydrogen bond network, which stabilises it further. PCA of the MD trajectory indicates the transition of active-hole to s2 to be a process with at least 2 steps, the first being the salt bridge formation. Evolutionary conservation analysis shows that the crucial residues involved in the s2-specific interactions are not reliably conserved across PKAs of other organisms. However, convergence to s2 may still be feasible through other courses of stabilising interactions. From a functional perspective, the s2 conformation opens up the age-helix away from the kinase core and mildly rearranges the catalytic cleft, thereby unmasking a hotspot for sub-strata binding that was absent in the initial structure. In an attempt to replicate the s2 conformation, we performed 4 repeat simulations of the same active-hole starting structure for 1 ms each. Although two of these independent simulations achieved the K73-E106 salt bridge, none of them cloned the complete extent of transition and con-mergence to s2. Instead, we sampled another set of novel conformations, s3, in one of the repeat simulations indicating a disposition for the ATP bound PKA to sample different conformations. Comparative analysis suggests a potential role of C-terminal tail in stabilising the active-hole conformation in physiological conditions. Chapter 5 characterises the extent of conservation of structural fluctuations in ho-mologous STY kinases and interprets the observations in the light of their regulatory diversity. Upon establishing that structural fluctuations of STY kinases carry activity-specific information (Chapter 2) and affirming the same using MD simulations (Chap-ter 3), we hypothesised that the mobility of STY kinases is an important consider-action to understand the basis of their regulatory features as well. In that case, one would expect the structural fluctuations to be better conserved in closely related STY kinases than distantly related ones. To test our hypothesis, we collated 73 crystal structures containing an STY kinase domain in the active conformation and subjected them to GNM based NMA as described above. The global mode structural fluctuations of pairs of STY kinases of varying evolutionary divergence (same-protein, within-subfamily, within-family, within-group and across-groups) were analysed. We found that the closely related STY kinase pairs (of same-protein and within-subfamily cate-goriest) have more conserved and better correlated structural fluctuations than those that were distantly related (of within-group and across-group categories). This con-serration of flexibility did not trivially follow from sequence/structure conservation, since a substantial 65.4% of variation in fluctuations was not accounted by variations in sequences and/or structures. Across the 73 active STY kinases belonging to different groups, we identified a conserved flexibility signature defined by low magnitude fluctuations of residues in and around the catalytic loop. Interestingly, we also identified sub-structural residue-specific fluctuation profiles characteristic of kinases of different categories. Specifically, fluctuation patterns that are statistically unique to kinase groups (AGC, TK) and families (PKA, CDK) were recognised. These fluctuation signatures localise in sites known to participate in protein-protein interactions typical of the kinase group and family concerned. Thus, we report for the first time that residues characterised by fluctuations that are differentially conserved within a group/family are involved in interactions specific to the group/family. Upon the validation of structural fluctuation as an indicative tool to understand kinase-specific interactions, we elucidate an application of this understanding. In SC kinase, we identified local regions around the age-helix to be exhibiting conserved differential fluctuations in comparison to its close relatives EGFR and Abl. Following from the learning that specific fluctuations are correlated with specific binding, we propose this as an attractive target for drug design, with minimal cross-reactivity. Overall, this chapter demonstrates the conservation of fluctuation in STY kinases and underscores the importance of consideration of fluctuations, over and above sequence and structural features, in understanding the roles of sites characteristic of kinases. Chapter 6 documents the frequency of substitution of structural fluctuations in STY kinases over the course of divergent evolution. So far, we had established that structural fluctuations are evidently distinct in the varied functional