Characterization of hydrophobin proteins at interfaces and in solutions using x rays

Hydrophobins are a group of particularly surface active proteins. The surface activity is demonstrated in the ready adsorption of hydrophobins to hydrophobic/hydrophilic interfaces such as the air/water interface. Adsorbed hydrophobins self-assemble into ordered films, lower the surface tension of water, and stabilize air bubbles and foams. Hydrophobin proteins originate from filamentous fungi. In the fungi the adsorbed hydrophobin films enable the growth of fungal aerial structures, form protective coatings and mediate the attachment of fungi to solid surfaces. This thesis focuses on hydrophobins HFBI, HFBII, and HFBIII from a rot fungus Trichoderma reesei. The self-assembled hydrophobin films were studied both at the air/water interface and on a solid substrate. In particular, using grazing-incidence x-ray diffraction and reflectivity, it was possible to characterize the hydrophobin films directly at the air/water interface. The in situ experiments yielded information on the arrangement of the protein molecules in the films. All the T. reesei hydrophobins were shown to self-assemble into highly crystalline, hexagonally ordered rafts. The thicknesses of these two-dimensional protein crystals were below 30 Å. Similar films were also obtained on silicon substrates. The adsorption of the proteins is likely to be driven by the hydrophobic effect, but the self-assembly into ordered films involves also specific protein-protein interactions. The protein-protein interactions lead to differences in the arrangement of the molecules in the HFBI, HFBII, and HFBIII protein films, as seen in the grazing-incidence x-ray diffraction data. The protein-protein interactions were further probed in solution using small-angle x-ray scattering. Both HFBI and HFBII were shown to form mainly tetramers in aqueous solution. By modifying the solution conditions and thereby the interactions, it was shown that the association was due to the hydrophobic effect. The stable tetrameric assemblies could tolerate heating and changes in pH. The stability of the structure facilitates the persistence of these secreted proteins in the soil.

Structural studies on members of the picornavirus superfamily

The pathogenic members of the picornavirus superfamily have adverse effects on humans, their crops and their livestock. As structure is related to function, detailed structural studies on these viruses are important not only for fundamental understanding of the viral life cycle, but also for the rational design of vaccines and inhibitors for disease control. These viruses have positive sense, single-stranded RNA genomes enclosed in a protein capsid. X-ray crystallography and cryo-electron microscopy studies have revealed that the isometric members of this group have icosahedrally-symmetric capsids made up of 60 copies of each of the structural proteins. The members that infect animal cells often employ one or more cellular receptors to facilitate cell entry which in some cases is known to initiate the uncoating sequence of the genome. The nature of the interactions between individual viruses and alternative cellular receptors has rarely been probed. The capsid assembly of the members of the picornavirus superfamily is considered to be cooperative and the interactions of RNA and capsid proteins are thought to play an important role in orchestrating virus assembly. The major aims of this thesis were to solve the structures of blackcurrant reversion virus (BRV), human parechovirus 1 (HPEV1) and coxsackievirus A7 (CAV7), as well as the structure of HPEV1 complexed with two of its cellular receptors using cryo-electron microscopy, three-dimensional image reconstruction and homology modeling. Each of the selected viruses represents a taxonomic group where little or no structural data was previously available. The results enabled the detailed comparison of the new structures to those of known picornaviruses, the identification of surface-exposed epitopes potentially important for host interaction, the mapping of RNA-capsid protein interactions and the elucidation of the basis for the specificity of two different receptor molecules for the same capsid. This work will form the basis for further studies on the influence of RNA on parechovirus assembly as a potential target for drug design.

Comparison of promoter-specific events during transcription initiation in mycobacteria

DNA protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNA polymerase (RNAP) binds to promoters in a sequence-specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. We present here the first detailed kinetic analysis of promoter RNAP interactions during transcription initiation in the sigma(A)-dependent promoters P-rrnAPCL1, P-rrnB and P-gyr of Mycobacterium smegmatis. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to distinct kinetic patterns, each one of the housekeeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation.

Sequential Assembly of an Active RNA Polymerase Molecule at the Air-Water Interface

At the heart of understanding cellular processes lies our ability to explore the specific nature of communication between sequential information carrying biopolymers. However, the data extracted from conventional solution phase studies may not reflect the dynamics of communication between recognized partners as they occur in the crowded cellular milieu. We use the principle of immobilization of histidine-tagged biopolymers at a Ni(II)-encoded Langmuir monolayer to study sequence-specific protein-protein interactions in an artificially crowded environment The advantage of this technique lies in increasing the surface density of one of the interacting partners that allows us to study macromolecular interactions in a controlled crowded environment, but without compromising the speed of the reactions. We have taken advantage of this technique to follow the sequential assembly process of the multiprotein complex Escherichia coil RNA polymerase at the interface and also deciphered the role of one of the proteins, omega (omega), in the assembly pathway. Our reconstitution studies indicate that in the absence of molecular chaperones or other cofactors, omega (omega) plays a decisive role in refolding the largest protein beta prime (beta') and its recruitment into the multimeric assembly to reconstitute an active RNA polymerase. It was also observed that the monolayer had the ability to distinguish between sequence-specific and -nonspecific interactions despite the immobilization of one of the biomacromolecules. The technique provides a universal two-dimensional template for studying protein-ligand interactions while mimicking molecular crowding.

