Academic literature on the topic 'Protein folding; Proline isomerisation'

SH2 domains are protein domains that modulate protein–protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the βE and βF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.

The protein-folding mechanism remains a major puzzle in life science. Purified soluble activation-induced cytidine deaminase (AID) is one of the most difficult proteins to obtain. Starting from inclusion bodies containing a C-terminally truncated version of AID (residues 1–153; AID153), an optimizedin vitrofolding procedure was derived to obtain large amounts of AID153, which led to crystals with good quality and to final structural determination. Interestingly, it was found that the final refolding yield of the protein is proline residue-dependent. The difference in the distribution ofcisandtransconfigurations of proline residues in the protein after complete denaturation is a major determining factor of the final yield. A point mutation of one of four proline residues to an asparagine led to a near-doubling of the yield of refolded protein after complete denaturation. It was concluded that the driving force behind protein folding could not overcome thecis-to-transproline isomerization, orvice versa, during the protein-folding process. Furthermore, it was found that successful refolding of proteins optimally occurs at high pH values, which may mimic protein foldingin vivo. It was found that high pH values could induce the polarization of peptide bonds, which may trigger the formation of protein secondary structures through hydrogen bonds. It is proposed that a hydrophobic environment coupled with negative charges is essential for protein folding. Combined with our earlier discoveries on protein-unfolding mechanisms, it is proposed that hydrogen bonds are a primary driving force forde novoprotein folding.

Due to the heterocyclic structure and distinct conformational profile, proline is unique in the repertoire of the 20 amino acids coded into proteins. Here, we summarize the biochemical work on the replacement of proline with (4R)- and (4S)-fluoroproline as well as 4,4-difluoroproline in proteins done mainly in the last two decades. We first recapitulate the complex position and biochemical fate of proline in the biochemistry of a cell, discuss the physicochemical properties of fluoroprolines, and overview the attempts to use these amino acids as proline replacements in studies of protein production and folding. Fluorinated proline replacements are able to elevate the protein expression speed and yields and improve the thermodynamic and kinetic folding profiles of individual proteins. In this context, fluoroprolines can be viewed as useful tools in the biotechnological toolbox. As a prospect, we envision that proteome-wide proline-to-fluoroproline substitutions could be possible. We suggest a hypothetical scenario for the use of laboratory evolutionary methods with fluoroprolines as a suitable vehicle to introduce fluorine into living cells. This approach may enable creation of synthetic cells endowed with artificial biodiversity, containing fluorine as a bioelement.

ABSTRACT Since, like other osmolytes, proline can act as a protein stabilizer, we investigated the thermoprotectant properties of proline in vitro and in vivo. In vivo, elevated proline pools in Escherichia coli (obtained by altering the feedback inhibition by proline of γ-glutamylkinase, the first enzyme of the proline biosynthesis pathway) restore the viability of a dnaK-deficient mutant at 42°C, suggesting that proline can act as a thermoprotectant for E. coli cells. Furthermore, analysis of aggregated proteins in the dnaK-deficient strain at 42°C by two-dimensional gel electrophoresis shows that high proline pools reduce the protein aggregation defect of the dnaK-deficient strain. In vitro, like other “chemical chaperones,” and like the DnaK chaperone, proline protects citrate synthase against thermodenaturation and stimulates citrate synthase renaturation after urea denaturation. These results show that a protein aggregation defect can be compensated for by a single mutation in an amino acid biosynthetic pathway and that an ubiquitously producible chemical chaperone can compensate for a defect in one of the major chaperones involved in protein folding and aggregation.

Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide’s vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein’s amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein’s amino acid sequence, its cotranslational nascent-chain elongation rate, and folding.

