The unique native structure is a basic requirement for normal functioning of most proteins. Many diseases stem from mutations in proteins that destabilize the protein structure thereby resulting in impairment or loss of function (Sunyaev et al. 2000). Therefore, it is important from both fundamental and applied points of view, to elucidate the sequence determinants of protein structure and function. With the advent of recombinant DNA techniques for modifying protein sequences, studies on the effect of amino acid replacements on protein structure and function have acquired momentum. It is well established from previous mutagenesis studies that buried residues in a protein are important determinants of protein structure or stability while surface residues are involved in protein function (Rennell et al. 1991; Terwilliger et al. 1994; Axe et al. 1998). Inspite of this, there is no universally accepted definition and probe to distinguish and identify buried residues from exposed residues. A part of this thesis aims to examine the feasibility of using scanning mutagenesis to distinguish between buried and exposed positions in the absence of three-dimensional structure and also to arrive at an experimental definition of the appropriate accessibility cut-off to distinguish between buried and exposed residues. Proline, being an unusual amino acid is usually exploited to determine sites in a protein important for protein stability (Sauer et al. 1992). This thesis also explores the use of proline scanning mutagenesis to make inferences about protein structure and stability. Temperature sensitive mutant proteins, which result from single amino acid substitutions, are particularly useful in elucidating the determinants of protein folding and stability (Grutter et al. 1987; Sturtevant et al. 1989). Temperature sensitive (ts) mutants are an important class of conditional mutants which are widely used to study gene function in vivo and in cell culture (Novick and Schekman 1979; Novick and Botstein 1985). They display a marked drop in the level or activity of the gene product when the gene is expressed above a certain temperature (restrictive temperature). Below this temperature (permissive temperature), the level or activity of the mutant is very similar to that of the wild type. Inspite of their widespread use, little is known about the molecular mechanisms responsible for generating a Ts phenotype. A part of this thesis discusses a set of sequence/structure-based strategies for the successful design and isolation of ts mutants of a globular protein, inferred from saturation mutagenesis of CcdB. The experimental system, CcdB (Controller of Cell Division or Death B protein), is a 101 residue, homodimeric protein encoded by F plasmid. The protein is an inhibitor of DNA gyrase and is a potent cytotoxin in E.coli (Bernard et al. 1993). Crystallographic structures of CcdB in the free and gyrase bound forms (Loris et al. 1999; Dao-Thi et al. 2005) are also available. Expression of the CcdB functional protein results in cell death, thus providing a rapid and easy assay for the protein (Chakshusmathi et al. 2004). This dissertation focuses on understanding the determinants of globular protein stability and temperature sensitivity using saturation mutagenesis of E.coli CcdB. Towards this objective, we attempted to replace each of the 101 residues of CcdB with 19 other amino acids using high throughput mutagenesis tools. A total of 1430 (~75%) of all possible single site mutants of the CcdB saturation mutagenesis library could be isolated. These mutants were characterized in terms of their activity at different expression levels. The correlation between the observed mutant phenotypes with residue burial, nature of substitution and expression level was examined. The introductory chapter (Chapter 1) describes the use of mutagenesis as a tool to understand the relationship between protein sequence, structure and function. It represents an overview of previous large scale mutagenesis studies from the literature. It also addresses the motivation behind this work and problems which we have attempted to address in these studies. Chapter 2 discusses mutagenesis based definitions and probes for residue burial in proteins as derived from alanine and charged scanning mutagenesis of CcdB. Every residue of the 101 amino acid E. coli toxin CcdB was substituted with Ala, Asp, Glu, Lys and Arg using site directed mutagenesis. The activity of each mutant in vivo was characterized as a function of CcdB transcriptional level. The mutation data suggest that an accessibility value of 5% is an appropriate cutoff for definition of buried residues. At all buried positions, introduction of Asp results in an inactive phenotype at all CcdB transcriptional levels. The average amount of destabilization upon substitution at buried positions decreases in the order Asp>Glu>Lys>Arg>Ala. Asp substitutions at buried sites in two other