Modeling of angiogenin-3 '-NMP complex

Angiogenin belongs to the Ribonuclease superfamily and has a weak enzymatic activity that is crucial for its biological function of stimulating blood vessel growth. Structural studies on ligand bound Angiogenin will go a long way in understanding the mechanism of the protein as well as help in designing drugs against it. In this study we present the first available structure of nucleotide ligand bound Angiogenin obtained by computer modeling. The importance of this study in itself notwithstanding, is a precursor to modeling a full dinucleotide substrate onto Angiogenin. Bovine Angiogenin, the structure of which has been solved at a high resolution, was earlier subjected to Molecular Dynamics simulations for a nanosecond. The MD structures offer better starting points for docking as they offer lesser obstruction than the crystal structure to ligand binding. The MD structure with the least serious short contacts was modeled to obtain a steric free Angiogenin - 3' mononucleotide complex structure. The structures were energetically minimized and subjected to a brief spell of Molecular Dynamics. The results of the simulation show that all the li,ligand-Angiogenin interactions and hydrogen bonds are retained, redeeming the structure and docking procedure. Further, following ligand - protein interactions in the case of the ligands 3'-CMP and 3'-UMP we were able to speculate on how Angiogenin, a predominantly prymidine specific ribonuclease prefers Cytosine to Uracil in the first base position.

Estimating the Threshold Surface Density of Gp120-CCR5 Complexes Necessary for HIV-1 Envelope-Mediated Cell-Cell Fusion

Reduced expression of CCR5 on target CD4(+) cells lowers their susceptibility to infection by R5-tropic HIV-1, potentially preventing transmission of infection and delaying disease progression. Binding of the HIV-1 envelope (Env) protein gp120 with CCR5 is essential for the entry of R5 viruses into target cells. The threshold surface density of gp120-CCR5 complexes that enables HIV-1 entry remains poorly estimated. We constructed a mathematical model that mimics Env-mediated cell-cell fusion assays, where target CD4(+)CCR5(+) cells are exposed to effector cells expressing Env in the presence of a coreceptor antagonist and the fraction of target cells fused with effector cells is measured. Our model employs a reaction network-based approach to describe protein interactions that precede viral entry coupled with the ternary complex model to quantify the allosteric interactions of the coreceptor antagonist and predicts the fraction of target cells fused. By fitting model predictions to published data of cell-cell fusion in the presence of the CCR5 antagonist vicriviroc, we estimated the threshold surface density of gp120-CCR5 complexes for cell-cell fusion as similar to 20 mu m(-2). Model predictions with this threshold captured data from independent cell-cell fusion assays in the presence of vicriviroc and rapamycin, a drug that modulates CCR5 expression, as well as assays in the presence of maraviroc, another CCR5 antagonist, using sixteen different Env clones derived from transmitted or early founder viruses. Our estimate of the threshold surface density of gp120-CCR5 complexes necessary for HIV-1 entry thus appears robust and may have implications for optimizing treatment with coreceptor antagonists, understanding the non-pathogenic infection of non-human primates, and designing vaccines that suppress the availability of target CD4(+)CCR5(+) cells.

Amino acid interaction preferences in helical membrane proteins

Membrane proteins are involved in a number of important biological functions. Yet, they are poorly understood from the structure and folding point of view. The external environment being drastically different from that of globular proteins, the intra-protein interactions in membrane proteins are also expected to be different. Hence, statistical potentials representing the features of inter-residue interactions based exclusively on the structures of membrane proteins are much needed. Currently, a reasonable number of structures are available, making it possible to undertake such an analysis on membrane proteins. In this study we have examined the inter-residue interaction propensities of amino acids in the membrane spanning regions of the alpha-helical membrane (HM) proteins. Recently we have shown that valuable information can be obtained on globular proteins by the evaluation of the pair-wise interactions of amino acids by classifying them into different structural environments, based on factors such as the secondary structure or the number of contacts that a residue can make. Here we have explored the possible ways of classifying the intra-protein environment of HM proteins and have developed scoring functions based on different classification schemes. On evaluation of different schemes, we find that the scheme which classifies amino acids to different intra-contact environment is the most promising one. Based on this classification scheme, we also redefine the hydrophobicity scale of amino acids in HM proteins.