states assumed by a single STY kinase (Chapter 2-3). In addition, fluctuations are differentially conserved within closely related kinases, but systematically vary across families (Chapter 5). In this chapter, we quantify the structural fluctuation variations in all residues of STY kinases put together. In a sense, this is the fluctuation space available for STY kinases across their functional states, binding modes, and regulatory mechanisms. To accomplish this, we systematically compiled all known eukaryotic kinase domain structures solved at resolutions better than 3 A˚. These structures were then divided into wild-type (harbouring no mutations and having typical amino acids at critical functional sites), pseudo-kinase (harbouring no mutations, but having unconventional amino acids at critical functional sites), disease mutant (harbouring mutations that have imp-plications in diseases) and mutant of unknown effect (harbouring mutations whose physiological manifestation is unknown) categories. Global mode structural fluctuations were determined for every kinase catalytic domain structure in each of the 4 enlisted categories. Similar to Benioff and Benioff’s BLOSUM that summarised the probability of all possible amino acid substitutions in homologous proteins, we documented a ma tricks of fluctuation substitution frequency in the conserved regions of wild-type kinases (named FLOSUM). We observe a positive correlation between the mean and variance of structural fluctuations at equivalent residue positions in wild-type kinase structures (Spearman rank order correlation, r = 0.69, p value < 1e 139). This implies that the residues with low flexibility, like catalytic loop, do not adopt diverse fluctuations in different functional states or across kinases. Substitution with any other fluctuation is heavily disfavoured at the lower range of flexibility than at the higher range. While we did not detect apparent differences in the FLOSUMs of wild-type, disease mutant and mutants of unknown effect structures, there is a remarkable distinction in the FLOSUM of pseudo-kinases. Fluctuation substitutions that are traditionally unfavourable in wild-type kinases are freely allowed in pseudo-kinases, thus exhibit-in poor conservative substitution. Over and above the lack of conventional amino acids, poor conservation of structural fluctuations and favourable substitution of de-viand fluctuations could render auxiliary functional character to the kinase domain in pseudo-kinases, despite their structural similarity to STY kinases. Taken together, this study summarises the structural fluctuation landscape of STY kinases in the form of a substitution matrix, which can serve as a model of flexibility substitution during protein evolution. Encouraged by structural fluctuations being differentially conserved in closely re-lasted kinases (Chapter 5) and conservatively substituted across kinases (Chapter 6), we extended this principle to the sequences of STY kinases in Chapter 7. This chapter reports the development of a method to predict the sites of functional specialisation in kinases, which differentiate one kinase from another, and applies it to all known STY kinase families. These are correlates of kinase-specific functional and regulatory attributes like specific protein-protein interactions, cognate substrate recognition and response to specific signals. Two cardinal properties of family-specific functional sites, viz., differential conservation and discriminatory ability, were used to identify them. We systematically compiled a data set of 5488 kinase catalytic domain sequences be-longing to 107 families. After aligning them into a single multiple sequence alignment, we comparatively analysed the amino acid distributions in topologically equivalent positions of different families. Based on 3 different analytical measures, physicochemical property, Shannon’s entropy and random probability, we scored the differential conservation of every alignment position in each family. By maximising the disc rim-inability between the kinase families, we integrated the results of the three measures and devised a single unified scoring scheme called ID score. This integrated scoring method could distinguish the 107 families from one another with an accuracy of 99.2%. Each site in every STY kinase family was given a score in the range 0 to 1, with 0 indicating no functional specialisation and 1 indicating maximum functional spa-canalisation, by the ID score. Several validations of the method were carried out to assess its competence. First, we selected