proteins, MBP and Thioredoxin were also shown to be severely destabilizing. Ala and Asp scanning mutagenesis, in combination with dose dependent expression phenotypes, was shown to yield important information on protein structure and activity. These results also suggest that such scanning mutagenesis data can be used to rank order sequence alignments and their corresponding homology models, as well as to distinguish between correct and incorrect structural alignments. When incorporated into a polypeptide chain, Proline (Pro) differs from all other naturally occurring amino acids in two important respects. The  dihedral angle of Pro is constrained to values close to –65o and Pro lacks an amide hydrogen. Chapter 3 describes a procedure to accurately predict the effects of proline introduction on protein stability. 77 of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to proline and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or non-perturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties, including main chain dihederal angle and main chain amide hydrogen bonds, were determined from 3D models of the mutant proteins built using MODELLER. The performance of the decision tree was assessed on 74 nsSNPs and 37 other proline substitutions from the literature. The overall accuracy of this algorithm was found to be 89% in case of CcdB, 71% in case of nsSNPs and 83% in case of other proline substitution data. Contrary to previous assertions, Proline scanning mutagenesis cannot be reliably used to make secondary structural assignments in proteins. The studies will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design. Mutants of CcdB were also characterized in terms of their activity at two different temperatures (30oC and 37oC) to screen for temperature sensitive (ts) mutants. The isolation and structural analysis of Ts mutants of CcdB is dealt with in Chapter 4. Of the total 1430 single site mutants, 12% showed a ts phenotype and were mapped onto the crystal structure of the protein. Almost all the ts mutants could be interpreted in terms of the wild type, native structure. ts mutants were found at all buried sites and all active sites (except one). ts mutants were also obtained at sites in close proximity to active site residues where polar side-chains were involved in H-bonding interaction with active site residues. Several proline substitutions also displayed a ts phenotype. The effect of expression level on ts phenotype was also studied. 78% of the mutants that showed an inactive phenotype at the lowest expression level and an active phenotype at highest expression level, resulted in a ts phenotype at an intermediate expression level. The molecular determinant responsible for the ts phenotype of buried site ts mutant is suggested to be the thermodynamic destabilization of the protein which results in a reduced steady state in vivo level of soluble, functional protein relative to wild type. The active site ts mutants probably lower the specific activity of the protein and hence the total activity relative to wild type. However these effects might be less severe at lower temperature. Specific structure/function based mutagenesis strategies are suggested to design ts mutant of a protein. These studies will simplify the design of ts mutants for any globular protein and will have applications in diverse biological systems to study gene function in vivo. Chapter 5 represents the structural and sequence correlations of a CcdB saturation mutagenesis library which was obtained by replacing each of 101 amino acid residues with 19 other amino acids. Polar substitutions i.e. Asn, Gln, Ser, Thr and His were poorly tolerated at buried sites at lower expression levels. Aromatic substitutions and Gly were also not well tolerated at buried positions at lower expression levels. Trp was poorly tolerated at residues with accessibility <15%. However, most of the surface exposed residues with accessibility >40% (except functional ones) could tolerate all kinds of substitutions. Chapter 6 deals with the thermodynamic characterization of monomeric and dimeric forms of CcdB. The stability and aggregation state of CcdB have been characterized as a function of pH and temperature. Size exclusion chromatography revealed that the protein is a dimer at pH 7.0, but a monomer at pH 4.0. CD analysis and fluorescence spectroscopy showed that the monomer is well folded, and has similar tertiary structure to the dimer. Hence intersubunit interactions are not required for folding of individual subunits. The oligomeric status of CcdB at pH 7.0 at physiologically relevant low concentrations of protein, was characterized by labeling the protein with two different pairs of donor and acceptor fluorescent dyes (Acrylodan-Pyrene and IAF-IAEDANS) separately and carrying out fluorescence resonance energy transfer (FRET) measurements by mixing them together. CcdB exists in a dimeric state even at nanomolar