A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival

After initiation of transcription, a number of proteins participate during elongation and termination modifying the properties of the RNA polymerase (RNAP). Gre factors are one such group conserved across bacteria. They regulate transcription by projecting their N-terminal coiled-coil domain into the active center of RNAP through the secondary channel and stimulating hydrolysis of the newly synthesized RNA in backtracked elongation complexes. Rv1080c is a putative gre factor (MtbGre) in the genome of Mycobacterium tuberculosis. The protein enhanced the efficiency of promoter clearance by lowering abortive transcription and also rescued arrested and paused elongation complexes on the GC rich mycobacterial template. Although MtbGre is similar in domain organization and shares key residues for catalysis and RNAP interaction with the Gre factors of Escherichia coli, it could not complement an E. coli gre deficient strain. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein-protein interactions for transcript cleavage. Decrease in the level of MtbGre reduced the bacterial survival by several fold indicating its essential role in mycobacteria. Another Gre homolog, Rv3788 was not functional in transcript cleavage activity indicating that a single Gre is sufficient for efficient transcription of the M. tuberculosis genome.

Structure and nucleotide specificity of Staphylococcus aureus dihydrodipicolinate reductase (DapB)

Lysine biosynthesis proceeds by the nucleotide-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) by dihydrodipicolinate reductase (DHDPR). The S. aureus DHDPR structure reveals different conformational states of this enzyme even in the absence of a substrate or nucleotide-cofactor. Despite lacking a conserved basic residue essential for NADPH interaction, S. aureus DHDPR differs from other homologues as NADPH is a more preferred co-factor than NADH. The structure provides a rationale-Lys35 compensates for the co-factor site mutation. These observations are significant for bi-ligand inhibitor design that relies on ligand-induced conformational changes as well as co-factor specificity for this important drug target. Structured summary of protein interactions: DHDPR binds to DHDPR by molecular sieving (View interaction). DHDPR binds to DHDPR by dynamic light scattering (View interaction). DHDPR binds to DHDPR by X-ray crystallography (View interaction). (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The Structure of the Thioredoxin-Triosephosphate Isomerase Complex Provides Insights into the Reversible Glutathione-Mediated Regulation of Triosephosphate Isomerase

Protein-protein interactions are crucial for many biological functions. The redox interactome encompasses numerous weak transient interactions in which thioredoxin plays a central role. Proteomic studies have shown that thioredoxin binds to numerous proteins belonging to various cellular processes, including energy metabolism. Thioredoxin has cross talk with other redox mechanisms involving glutathionylation and has functional overlap with glutaredoxin in deglutathionylation reactions. In this study, we have explored the structural and biochemical interactions of thioredoxin with the glycolytic enzyme, triosephosphate isomerase. Nuclear magnetic resonance chemical shift mapping methods and molecular dynamics-based docking have been applied in deriving a structural model of the thioredoxin-triosephosphate isomerase complex. The spatial proximity of active site cysteine residues of thioredoxin to reactive thiol groups on triosephosphate isomerase provides a direct link to the observed deglutathionylation of cysteine 217 in triosephosphate isomerase, thereby reversing the inhibitory effect of S-glutathionylation of triosephosphate isomerase.

The Structure of the Thioredoxin−Triosephosphate Isomerase Complex Provides Insights into the Reversible Glutathione-Mediate Regulation of Triosephosphate Isomerase

Protein−protein interactions are crucial for many biological functions. The redox interactome encompasses numerous weak transient interactions in which thioredoxin plays a central role. Proteomic studies have shown that thioredoxin binds to numerous proteins belonging to various cellular processes, including energy metabolism. Thioredoxin has cross talk with other redox mechanisms involving glutathionylation and has functional overlap with glutaredoxin in deglutathionylation reactions. In this study, we have explored the structural and biochemical interactions of thioredoxin with the glycolytic enzyme, triosephosphate isomerase. Nuclear magnetic resonance chemical shift mapping methods and molecular dynamics-based docking have been applied in deriving a structural model of the thioredoxin−triosephosphate isomerase complex. The spatial proximity of active site cysteine residues of thioredoxin to reactive thiol groups on triosephosphate isomerase provides a direct link to the observed deglutathionylation of cysteine 217 in triosephosphate isomerase, thereby reversing the inhibitory effect of S-glutathionylation of triosephosphate isomerase.

