Using statistical physics principles to solve problems in biology is one of the most promising directions due to the complexity and non-equilibrium fluctuations in biological systems. In this work, we try to describe the dynamics at both cellular and molecular levels. Microtubule dynamics and dynamic disorder of enzyme proteins are two of the examples we investigated. The dynamics of microtubules and the mechanical properties of these polymers are essential for many key cellular processes. However, critical discrepancies between experimental observations and existing models need to be resolved before further progress towards a complete model can be made. We carried out computational studies to compare the mechanical properties of two alternative models, one corresponding to the existing, conventional model, and the other considering an additional type of tubulin lateral interaction described in a cryo-EM structure of a proposed trapped intermediate in the microtubule assembly process. Our work indicates that a class of sheet structures is transiently trapped as an intermediate during the assembly process in physiological conditions. In the second part of the work, we analyzed enzyme slow conformational changes in the context of regulatory networks. A single enzymatic reaction with slow conformational changes can serve as a basic functional motif with properties normally discussed with larger networks in the field of systems biology. The work on slow enzyme dynamics fills the missing gap between studies on intramolecular and network dynamics. We also showed that enzyme fluctuations could be amplified into fluctuations in phosphorylation networks. This can be used as a novel biochemical "reporter" for measuring single enzyme conformational fluctuation rates. / Ph. D.

No / The growth of crystals of a-resorcinol from the vapor phase is asymmetric along the polar axis. By means of molecular-dynamics simulations, the slower growth at the (011) polar surface is traced back to conformational change of the molecule and to surface reconstruction, which may be a general phenomenon in polar crystals.

No / Various host-guest peptide series are used by experimentalists as reference conformational states. One such use is as a baseline for random-coil NMR chemical shifts. Comparison to this random-coil baseline, through secondary chemical shifts, is used to infer protein secondary structure. The use of these random-coil data sets rests on the perception that the reference chemical shifts arise from states where there is little or no conformational bias. However, there is growing evidence that the conformational composition of natively and nonnatively unfolded proteins fail to approach anything that can be construed as random coil. Here, we use molecular dynamics simulations of an alanine-based host-guest peptide series (AAXAA) as a model of unfolded and denatured states to examine the intrinsic propensities of the amino acids. We produced ensembles that are in good agreement with the experimental NMR chemical shifts and confirm that the sampling of the 20 natural amino acids in this peptide series is be far from random. Preferences toward certain regions of conformational space were both present and dependent upon the environment when compared under conditions typically used to denature proteins, i.e., thermal and chemical denaturation. Moreover, the simulations allowed us to examine the conformational makeup of the underlying ensembles giving rise to the ensemble-averaged chemical shifts. We present these data as an intrinsic backbone propensity library that forms part of our Structural Library of Intrinsic Residue Propensities to inform model building, to aid in interpretation of experiment, and for structure prediction of natively and nonnatively unfolded states. / NIH GM 50789, Ministry of Education, Youth and Sports (MSMT) of the Czech Republic LH11020

No / D-amino acids are useful building blocks for de novo peptide design and they play a role in aging-related diseases associated with gradual protein racemization. For amino acids with achiral side chains, one should be able to presume that the conformational propensities of L- and D-amino acids are a reflection of one another due to the straightforward geometric inversion at the Cα atom. However, this presumption does not account for the directionality of the backbone dipole and the inverted propensities have never been definitively confirmed in this context. Furthermore, there is little known of how alternative side chain chirality affects the backbone conformations of isoleucine and threonine. Using a GGXGG host-guest pentapeptide system, we have completed exhaustive sampling of the conformational propensities of the D-amino acids, including D-allo-isoleucine and D-allo-threonine, using atomistic molecular dynamics simulations. Comparison of these simulations with the same systems hosting the cognate L-amino acids verifies that the intrinsic backbone conformational propensities of the D-amino acids are the inverse of their cognate L-enantiomers. Where amino acids have a chiral center in their side chain (Thr, Ile) the β-configuration affects the backbone sampling, which in turn can confer different biological properties. / NIH

No description available.