MYC is a family of three regulator genes that codes for transcription factors. Expression of Myc proteins from MYC genes is found to be deregulated in 70 % of all cancer forms. The three human homologs C-Myc, N-Myc and L-Myc are mainly associated with cancer in the lymphatic system, nerve tissues and lung cancer, respectively. Even though N-Myc is associated with Neuroblastoma, the cancer variant that is most common among children, the field is focused towards C-Myc. The activation of C-Myc begins with phosphorylation of Serine 62, followed by trans-to-cis isomerisation of Proline 63. Then Threonine 58 becomes phosphorylated leading to that Serine 62 is dephosphorylated and subsequent cis-to-trans isomerisation of Proline 63, and C-Myc is marked for degradation. Cis-trans isomerisation is necessary for regulation of gene expression, and is therefore important to understand. Since N-Myc and C-Myc have identical sequences between residues 47 to residue 69, the hypothesis is that N-Myc is activated in the same manner, but this has not been confirmed. In this project the first 69 amino acids of N-Myc were analysed with NMR spectroscopy. This resulted in a near complete assignment of the major conformation, and of the alternative minor conformations as well. The traditional assignment experiments HNCACB, HN(CO)CACB, HNCO, HN(CA)CO in combination with CCH-TOCSY and HN(CCO)C revealed that the majority of the minor configurations can be explained by cis/trans isomerisation of prolines. In addition, the protein was analysed with direct carbon detected NMR spectroscopy to be able to detect the prolines.

Die vorliegende Arbeit untersucht mit bioinformatischen Methoden die biologische Bedeutung von Ähnlichkeiten in Kleinstrukturen und peptidischen Sequenzmotiven sowie lokaler und globaler Sequenzähnlichkeit. Der erste Teil der Arbeit behandelt chemische Ähnlichkeiten. Ausgehend von bekannten Inhibitoren der Fehlfaltung des Prionproteins wurde eine Datenbank pharmakologischer Wirkstoffe nach chemisch und strukturell ähnlichen Substanzen durchsucht und 16 Substanzen als neue potentielle Inhibitoren der Fehlfaltung vorgeschlagen. Der nächste Teil untersucht Ähnlichkeiten in Sequenzmotiven, die eine Interaktion mit Pex19, dem Importrezeptor für peroxisomale Membranproteine, vermitteln. In Zusammenarbeit mit einer experimentellen Arbeitsgruppe konnte die Bindestelle charakterisiert und Präferenzen für bestimmte Aminosäuren herausgearbeitet werden. Das Bindemotiv ist eine vermutlich helikale Region mit verzweigtkettigen aliphatischen und basischen Aminosäuren. Aus experimentellen Daten konnte eine positionsabhängige Vorhersagematrix erstellt und validiert werden. Die Beziehung zwischen lokalen Sequenzähnlichkeiten und der Konformation von Prolylbindungen in Proteinen ist Thema des dritten Teils. Die Aminosäurepräferenzen in der Nachbarschaft von cis- und trans-Prolylresten unterscheiden sich, und beide zeigen unterschiedliche Austauschpräferenzen bei Mutationen. Im Gegensatz zu lokaler Sequenzähnlichkeit ist eine globale Sequenzähnlichkeit von nur 20% ein wesentlich besserer Indikator für das Auftreten von cis-Prolylbindungen. Der letzte Teil befaßt sich mit inverser Sequenzähnlichkeit zwischen Proteinen, die wesentlich öfter auftritt als erwartet. Proteine aus einem nichtredundanten Datensatz wurden gleich- und gegenläufig aligniert und strukturelle Ähnlichkeiten zwischen den aufgefundenen Proteinpaaren untersucht. Es konnte gezeigt werden, daß bis auf kurze Sekundärstruktur-Einheiten eine inverse Sequenzähnlichkeit zwischen Proteinen keine strukturelle Ähnlichkeit impliziert.
This work is dealing with the biological impact of similarities between chemical structures, protein sequence motifs and local sequence surrounding as well as global sequence similarity. All four aspects are analyzed by computational methods. The first part is dealing with chemical similarities. Based on a recently published set of prion protein misfolding inhibitors, a data base of approved drugs has been screened for compounds with chemical and structural similarities to these substances. 16 drugs are proposed as new potential inhibitors of prion protein aggregation. The next part addresses similarities of sequence motifs which mediate the interaction with the peroxisomal membrane protein import receptor Pex19. In cooperation with an experimental group, the binding site could be characterized, and amino acid preferences of the different positions of the motif have been determined. The binding motif is a probably helical region of target proteins bearing branched aliphatic and basic residues. A position specific scoring matrix for the prediction of Pex19 binding sites could be generated and validated. The relation between local sequence similarity and prolyl bond conformation is examined in the third part. Amino acid preferences of neighboring residues differ between cis and trans prolyl residues, and both species show different amino acid exchange patterns upon mutation. In contrast to local sequence similarity, overall sequence similarity between proteins as low as 20% is a much better indicator for the occurrence of cis prolyl bonds. The last part focuses on inverse sequence similarity between proteins which occurs far more often than expected by chance. Proteins from a nonredundant data set have been aligned in parallel and antiparallel, and structural similarities between the detected protein pairs have been examined. It could be shown that, with the exception of short secondary structural elements, inverse sequence similarity does not imply structural similarity.