those residue positions which have consistently high ID scores across most families. Using these hotspot alignment positions that render specificity to the kinase, we clustered the kinase sequences into groups and families. We found that the ID score predicted sites clustered the kinases better than the traditional clustering using the entire alignment. Despite reduction in information, the increase in accuracy of clustering is feasible because of efficient filtering of non-discriminatory sites by ID score. Second, a linear discriminant classifier was observed to predict the kinase family, based on the ID score predicted sites, better than traditional methods. Third, family-specific protein-protein interaction sites in CDK and substrate recognising distal sites in MAPK were scored significantly higher than other residues by ID score (Two-tailed unpaired t-test, p value < 0.05). Fourth, family-specific oncogenic driver mutation sites in 8 different kinase families were identified confidently by the ID score. Finally, we demonstrate one feasible application of the ID score method in the prediction of specific protein-protein interaction sites. In summary, we developed an integrated discriminatory method to identify regions of functional specialisation in all known kinases, validated the results for known cases and elucidate a potential application of the method. The learning from the entire thesis work is summarised in Chapter 8, which positions the work in the larger context of functioning of the kinase domain and the use of dynamics to interpret protein functions. The validity of the simple, yet use-full, NMA of proteins and complementary MD simulations to understand basic mechanistic and dynamic properties of proteins is highlighted. Similar to sequence and structure, dynamics is now recognised as a crucial feature holding information about protein function. The main learning of the thesis that the flexibility and mobility of STY kinases is conserved and conservatively substituted at different levels of hierarchy (different functional forms within a kinase, across kinase families and across the entire STY kinase superfamily) is discussed. The contributions of the work in fur-the ring the knowledge of specificity determinants in kinases, which dictate precise regulatory and control mechanisms, are presented. Supplementary information helpful in understanding of the results of individual chapters, but could not be printed in the thesis due to its length, are provided in an optical disk attached to the thesis. The material in the optical disk is referred to in appropriate places in the individual chapters

Carcinomatose péritonéale est un terme utilisé pour désigner la dissémination métastatique généralisée du cancer dans la cavité péritonéale. Il se caractérise par l’accumulation de liquide appelé « ascite » et est considéré comme étant au stade terminal du cancer, car il est difficile à traiter. L'ascite accumulée dans la PC comprend des cellules tumorales, cytokines et cellules immunitaires. Les cellules cancéreuses expriment des protéines spécifiques qui les aident à supprimer les cellules immunitaires et à survivre, appelées points de contrôle immunitaires. Des points de contrôle immunitaires sont présents pour réguler le système immunitaire et sont cruciaux contre la tolérance de soi. PD-1 / PD-L1 et CTLA-4 sont des voies de contrôle immunitaire bien établies adaptées au cancer pour échapper à l'immunité. Récemment, HLA-G a été reconnu comme un point de contrôle et il a été constaté que la survie globale était diminuée dans plusieurs types de cancers solides.Au cours de ma thèse, nous avons évalué l'expression de HLA-G dans la carcinomatose ovarienne. Nous avons constaté que les cellules cancéreuses dans l'ascite de presque tous les patients atteints de carcinomatose ovarienne exprimaient HLA-G. De plus, des taux croissants de sHLA-G1 et de HLA-G5 ont été trouvés dans les ascites. Cette présence de sHLA-G s'est révélée être corrélée positivement avec les Tregs et en corrélation négative avec les cellules T cytotoxiques (CD8) et les cellules NK. De plus, nous avons constaté que les ascites peuvent induire l’expression de HLA-G dans des «Hospicells» via des cytokines inflammatoires. Parmi les cytokines inflammatoires, le TGF-β et IL-1β ont une importance capitale dans l’induction de HLA-G. En outre, nous avons constaté que IL-1β implique la voie NF-κB. Dans une cohorte distincte de carcinomatose péritonéale, composée de patients atteints de PC d'origine différente, nous avons constaté que le groupe de cellules