A família proteína quinases C (PKC) é composta por dez isoenzimas, as quais são capazes de fosforilar resíduos de serina e treonina. A ativação dessas quinases envolve mudanças conformacionais, como a remoção do pseudo-substrato do sítio ativo e associação dessas enzimas com lipídeos em membranas biológicas. Além disso, três fosforilações são importantes para a maturação/ enovelamento da enzima e não estão associadas com o estado de ativação das cPKCs. Apesar dessas quinases estarem envolvidas em vários processos patológicos, como carcinogênese e doenças cardiovasculares, ainda não se estabeleceu a relação entre estado de ativação das PKCs com essas doenças. Isso se deve, em parte, à ausência de ferramentas que possibilitam a distinção das formas ativas e inativas das PKCs. Na presente tese, baseando-se em mudanças conformacionais sofridas pelas PKCs durante o processo de ativação, dois anticorpos contra cPKCs ativas foram racionalmente desenvolvidos, sendo um anticorpo policlonal (anti-C2Cat) e outro monoclonal (4.8E). O anticorpo anti-C2Cat foi desenvolvido a partir de imunização de coelhos com um peptídeo localizado na região de interação entre os domínios C2 e catalítico na PKC inativa. Já o anticorpo monoclonal 4.8E foi produzido após a imunização de camundongos Balb/ C com extrato de proteínas proveniente de células HEK293T superexpressando formas constitutivamente ativas da PKCβI. A seletividade de anti-C2Cat e 4.8E por cPKCs ativas foi demonstrada por ensaios de ELISA e de imunoprecipitação, sendo que os anticorpos sempre apresentaram maior afinidade por cPKCs ativas purificadas, superexpressas ou mesmo as endógenas. O anticorpo anti-C2Cat foi capaz de monitorar a dinâmica espaço-temporal da ativação das cPKCs em linhagens de neuroblastoma (Neuro-2A e SK-N-SH) estimuladas com PMA, morfina, ATP ou glutamato por diferentes tempos. Ainda, um maior conteúdo de cPKCs ativas foi detectado por anti-C2Cat na linhagem de câncer de mama MDA-MB-231 (triplo- negativa) do que em células MCF-7 (ER+). Em acordo com esses dados, anti-C2Cat identificou uma maior ativação de cPKCs em tumores mais agressivos de câncer de mama (subtipo triplo-negativo) do que em tumores menos agressivos (ER+, subtipo luminal). Os anticorpos conformacionais anti-C2Cat e 4.8E foram aplicados para elucidar vias de sinalização que levam à carcinogênese em células MDA-MB-231, por meio da realização de ensaios de co-imunoprecipitação, seguida pela identificação das proteínas por espectrometria de massas. Usando essa abordagem, os resultados sugerem que as cPKCs ativas possam estar envolvidas com a tradução de proteínas envolvidas na migração celular, como actina. Em conjunto, os resultado obtidos na presente tese demonstram duas formas racionais de desenvolver anticorpos contra cPKCs ativas, sendo que algumas aplicações para estas ferramentas foram demonstradas. Estratégias baseadas em mudanças conformacionais, similares às apresentadas aqui, poderão ser utilizadas para a produção racional de anticorpos contra outras quinases ou proteínas / The protein kinase C family (PKC) is composed of ten isoenzymes, which are capable of phosphorylating serine and threonine amino acid residues. PKC activation involves conformational changes, such as removing the pseudo-substrate from the active site and binding of the enzyme to lipids in biological membranes. In addition, PKC undergoes three phosphorylations that are important for the maturation/ folding of the enzyme and are not linked with activation status. Despite the fact that these kinases are involved in various pathological processes, such as carcinogenesis and cardiovascular disease, a relationship between PKC activation status with these diseases has not yet been established. This is partly due to the lack of tools to detect active PKC in tissue samples. In this thesis, based on conformational changes suffered by PKC during its activation, two antibodies against active cPKCs were rationally developed; a polyclonal antibody (anti-C2Cat) and a monoclonal (4.8E). Anti-C2Cat was produced after immunization of rabbits with a peptide located at the interface between the C2 and catalytic domains of cPKCs in an inactive PKC. The monoclonal antibody 4.8E was produced after immunization of Balb/C mice with total lysates from HEK293T cells overexpressing constitutively active forms of PKCβI. The anti-C2Cat and 4.8E specificity by active cPKCs was demonstrated by ELISA and immunoprecipitation assays, where the antibodies always showed higher affinity to active cPKCs. Anti-C2Cat was able to detect the temporal and spatial dynamics of cPKC activation upon receptor (morphine, ATP or glutamate) or phorbol ester stimulation in neuroblastoma lines (Neuro-2A and SK-N-SH). Futhermore, anti-C2Cat is able to detect active PKC in human tissues. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Also, both antibodies were applied to study signaling pathways that lead to carcinogenesis in MDA-MB-231 cells by performing co-immunoprecipitation and mass spectrometry. Using this approach, the results suggest that active cPKCs may be involved in translation of proteins involved in cell migration, such as actin. Taken together, the results obtained in this thesis showed two rational ways to develop antibodies against active cPKCs and some applications for these tools were demonstrated. Strategies based on conformational changes, similar to those presented herein may be used for rational production of antibodies against other kinases and proteins.