Die Pektat-Lyasen gehören zu einer Proteinfamilie, die meistens von pflanzenpathogenen Mikroorganismen sekretiert werden. Die Enzyme katalysieren den Abbau von Polygalakturonsäure, einem Hauptbestandteil in
pflanzlichen Mittellamellen und Primärzellwänden. Der Abbau der alpha-1,4-verbrückten Galakturonsäurereste erfogt durch eine beta-Eliminierungsreaktion, dabei entsteht ein Produkt mit einer ungesättigten C4-C5 Bindung am nicht reduzierenden Ende, das durch spektroskopische Messungen beobachtet werden kann. Für die enzymatische Reaktion der Pektat-Lyasen ist Calcium nötig und das pH-Optimum der Reaktion liegt bei pH 8.5. Alle bis jetzt bekannten Strukturen der Pektat- und Pektin-Lyasen haben das gleiche Strukturmotiv - eine rechtsgängige parallele beta-Helix. Die Struktur der Pektat-Lyase aus Bacillus subtilis (BsPel) ist im Komplex mit Calcium gelöst worden. BsPel ist ein monomeres Protein mit einer ungefähren Molekularmasse von 43 kDa, das keine Disulfidbrücken enthält. Dies erlaubte sowohl eine effiziente rekombinante Expression des Wildtypproteins, als auch von destabilisierten Mutanten im Cytoplasma von E. coli. Parallele beta-Helices sind relativ große, jedoch verhältnismäßig einfach aufgebaute Proteine. Um detailliertere Informationen über die kritischen Schritte bei der in vitro-Faltung von parallelen beta-Helices zu erhalten, sollte in der vorliegenden Arbeit versucht werden, den Faltungsmechanismus dieses Proteins näher zu charakterisieren. Dabei sollte vor allem die Frage geklärt werden, welche Wechselwirkungen für die Stabilität dieses Proteins einerseits und für die Stabilität von essentiellen Faltungsintermediaten andererseits besonders wichtig sind.

Rückfaltung von BsPel, ausgehend vom guanidiniumchlorid-denaturierten Zustand, war bei kleinen Proteinkonzentrationen und niedrigen Temperaturen vollständig möglich. GdmCl-induzierte Faltungsübergänge waren aber nicht reversibel und zeigten eine apparente Hysterese. Kinetische Messungen des Fluoreszenz- und CD-Signals im fernen UV ergaben eine extreme Denaturierungsmittelabhängigkeit der Rückfaltungsrate im Bereich des Übergangmittelpunktes. Der extreme Abfall der Rückfaltungsraten mit steigender Denaturierungsmittelkonzentration kann als kooperative
Entfaltung eines essentiellen Faltungsintermediats verstanden werden. Dieses Faltungsintermediat ist temperaturlabil und kann durch den Zusatz Glycerin im Renaturierungspuffer stabilisiert werden, wobei sich die Hysterese verringert, jedoch nicht vollständig aufgehoben wird. Durch reverse Doppelsprungexperimente konnten zwei transiente Faltungsintermediate nachgewiesen werden, die auf zwei parallelen Faltungswegen liegen und beide zum nativen Zustand weiterreagieren können. Fluoreszenzemissionsspektren der beiden Intermediate zeigten, daß beide schon nativähnliche Struktur aufweisen. Kinetische Daten von Prolin-Doppelsprungexperimenten zeigten, daß Prolinisomerisierung den geschwindigkeitsbestimmenden Schritt in der Reaktivierung des denaturierten Enzyms darstellt. Desweiteren konnte durch Prolin-Doppelsprungexperimenten an Mutanten mit Substitutionen im Prolinrest 281 gezeigt werden, daß die langsame Renaturierung von BsPel nicht durch die Isomerisierung der einzigen cis-Peptidbindung an Prolin 281 verursacht wird, sondern durch die Isomerisierung mehrerer trans-Proline. Die beiden beobachteten transienten Faltungsintermediate sind somit wahrscheinlich zwei Populationen von Faltungsintermediaten mit nicht-nativen X-Pro-Peptidbindungen, wobei sich die Populationen durch mindestens eine nicht-native X-Pro-Peptidbindung unterscheiden.