concentrations, thus indicating that the dimeric form is likely to be the physiologically active form of CcdB. The stability of the dimeric form at pH 7.0 and the monomeric form at pH 4.0 was characterized by isothermal denaturant unfolding and calorimetry. The free energies of unfolding were found to be 9.2 kcal/mol (1 cal=4.184 J) and 21 kcal/mol at 298 K for the monomer and dimer respectively. The denaturant concentration at which one-half of the protein molecules are unfolded (Cm) for the dimer is dependent on protein concentration, whereas the Cm of the monomer is independent of protein concentration, as expected. Although thermal unfolding of the protein in aqueous solution is irreversible at neutral pH, it was found that thermal unfolding is reversible in the presence of GdnCl (guanidinium chloride). Differential scanning calorimetry in the presence of low concentrations of GdnCl in combination with isothermal denaturation melts as a function of temperature were used to derive the stability curve for the protein. The value of Cp (representing the change in excess heat capacity upon protein denaturation) is 2.8 ± 0.2 kcalmol-1K-1 for unfolding of dimeric CcdB, and only has a weak dependence on denaturant concentration. These studies advanced the understanding of protein folding of oligomeric proteins. The concluding section summarizes all the chapters in a nutshell and addresses the future directions provided by these investigations.

The MCM helicase plays an important role in eukaryotic DNA replication, unwinding double stranded DNA ahead of the replication fork. MCM is a hetero-hexamer consisting of the six related proteins, Mcm2-Mcm7. The distantly related MCM-binding protein (MCM-BP) was first identified in a screen for proteins interacting with MCM2-7 in human cells and was found to specifically interact with Mcm3-7 but not Mcm2. It is conserved in most eukaryotes and seems to play an important role in DNA replication but its exact function is not clear yet. This study contributes to the understanding of the fission yeast homologue of MCM-BP, named Mcb1, but also of MCM-BP in general. Results presented in this thesis document the initial biochemical characterisation of the complex Mcb1 forms with Mcm proteins, the MCMᴹᶜᵇ¹ complex. Interactions of Mcb1 with Mcm proteins, potential interaction sites between the proteins and the size of the complex were analysed using a variety of methods, including tandem affinity purification, co-immunoprecipitation, sucrose gradients and in vitro pull-down assays. Sequence analysis and structure prediction were utilised to gain some insight into Mcb1 and MCM-BP ancestry and structure. Results presented here indicate that fission yeast Mcb1 shares homology with Mcm proteins and forms a complex with Mcm3-Mcm7 but not Mcm2 and thus replaces the latter in an alternative high molecular weight complex that is likely to have an MCM-like appearance. Deletion of mcb1⁺ showed that Mcb1 is essential in fission yeast. To examine the cellular function of the protein, temperature-sensitive mutants were generated. Inactivation of Mcb1 leads to an increase in DNA damage and cell cycle arrest in G2-phase depending on the activation of the Chk1 dependent DNA damage checkpoint. Similar observations were made when Mcb1 was overexpressed, indicating that certain levels of the protein are important for accurate DNA replication. Construction of truncated versions of Mcb1 suggested that almost the full-length protein is needed for proper function.

Gene deletions are a powerful method to uncover the cellular functions of a given gene in living systems. A limitation to this methodology is that it is not applicable to essential genes. Even for non-essential genes, gene knockouts cause complete absence of gene product thereby limiting genetic analysis of the biological pathway. Alternatives to gene deletions are mutants that are conditional, for e.g, temperature sensitive (ts) mutants are robust tools to understand temporal and spatial functions of genes. By definition, products of such mutants have near normal activity at a lower temperature or near-optimal growth temperature which is called as the permissive temperature and reduced activity at a higher, non-optimal temperature called as the non-permissive temperature. Generation of ts alleles in genes of interest is often time consuming as it requires screening a large population of mutants to identify those that are conditional. Often many essential proteins do not yield ts such alleles even after saturation mutagenesis and extensive screening (Harris et al., 1992; Varadarajan et al., 1996). The limited availability of such mutants in many essential genes prompted us to adopt a biophysical approach to design temperature-sensitive missense mutants in an essential gene of fission yeast. Several studies report that mutations in buried or solvent-inaccessible amino acids cause extensive changes in the thermal stability