Saccharomyces cerevisiae NineTeen Complex (NTC)-associated Factor Bud31/Ycr063w Assembles on Precatalytic Spliceosomes and Improves First and Second Step Pre-mRNA Splicing Efficiency

Pre-mRNA splicing occurs in spliceosomes whose assembly and activation are critical for splice site selection and catalysis. The highly conserved NineTeen complex protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NineTeen complex-associated proteins is the nonessential protein Bud31/Ycr063w, which is also a component of the Cef1p subcomplex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors, but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed that its earliest association is with precatalytic B complex and that the interaction continues in catalytically active complexes with stably bound U2, U5, and U6 small nuclear ribonucleoproteins. In complementary experiments, wherein precatalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Cross-linking of proteins to pre-mRNAs with a site-specific 4-thio uridine residue at the -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a similar to 25-kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.

Cis-trans peptide variations in structurally similar proteins

The presence of energetically less favourable cis peptides in protein structures has been observed to be strongly associated with its structural integrity and function. Inter-conversion between the cis and trans conformations also has an important role in the folding process. In this study, we analyse the extent of conservation of cis peptides among similar folds. We look at both the amino acid preferences and local structural changes associated with such variations. Nearly 34% of the Xaa-Proline cis bonds are not conserved in structural relatives; Proline also has a high tendency to get replaced by another amino acid in the trans conformer. At both positions bounding the peptide bond, Glycine has a higher tendency to lose the cis conformation. The cis conformation of more than 30% of beta turns of type VIb and IV are not found to be conserved in similar structures. A different view using Protein Block-based description of backbone conformation, suggests that many of the local conformational changes are highly different from the general local structural variations observed among structurally similar proteins. Changes between cis and trans conformations are found to be associated with the evolution of new functions facilitated by local structural changes. This is most frequent in enzymes where new catalytic activity emerges with local changes in the active site. Cis-trans changes are also seen to facilitate inter-domain and inter-protein interactions. As in the case of folding, cis-trans conversions have been used as an important driving factor in evolution.

Annexin A2 and PSF proteins interact with p53 IRES and regulate translation of p53 mRNA

p53 mRNA has been shown to be translated into two isoforms, full-length p53 (FL-p53) and a truncated isoform Delta N-p53, which modulates the functions of FL-p53 and also has independent functions. Previously, we have shown that translation of p53 and Delta N-p53 can be initiated at Internal Ribosome Entry Sites (IRES). These two IRESs were shown to regulate the translation of p53 and Delta N-p53 in a distinct cell-cycle phase-dependent manner. Earlier observations from our laboratory also suggest that the structural integrity of the p53 RNA is critical for IRES function and is compromised by mutations that affect the structure as well as RNA protein interactions. In the current study, using RNA affinity approach we have identified Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) as novel ITAFs for p53 IRESs. We have showed that the purified Annexin A2 and PSF proteins specifically bind to p53 IRES elements. Interestingly, in the presence of calcium ions Annexin A2 showed increased binding with p53 IRES. Immunopulldown experiments suggest that these two proteins associate with p53 mRNA ex vivo as well. Partial knockdown of Annexin A2 and PSF showed decrease in p53 IRES activity and reduced levels of both the p53 isoforms. More importantly the interplay between Annexin A2, PSF and PTB proteins for binding to p53mRNA appears to play a crucial role in IRES function. Taken together, our observations suggest pivotal role of two new trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.

Euglena sp as a suitable source of lipids for potential use as biofuel and sustainable wastewater treatment

Prolific algal growth in sewage ponds with high organic loads in the tropical regions can provide cost-effective and efficient wastewater treatment and biofuel production. This work examines the ability of Euglena sp. growing in wastewater ponds for biofuel production and treatment of wastewater. The algae were isolated from the sewage treatment plants and were tested for their nutrient removal capability. Compared to other algae, Euglena sp. showed faster growth rates with high biomass density at elevated concentrations of ammonium nitrogen (NH4-N) and organic carbon (C). Profuse growth of these species was observed in untreated wastewaters with a mean specific growth rate (mu) of 0.28 day(-1) and biomass productivities of 132 mg L-1 day(-1). The algae cultured within a short period of 8 days resulted in the 98 % removal of NH4-N, 93 % of total nitrogen 85 % of ortho-phosphate, 66 % of total phosphate and 92 % total organic carbon. Euglenoids achieved a maximum lipid content of 24.6 % (w/w) with a biomass density of 1.24 g L-1 (dry wt.). Fourier transform infrared spectra showed clear transitions in biochemical compositions with increased lipid/protein ratio at the end of the culture. Gas chromatography and mass spectrometry indicated the presence of high contents of palmitic, linolenic and linoleic acids (46, 23 and 22 %, respectively), adding to the biodiesel quality. Good lipid content (comprised quality fatty acids), efficient nutrient uptake and profuse biomass productivity make the Euglena sp. as a viable source for biofuel production in wastewaters.