Der Austausch des Prolinrestes 281 gegen verschiedene Aminosäuren (Ala, Ile, Leu, Phe, Gly) führte zu einer starken Destabilisierung des nativen Proteins und daneben auch zu einer Reduktion in der Aktivität, da die Mutationsstelle in der Nähe der putativen Substratbindetasche liegt. Die Rückfaltungskinetiken der Prolinmutanten war bei 10°C annähernd gleich zum Wildtyp und die geschwindigkeitsbestimmenden Schritte der Faltung waren durch die Mutation nicht verändert. Die durch die Mutation verursachte drastische Destabilisierung des nativen Zustands führte zu einem reversiblen Entfaltungsgleichgewicht bei pH 7 und 10°C. GdmCl-induzierte Faltungsübergänge der Mutante P281A zeigten bei Messungen der Tryptophanfluoreszenzemission und der Aktivität einen kooperativen Phasenübergang mit einem Übergangsmittelpunkt bei 1.1 M GdmCl. Durch die Übereinstimmung der Faltungsübergänge bei beiden Messparametern konnten die Faltungsübergänge nach dem Zwei-Zustandsmodell ausgewertet werden. Dabei wurde eine freie Sabilisierungsenthalpie der Faltung für die Mutante von - 64.2 ± 0.4 kJ/mol und eine Kooperativität des Übergangs
von - 58.2 ± 0.3 kJ/(mol·M) bestimmt.


BsPel enthält, wie die meisten monomeren rechtsgängigen parallelen beta-Helix-Proteine, einen internen Stapel wasserstoffverbrückter Asparagin-Seitenketten. Die Mehrheit der erzeugten Mutanten mit Substitutionen im Zentrum der Asn-Leiter (N271X) waren als enzymatisch aktives Protein zugänglich. Die Auswirkung der Mutation auf die Stabilität und Rückfaltung wurde an den Proteinen BsPel-N271T und BsPel-N271A näher analysiert. Dabei führte die Unterbrechung des Asparaginstapels im Inneren der beta-Helix zu keiner drastischen Destabilisierung des nativen Proteins. Allerdings führten diese Mutationen zu einem temperatur-sensitiven Faltungsphänotyp und die Hysterese im Denaturierungsübergang wurde verstärkt. Offenbar wird durch die Unterbrechung des Asparaginstapel ein essentielles, thermolabiles Faltungsintermediat destabilisiert. Der Asparaginstapel wird somit bei der Faltung sehr früh ausgebildet und ist wahrscheinlich schon im Übergangszustand vorhanden.
Pectate lyases belong to a family of proteins secreted by plant pathogenic microbes. The enzymes cleave alpha-1,4 linked galacturonic acid by a beta-elimination that results in an unsaturated product, which can be quantified spectrophotometrically. Calcium is essential for the activity and the pH-optimum is near 8.5. All known structures of pectate and pectin lyases have the same structural motif - a right handed parallel beta-helix. The structure of pectate lyase from Bacillus subtilis (BsPel) has been solved in complex with calcium. It is a monomeric protein, with a molecular mass of about 43 kDa and without disulfide bonds. This allows its high-yield recombinant expression in the cytoplasm of Escherichia coli. Parallel beta-helices are relative large proteins, however with a simple folding topology. The objective of this work was to characterize the folding mechanism of BsPel. In particular we investigated the role of the interactions of certain residues in the parallel beta-helix for the stability of the native protein and the stability of essential folding intermediates.