cancéreuses dans l'ascite avait une expression génique hétérogène de PD-L1, CTLA-4 et HLA- G. En outre, nous avons constaté que tous les patients présentaient des taux solubles de HLA-G et PD-L1 dans leur ascite. Cependant, seulement 5 patients présentaient des taux de CTLA-4 solubles dans leur ascite. De plus, nous avons trouvé une très forte corrélation positive entre le niveau de gène de PD-L1 et de CTLA-4, alors qu'aucune corrélation n'a été trouvée pour HLA-G avec PD-11 et CTLA-4 suggérant que HLA-G agit indépendamment des deux points de contrôle immunitaires. En outre, nous avons évalué l'expression de ces points de contrôle immunitaires par des nodules de cancer présents sur la membrane péritonéale. Nous avons trouvé une faible expression de HLA-G et PD-L1, mais la moitié des échantillons étaient fortement positifs pour sHLA-G. Nous avons également constaté que le sHLA-G pouvait être absorbé par l'ascite par la couche mésothéliale. Cette sHLA-G absorbée peut fournir un environnement immunosuppresseur pour la fixation des grappes de cellules cancéreuses à la membrane péritonéale. In vitro, nous avons constaté que l'ascite peut exercer une action immunosuppressive et retarder la lyse des cellules cancéreuses par les cellules immunitaires.De plus, nous avons constaté que la différenciation des cellules cancéreuses se traduit par une augmentation des propriétés immunosuppressives par une expression accrue de HLA-G ou PD-L1. En outre, l'expression de HLA-G et PD-L1 dépend de la phase du cycle cellulaire. Les cellules cancéreuses, si elles sont bloquées dans les cellules mitotiques, expriment des niveaux élevés de HLA-G et de PD-L1, tandis qu'une expression plus faible a été observée en phase G1. Par conséquent, nous suggérons d’éviter l’utilisation d’inhibiteurs de la mitose car ils pourraient augmenter la suppression immunitaire du cancer. De plus, le Ki-67 étant directement lié à l'index mitotique, nous suggérons de développer une échelle de Ki-67 pour évaluer le profil d'immunosuppresseur des patients cancéreux. / Peritoneal carcinomatosis (PC) is a term used for widespread metastatic dissemination of cancer to the peritoneal cavity. It is characterized by the accumulation of fluid called “ascites” and is considered a terminal stage of cancer, as it is hard to treat. The overall survival rate for untreated patients is six-months. However, owing to modern techniques like HIPEC, the survival rate can be increased up to five years. The ascites accumulated in PC, consists of tumor cells, cytokines and immune cells. Cancer cells express specific proteins to suppress immune cells activity and their attack, known as immune checkpoints. PD-1/PD-L1 and CTLA-4 are well established immune checkpoint pathways adapted by cancer in evading immunity. Recently, HLA-G has been recognized as an immune checkpoint and has been found to decrease overall survival in several types of solid cancers. We evaluated the expression of HLA-G in ascites from ovarian carcinomatosis. We found that HLA-G is expressed by cancer cells in ascites from all of the patients(n=16) with ovarian carcinomatosis. Moreover, increased levels of sHLA-G1 and HLA-G5 were found in ascites. This presence of sHLA-G isoforms was found to be positively correlated with Tregs and negatively correlated with cytotoxic T-cells (CD8) and NK-cells suggesting the role of HLA-G in immune suppression. Further, we found that ascites can induce the expression of HLA-G in “Hospicells” via inflammatory cytokines. Among the inflammatory cytokines, TGF-β and IL-1β are of crucial importance in HLA-G induction with IL-1β being more potent compared to TGF-β. Further, we found that IL-1β induces HLA-G expression through NF-κB pathway.In a separate cohort of peritoneal carcinomatosis(n=27), consisting of patients with cancer from a different origin, we found that cancer cell cluster in ascites (n=23) had a heterogeneous gene expression of PD-L1, CTLA-4 and HLA-G. Further, we found that all of the patients presented soluble levels of HLA-G in their ascites. However, one patient was negative for soluble PD-L1 and only 5 patients presented soluble CTLA-4 levels in their ascites. This heterogeneity explains why some of the patients respond to immune therapy while others don’t. This also suggests the need for prescreening patients before immune therapy. Moreover, we found a very strong positive correlation (rs=0.793) between gene level of PD-L1 and CTLA-4, while no correlation was found for HLA-G with PD-L1 and CTLA-4 suggesting that HLA-G acts independently of both the immune checkpoints. Also, we