of proteins and specific substitutions create temperature-sensitive mutants (Rennell et al., 1991; Sandberg et al., 1995). We used the above approach to generate conditional mutants in the fission yeast gene spprp18+encoding an essential predicted second splicing factor based on its homology with human and S. cerevisiae proteins. We have used a missense mutant coupled with a conditional expression system to elucidate the cellular functions of spprp18+. Further, we have employed the same biophysical principle to generate a missense mutant in spago1+ RNA silencing factor that is non-essential for viability but has critical functions in the RNAi pathway of fission yeast. Fission yeast pre-mRNA splicing: cellular functions for the protein factor SpPrp18 Pre-mRNA splicing is an evolutionarily conserved process that excises introns from nascent transcripts. Splicing reactions are catalyzed by the large ribonuclear protein machinery called the spliceosome and occur by two invariant trans-esterification reactions (reviewed in Ruby and Abelson, 1991; Moore et al., 1993). The RNA-RNA, RNA–protein and protein-protein interactions in an assembly of such a large protein complex are numerous and highly dynamic in nature. These interactions in in vitro splicing reactions show ordered recruitment of essential small nuclear ribonucleic particles snRNPs and non–snRNP components on pre-mRNA cis-elements. Further these trans acting factors recognize and poise the catalytic sites in proximity to identify and excise introns. The precision of the process is remarkable given the diversity in architecture for exons and introns in eukaryotic genes (reviewed in Burge et al., 1999; Will and Luhrmann, 2006). Many spliceosomal protein components are conserved across various organisms, yet introns have diverse features with large variations in primary sequence. We hypothesize that co-evolution of splicing factor functions occurs with changes in gene and intron architectures and argue for alternative spliceosomal interactions for spliceosomal proteins that thus enabling splicing of the divergent introns. In vitro biochemical and genetic studies in S. cerevisiae and biochemical studies with human cell lines have indicated that ScPRP18 and its human homolog hPRP18 function during the second catalytic reaction. In S. cerevisiae, ScPrp18 is non-essential for viability at growth temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Horowitz and Abelson, 1993b). The concerted action of ScSlu7 - ScPrp18 heteromeric complex is essential for proper 3’ss definition during the second catalytic reaction (Zhang and Schwer, 1997; James et al., 2002). These in vitro studies also hinted at a possible intron -specific requirement for ScPrp18 and ScSlu7 factors as they were dispensable for splicing of intron variants made in modified ACT1 intron containing transcripts (Brys and Schwer, 1996; Zhang and Schwer, 1997). A short spacing distance between branch point adenosine to 3’splice site rendered the substrate independent of Prp18 and Slu7 for the second step (Brys and Schwer, 1996; Zhang and Schwer, 1997). Extensive mutational analyses of budding yeast ScPrp18 identified two functional domains and suggested separate roles during splicing (Bacikova and Horowitz, 2002; James et al., 2002). Fission yeast with its genome harboring multiple introns and degenerate splice signals has recently emerged as a unique model to study relationships between splicing factors and their role in genomes with short introns. Previously, studies in our lab had initiated genetic and mutational analysis of S. pombe Prp18, the predicted homolog of budding yeast Prp18. Genetic analysis showed its essentiality, but a set of missense mutants based on studies of budding yeast ScPrp18 (Bacikova and Horowitz, 2002) gave either inactive null or entirely wild type phenotype for the fission yeast protein. In this study, we have extended our previous mutational analysis of fission yeast Prp18 by adopting biophysical and computational approaches to generate temperature-sensitive mutants. A missense mutant was used to understand the splicing functions and interactions of SpPrp18 and the findings are summarized below. Fission yeast SpPrp18 is an essential splicing factor with transcript-specific functions and links efficient splicing with cell cycle progression We initiated our analysis of SpPrp18 by adopting a biophysical approach to generate ts mutants. We used the PREDBUR algorithm to predict a set of buried residues, which when mutated could result in a temperature-sensitive phenotype that complements the null allele at permissive temperature. These predictions are based upon two biophysical properties of amino acids: 1) Hydrophobicity, which is calculated in a window of seven amino acids 2) Hydrophobic moment, which is calculated in a sliding window of nine amino acids in a given protein sequence. Several studies correlate these properties to protein