Refolding of BsPel was possible at low protein concentrations and low temperature. However, denaturation of BsPel was not freely reversible. De- and renaturation curves showed a large apparent hysteresis. Furthermore, the folding rate constant deduced from fluorescence and circulardichroism measurements showed a very strong dependence on denaturant concentrations near the midpoint of the renaturation transition. This can be explained with a cooperative unfolding of an essential folding intermediate. Upon stabilisation of the temperature-sensitive intermediate by addition of glycerol in the renaturation buffer, the hysteresis is reduced, but does not disappear. Reverse double mixing kinetic experiments have shown that two transient folding intermediates are on the folding pathway. These intermediates are on parallel pathways and both can fold to the native state. Fluorescence emission spectra have shown the native-like structure of both intermediates. Furthermore, data from proline double mixing kinetic experiments revealed that isomerization of peptidyl-prolyl bonds was responsible for the slow kinetics in the reactivation of the enzyme. However, the isomerization of the single cis-peptidyl-prolyl bond at Pro281 was not responsible for the slowest folding phase observed, but rather the isomerization of other trans-peptidyl-prolyl bonds. Thus, both transient folding intermediates observed probably represent two populations of folding intermediates with non-native X-Pro-peptide bonds. The difference of the two populations is at least one non-native X-Pro-peptide bond.

Mutations of the proline 281 against various residues (Ala, Ile, Leu, Phe, Gly) resulted in a strong destabilization of the native protein. Also, the activity of the mutant proteins was strong reduced due to the position of the mutation site near the putative active center of the protein. At 10°C the kinetic folding behavior of the proline mutants was not significant changed. However, the strong destabilization of the native state in the proline mutants resulted in a reversible folding equilibrium at pH 7 and 10°C. The unfolding of the P281A mutant was reversible as determined by fluorescence emission and enzyme activity measurements. The coincidence of these detected transitions is consistent with a two-state equilibrium transition. At pH 7 and 10°C the delta G°(H₂O) for folding of P281A was - 64.2 ± 0.4 kJ/mol, with a midpoint of the transition at 1.1 M GdmCl and a cooperativity of - 58.2 ± 0.3 kJ/(mol·M).

BsPel has an asparagine ladder in turn 2 of the parallel beta-helix with extensive network of side-chain hydrogen bonds between the Asn residues. Such an Asn-ladder is a conserved feature of many monomeric beta-helices crystallized so far. The middle Asn residue (271) was selected and exchanged for various residues. Most of the mutants were expressed at 25°C as soluble and active proteins but with a significant reduction in yield. Mutants N271T and N271A were selected to study the stability and refolding of these proteins in comparison with the wild-type protein. The substitution in the Asn-ladder did not drastically destabilize the native protein, but caused a temperature-sensitive-folding (tsf) phenotype with an increased hysteresis in the de- and renaturation transition curves. In addition, the disruption of the Asn-ladder resulted in destabilization of an essential, thermosensitive folding intermediate. Thus, the Asn-ladder is formed very early during the folding, probably well before the transition state of folding.

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 group 2 late embryogenesis abundant (LEA) proteins, also known as the dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. In this work, we study the potential roles that dehydrins may have in stabilizing membranes and actin microfilaments during cold stress. We have cloned and expressed in E. coli two dehydrins from Thellungiella salsuginea, denoted TsDHN-1 (acidic) and TsDHN-2 (basic). These proteins were expressed as SUMO-fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and for structural analysis by CD and Fourier transform infrared (FTIR) spectroscopy. We show using transmission-FTIR spectroscopy that ordered secondary structure is induced and stabilized in these proteins by association with large unilamellar vesicles emulating the lipid compositions of plant plasma and organellar membranes. The increase in secondary structure by membrane association is further facilitated by the presence of Zn2+. Lipid composition and temperature have synergistic effects on the secondary structure. Our single molecule force spectroscopy studies also suggest tertiary folding of both TsDHN-1 and TsDHN-2 induced by association with lipids. From Langmuir-Blodgett monolayer compression studies, and from topographic studies using atomic force microscopy at variable temperature, we conclude that TsDHN-1 stabilizes the membrane at lower temperatures. Finally, we show that the conformations of TsDHN-1 and TsDHN-2 are affected by pH, interactions with cations and membranes, and phosphorylation. Actin assembly by these dehydrins was assessed by sedimentation assays, and viewed by transmission electron and atomic force microscopy. Phosphorylation enabled both dehydrins to polymerize actin filaments, a phenomenon that may occur in the cytosols of plant cells undergoing environmental stress. These results support the hypothesis that dehydrins stabilize plant organellar membranes and/or the cytoskeleton in conditions of stress, and further that phosphorylation may be an important feature of this stabilization.
NSERC