evaluated the expression of these immune checkpoints by cells in peritoneal tissue (n=20). We found low expression of HLA-G and PD-L1, but the majority of the samples were found strongly positive for sHLA-G presence. This sHLA-G can provide an immune-suppressive environment for the attachment of the cancer cell clusters to the peritoneal membrane to form cancer nodule. Additionally, we developed an in-vitro cytotoxicity assay to show that the ascites can provide the immune-suppressive action by interfering with immune cell interaction and delaying the lysis of cancer cells by the immune cells.In parallel, we found that the differentiation of the cancer cells results in increased expression of immune checkpoints like HLA-G or PD-L1. This may render these cells more immune resistant and can protect against immune attack. However, in-vivo mice model is needed to study the oncogenic potential of these differentiated cells. Further, we report that the expression of HLA-G and PD-L1 is dependent on the cell cycle phase. The cancer cells, if blocked in mitotic phase express high levels of HLA-G and PD-L1, while lowest expression was observed in G1-phase. Therefore, we suggest avoiding the use of mitotic inhibitors as it may increase the immune suppression of cancer. Moreover, as Ki-67 is directly related to the mitotic index, we suggest developing a Ki-67 scale to evaluate the immune-suppressive profile of cancer patients.

La température ambiante joue un rôle direct dans le fonctionnement et le développement de la plante. L'augmentation des températures ambiantes mondiales pose un défi important aux espèces de plantes sauvages et cultivées. La plupart des espèces de plantes ajustent leur cycle de reproduction et leur développement pour optimiser leur survie et leur forme par temps ambiant élevé (Barnabás et al., 2008; Fitter et Fitter, 2002; Willis et al., 2008). Ces adaptations conduisent généralement à des hypocotyles allongés, à une réduction du nombre de feuilles au moment de la floraison, à une transition accélérée des phases de croissance végétative à reproductive, à une réduction du nombre de graines, à des gousses plus petites et à une diminution de la surface foliaire. Face au changement climatique rapide, en particulier à l'augmentation de la température permissive à la croissance ambiante, il est urgent de régler la thermoréponse des usines pour adapter les installations aux changements climatiques et garantir la production alimentaire future.L'expression de PIF4 est contrôlée par le complexe du soir (EC), un complexe à 3 protéines comprenant ELF3, ELF4 et LUX, en fonction de la température. La CE est capable de réprimer l'expression de PIF4 en se liant à des motifs spécifiques sur le promoteur de PIF4 à une température de croissance ambiante inférieure. Cependant, cette répression de l'expression de PIF4 est éliminée à une température ambiante de croissance plus élevée, ce qui entraîne un vieillissement prématuré des plantes.RésultatsLes trois protéines de la CE ont été produites en utilisant une stratégie d’expression différente, ELF4 et LUX dans E. coli, et ELF3 dans des cellules d’insecte. Les méthodes de purification des protéines et les tampons ont été optimisés pour obtenir des ELF3 et LUX stables. Une chromatographie d'exclusion de taille a été réalisée pour vérifier les états oligomères des protéines. LUX étant la seule protéine de la CE à posséder un domaine de liaison à l'ADN connu, elle a été utilisée pour effectuer des tests de déplacement de la mobilité afin de comprendre l'affinité de liaison de LUX pour ses motifs ADN cibles obtenus à partir de tests de puces de liaison de protéines. D'après les tests de décalage de mobilité, il a été observé que le domaine de liaison à l'ADN (DBD) seul pouvait se lier aux motifs d'ADN de son substrat à des concentrations molaires nano alors qu'une quantité de protéine excédentaire de 10 fois était nécessaire pour que la longueur totale de LUX obtienne la même quantité de liaison. Des pistes de cristallisation à haut débit ont été réalisées pour LUX et LUX-DBD pleine longueur avec deux motifs de liaison différents. Aucun cristal n'a été obtenu pour le LUX complet, alors que des cristaux ont été obtenus pour le LUX DBD avec les deux motifs d'ADN. La structure de LUX DBD en complexe avec ses motifs cibles a été résolue par diffraction aux rayons X. À partir de la structure Crystal, il a été constaté que LUX DBD avait adopté une structure à 3 hélices, la seconde hélice étant responsable de la lecture de la base. Fait intéressant, il a été observé qu'un résidu d'arginine