stability and function (Varadarajan et al., 1996). One of the buried residue mutants V194R, in helix 1 of SpPrp18 conferred weak temperature- sensitivity and strong cold-sensitivity even when the protein was over expressed from a plasmid. Through semi-quantitative RT-PCR we showed splicing-defects for tfIId+ intron1 in these cells even when grown at permissive temperature. The primary phenotype was the accumulation of pre-mRNA. Further, we showed this splicing arrest is co-related with reduced levels of SpPrp18 protein, linking protein stability and splicing function. Next we examined the effects of this mutation on function by further reduction of protein levels. This was done by integrating the expression cassette nmt81:spprp18+/spprp18V194R at the leu1 chromosomal locus and by metabolic depletion of the integrated allele. Through RT-PCRs we demonstrated that depletion of wild type or missense protein has intron specific splicing defects. These findings showed its non-global and possibly substrate-specific splicing function. In the affected introns, precursor accumulation is the major phenotype, confirming prior data from our lab that hinted at its likely early splicing role. This contrasts with the second step splicing role of the human or budding yeast Prp18 proteins. Previous data from our lab showed loss of physical interaction between SpPrp18 and SpSlu7 by co-immunoprecipitation studies. This again differs from the strong and functionally important ScPrp18 and ScSlu7 interaction seen in budding yeast. We show the absence of charged residues in SpSlu7 interaction region formed by SpPrp18 helix1 and helix2 which can explain the altered associations for SpPrp18 in fission yeast. Importantly, as the V194R mutation in helix 1 shows splicing defects even at permissive temperature, the data indicate a critical role for helix 1 for splicing interactions, possibly one that bridges or stabilizes the proposed weak association of SpPrp18-SpSlu7 with a yet unknown splicing factor. We also investigated the effects of mutations in other helices; surprisingly we recovered only mutations with very subtle growth phenotypes and very mild splicing defects. Not surprisingly, stop codon at L239 residue predicted to form a truncated protein lacking helices 3, 4 and 5 conferred recessive but null phenotype implicating essential functions for other helices. Other amino acid substitutions at L239 position had near wild type phenotype at 30°C and 37°C. Helix 3 buried residue mutant I259A conferred strong cold-sensitivity when over expressed from plasmid, but semi quantitative analysis indicated no splicing defects for intron1 in the constitutively expressed transcript tfIId+. These findings indicate cold sensitivity either arises due to compromised splicing of yet unknown transcripts or that over-expressed protein has near wild type activity. We find mutations in the helix 5 buried residues L324 also conferred near WT phenotype. Earlier studies in the lab found that substitution of surface residues KR that are in helix 5 with alanine lead to null phenotypes (Piyush Khandelia and Usha Vijayraghavan unpublished data). We report stable expression of all of these mutant proteins; L239A, L239P, L239G, I259A, I259V, L324F, L324A as determined by our immunoblot analysis at 30°C and 37°C. The mild phenotypes of many buried residues can be attributed to orientation of their functional groups into a protein cavity between the helices. Lastly, our microscopic cellular and biochemical analysis of cellular phenotypes of spprp18 mutant provided a novel and direct role of this factor in G1-S transition of cell cycle. Our RT-PCR data suggest spprp18+ is required for efficient splicing of several intron containing transcripts involved in G1-S transition and subsequent activation of MBF complex (MluI cell cycle box-binding factor complex) during S-phase and shows a mechanistic link between cell cycle progression and splicing. A tool to study links between RNA interference, centromeric non-coding RNA transcription and heterochromatin formation S.pombe possesses fully functional RNA interference machinery with a single copy for essential RNAi genes ago1+, dcr1+ and rdp1+. Deletion of any of these genes causes loss of heterochromatinzation with abnormal cytokinesis, cell-cycle deregulation and mating defects (Volpe et al., 2002). In S.pombe, exogenous or endogenously generated dsRNA’s from transcription of centromeric repeats are processed by the RNaseIII enzyme dicer to form siRNA. These siRNA’s are loaded in Ago1 to form minimal RNA induced silencing complex (RISC) complex or specialized transcription machinery complex RNA induced transcriptional silencing (RITS) complex and target chromatin or complementary mRNAs for silencing. Thus as in other eukaryotes, fission yeast cells deploy RNAi mediated silencing machinery to regulate gene-expression and influence chromatin status. Several recent studies point to emerging new