The slow rates of peptide bond isomerization in imino acids and the substantial population of the cis peptide bond isomer in Xaa-Pro linkages in peptides were first recognized in NMR studies of proline-containing model compounds (Maia et al., 1971). The important role of this isomerization in protein stability and folding (reviewed by Kim and Baldwin, 1982, 1990; Schmid, 1993) were recognized several years later (Brandts et al., 1975) and the biological relevance of this process was substantiated by the discovery of a ubiquitous enzyme that catalyzes Xaa-Pro peptide bond isomerization (Fischer et al., 1984, 1989; Takahashi et al., 1989). The strict evolutionary conservation of some prolyl residues and the observation that the kinetics of interconversion between alternative functional forms of some systems is consistent with the time scale of proline isomerization suggest that proline isomerization may play a wide role in protein structure and function. Suggestive examples include the sodium pump of Escherichia coli, the disulfide isomerase/thioredoxin class of enzymes, concanavalin A, and bovine prothrornbin fragment I (Brown et al., 1977; Marsh et al, 1979; Dunker, 1982; Brandland Deber, 1986; Langsetmo et al, 1989). NMR spectroscopy is one of the most suitable tools for studying this isomerization reaction. The rates generally are slow on the time scale of NMR chemical shifts but, in favorable cases, are comparable to longitudinal relaxation rates so that the isomerization process can be investigated by chemical exchange spectroscopy. NMR data obtained on calbindin D9k (Chazin et al., 1989), insulin (Higgins et al., 1988), and staphylococcal nuclease (nuclease) as discussed below have shown that each exists in solution under native conditions as a mixture of slowly exchanging conformers. The fact that dynamic molecular heterogeneity in nuclease was first observed in the laboratory of Oleg Jardetzky, as manifested by splitting of the histidyl 1H ε1 resonance from His46 in one-dimensional 1H NMR spectra recorded at 100 MHz (Markley et al., 1970), makes this topic particularly appropriate to a volume celebrating his scientific contributions.

We present here the first report of the pressure dependence of pressure-jump relaxation kinetics for protein folding transitions. We have studied the relaxation kinetics for the unfolding/refolding of wild-type Staphylococcal nuclease and have found that the relaxation kinetics observed at high pressure are much slower than those observed by pH or denaturant jumps at atmospheric pressure. This indicates that these processes have large, positive values for the activation volumes, most likely stemming from exclusion of solvent from a transition state that is less well packed than the native state. We examined the pressure-jump relaxation kinetics of three single-site mutations in nuclease that lead to alterations in the interactions between the two domains of the protein and changes in the equilibrium constant for isomerization of the lysine-116 to proline 117 peptide bond away from the cis form that predominates in the wild-type enzyme. At comparable pressures, the relaxation times for these mutants were significantly shorter than those observed for the wild type, indicating lower values of the activation volumes. We propose that these mutations cause a decrease in the cooperativity of the unfolding of the two domains, leading to a decrease in the degree of solvent exclusion at the rate-limiting step. The mechanism by which a particular amino acid sequence determines the fold and stability of globular proteins remains one of the most interesting and important unresolved issues in biophysical chemistry. The approaches to increasing our understanding of this phenomenon typically have involved perturbation of the proteins by chemical means or by temperature extremes. The equilibrium or time-dependent responses to these perturbations are then monitored (using a spectroscopic signal, activity, or some other observable) to extract the energetic or kinetic aspects of the unfolding or refolding transitions. Another means of perturbing the system is to modify the protein itself, either chemically or by site-directed mutagenesis, and to assess the effects of modification on the equilibrium or kinetic folding or refolding profiles. This approach has generated a great deal of information about small globular proteins that denature reversibly.