présent au niveau de l'hélice n-terminale servait de pince pour interagir avec le motif ADN cible.En utilisant les tests de mutagenèse dirigée et de décalage de mobilité, il a été confirmé que cette arginine présente à la position 146 de la protéine est essentielle pour déterminer l'affinité. Il a été constaté que l'affinité de liaison était réduite d'un facteur 5 lorsque cet acide aminé était modifié. En outre pour comprendre son effet in planta. Les lignes de lux mutantes ont été complétées par une longueur totale de type sauvage et une version substituée par Arg14Ala de la longueur totale de LUX. Grâce à ces expériences, nous avons pu montrer que la complémentation complète du mutant R146A n’était pas observée, alors que la version de type sauvage était capable de compléter complètement le phénotype du mutant / Ambient temperature plays a direct role in plant functioning and development. Increase in global ambient temperatures poses a significant challenge to wild and cultivated plant species. Most plant species adjust reproductive timing and development to optimize survival and fitness in higher ambient temperatures (Barnabás et al., 2008; Fitter and Fitter, 2002; Willis et al., 2008). These adaptations generally lead to elongated hypocotyls, fewer leaves at time of flowering, accelerated transition from vegetative to reproductive growth phases, fewer seeds, smaller seed pods and decreased leaf area. In the face of rapid climate change, specifically increased ambient growth permissive temperatures, tuning plant thermoresponse is urgently needed to engineer plants for adaptation to climate change and for securing future food production.PIF4 expression is controlled by evening complex (EC), a 3 protein complex comprising of ELF3, ELF4 and LUX, in a temperature dependent manner. The EC is able to repress PIF4 expression by binding to specific motifs at the promoter of PIF4 at lower ambient growth temperature. However this repression of PIF4 expression is removed at higher ambient growth temperature leading to premature ageing of plantsResultsAll three proteins of EC were produced using different expression strategy, ELF4 and LUX in E. coli while ELF3 in insect cells. Protein purification methods and buffers were optimized to obtain stable ELF3 ELF4 and LUX. Size exclusion chromatography was done to verify oligomeric states of the proteins. Since LUX is the only protein in the EC which has known DNA binding domain, it was used for doing mobility shift assays to understand the binding affinity of LUX for its target DNA Motifs which were obtained from protein binding microarrays assays. From the mobility shift assays, it was observed that the DNA binding domain (DBD) alone could bind to its substrate DNA motifs at Nano molar concentrations while 10 fold excesses amount of protein was required for the full length LUX to obtain same amount of binding. High throughput crystallization trails were carried out for full length LUX and LUX- DBD with two different binding motifs. No crystals were obtained for full length LUX while Crystals were obtained for LUX DBD with both the DNA Motifs. Structure for LUX DBD in complex with its target motifs were solved through X-Ray diffraction. Using site directed mutagenesis and Mobility shift assays it was confirmed that this Arginine present at the 146 position of the protein is critical for determining affinity. It was found that the binding affinity was reduced by a factor of 5 when this amino acid was changed. Further to understand its effect in planta.. With this experiments we were able to show that with the R146A mutant full complementation wasn’t observed while the wildtype version was able to completely complement the mutant phenotype.To understand temperature based dynamics of the complex, ELF3, which is the most intrinsically disordered protein of the three proteins that constitute the complex, was studied for structural variation through CD spectroscopy and DLS experiments. From these experiments it was found that ELF3 attains a β-sheet like confirmation at higher temperature while a more globular confirmation at lower temperatures. It was found that this activity of ELF is reversible allowing for flexibility of the whole complex. We found that there were prion like domains in ELF3 protein which were primarily responsible for transition to β-sheet structure at higher temperature.In Order to engineer plants that could survive at higher ambient temperature, we decided to mutate promoter elements of PIF4 through CRISPR/Cas9 to obtain plants that can survive higher temperature.