roles of RNAi and its association with other RNA processes (Woolcock et al., 2011; Bayane et al., 2008; Kallgren et al., 2014). Many recent reports suggest physical interactions of RISC or RITS and RNA dependent RNA polymerase complex (RDRC) with either some factors of the spliceosomal machinery, heterochromatin machinery (CLRC complex) and the exosome mediated RNA degradation machinery (Bayne et al., 2008 and Chinen et al., 2010 ; Hiriart et al., 2012; Buhler et al., 2008; Bayne et al., 2010 ). Thus we presume conditional alleles in spago1+ will facilitate future studies to probe the genetic network between these complexes as most analyses thus far rely on ago1∆ allele or have been based on proteomic pull down analyses of RISC or RITS complexes. In this study, we employed biophysical and modeling approaches described earlier to generate temperature sensitive mutants in spago1+ and spdcr1+. We tested several mutants for their ability to repress two reporter genes in a conditional manner. Our modeling studies on SpAgo1 PAZ domain indicated structural similarities with human Ago1 PAZ domain. We created site-directed missense mutants at predicted buried residues or in catalytic residues. We also analyzed the effects of random amino acid replacements in specific predicted buried or catalytic residues of SpAgoI. These ago1 mutants were screened as pools for their effects on silencing of GFP or of ura4+ reporter genes. These assays assessed post transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) activity of these mutants. We obtained three temperature sensitive SpAgo1 mutants V324G, V324S and L215V while the V324E replacement was a null allele. Based upon our modeling, a likely explanation for the phenotype of these mutants is structural distortion or mis-orientation of the functional groups caused due to these mutations, which affect activity in a temperature dependent manner. This distortion in the PAZ domain may affect binding of siRNA and thereby lead to heterochromatin formation defects that we observed. Our data on the SpAgo1 V324 mutant shows conditional centromeric heterochromatin formation confirmed by semi quantitative RT-PCR for dh transcripts levels that shows temperature dependent increase in these transcripts. We find reduced H3K9Me2 levels at dh locus by chromatin immunoprecipitation (ChIP) assay, linking the association of siRNAs for establishment of heterochromatin at this loci. The data on PTGS of GFP transcripts show SpAgo1 V324G mutation has decreased slicing activity as semi-quantitative RT-PCR for GFP transcripts show increased levels at non permissive temperature. These studies point out the importance of siRNA binding to the PAZ domain and its effect on slicing activity of SpAgo1. The mutations in Y292 showed residue loss of centromeric heterochromatin formation phenotype. Thus, we ascribe critical siRNA binding and 3’ end recognition functions to this residue of SpAgo1. These studies point out functional and structural conservation across hAgo1 and SpAgo1. Adopting the aforementioned biophysical mutational approach, we generated mutants in spdcr1+ and screened for those with conditional activity. Our modeling studies on SpDcr1 helicase domain shows it adopts the conserved helicase domain structure seen for other DEAD Box helicases. Our data on mutational analysis of a conserved buried residue I143 in the walker motif B created inactive protein. The data confirm critical functions for dicer in generation of siRNAs and also in recognition of dsRNA ends. Mutants in buried residues L1130 and I1228 of RNase IIIb domain were inactive and the proximity of these residues to the catalytic core suggest that the critical structural alignment of catalytic residues is indispensable for carrying out dsRNA cleavage to generate siRNAs. We also attribute critical catalytic functions to SpDcr1 D1185 residue for generation of siRNA and heterochromatin formation as measured by our transcriptional gene silencing assay. Our studies employing biophysical and computational approaches to design temperature-sensitive mutants have been successfully applied to an essential splicing factor SpPrp18, which was refractory for ts mutants by other methods. Using a missense mutant, we showed its intron-specific splicing function for subsets of transcripts and deduced that its ubiquitous splicing role is arguable. We have uncovered a link between the splicing substrates of SpPrp18 and direct evidence of splicing based cell cycle regulation, thus providing a mechanistic link to the cell cycle arrest seen in some splicing factor mutants. The same methodology was applied to another important biological pathway, the RNAi machinery, where central factors SpAgoI and SpDcrI were examined We report the first instance of conditional gene silencing tool by designing Ago1 ts mutants which will be useful for future studies of the global interaction network between RNAi and other RNA processing events.