The insertion of ω- amino acid residues as guests into host α-peptide sequences permits expansion of the range of polypeptide secondary structures. The term ω- amino acid is used to refer to the entire family of residues generated by homologation of the backbone of α - amino acid residues. This explores the consequences of insertion of substituted β-residues (β3) , unsubstituted β-residues , unsubstituted γ-residues (gamma aminobutyric acid) and unsubstituted δ-residues (delta aminovaleric acid) into host α -peptide sequences. Chapter 1 provides an introduction to the conformational properties of β-peptides and reviews current literature on the structural features of peptides containing ω-amino acid residues. The available crystallographic information is summarized. The conformational properties of β- residues may be described by three degrees of torsional freedom : φ (N – Cβ) , θ (Cβ -Cα) and ψ (Cα-CO). Similarly, the conformational properties of γ -residues is based on four torsional parameters ( φ , θ1 , θ2, ψ) and the conformational properties of δ - residues is based on five degrees of freedom ( φ , θ1 , θ2, θ3,ψ). The rational use of β -residues in peptide design requires an understanding of the nature of local conformations, which are readily accessible. The conformational space for β -residues can be represented in a three dimensional plot. The observed distribution of φ , θ and ψ values for β -residues in peptide crystal structures presented in this section permits a correlation - between the torsion angle θ and the secondary structure context. The gauche (g+ and g ) conformations induce helical folding and the trans conformation is generally observed in the strands of a hairpin. The most striking feature of hybrid sequences is the observation of novel hydrogen bonded rings in peptide structures. Chapter 2 describes the effects of insertion of β-residues into specific positions in the strand segments of designed peptide hairpins. Insertion of β -residues into the strands of a hairpin changes the orientation of peptide bonds, resulting in a “polar sheet” arrangement. The conformational analysis of three designed peptide hairpins composed of α/β - hybrid segments are described: Boc-Leu-βPhe-Val-DPro-Gly- Leu-βPhe-Val-OMe (BBH8) , Boc-βLeu- Phe-βVal-DPro-Gly-βLeu-Phe-βVal-OMe (BAB8) and CF3COO-H3N+-Leu-Val-Val-βPhe-DPro-Gly-βPhe-Leu-Val-Val-OMe (BHFF10). All the peptides have been characterized by 500 MHz 1H-NMR spectroscopy and several crucial long range NOEs confirm a predominant population of β-hairpin conformations in CD3OH. X-ray diffraction studies on single crystals of peptide BBH8 reveal a β-hairpin conformation, stabilized by three cross-strand hydrogen bonds and a Type II′β-turn at the DPro-Gly turn segment. Designed β-hairpin peptide scaffolds may be used to probe cross-strand sidechain interactions in β-sheet structures. A previously reported peptide β-hairpin, Boc-Leu-Phe-Val-DPro-Gly-Leu-Phe-Val-OMe exhibited an anomalous far UV CD spectrum, which was interpreted in terms of interactions between facing aromatic chromophores, Phe 2 and Phe 7 (Zhao, C.; Polavarapu, P.L.; Das,C. and Balaram, P. J. Am. Chem. Soc., 2000, 122, 8228-8231). In BBH8 and BHFF10 the two cross-strand βPhe residues are at non-hydrogen bonding positions, with the benzyl sidechains pointing on opposite faces of the β- sheet. BBH8 yields a “hairpin –like” CD spectrum, with a minimum at 224 nm. The CD spectrum of BAB8 reveals a negative band at 234 nm and a positive band at 221 nm suggestive of an exciton split doublet. BHFF10 yields a “hairpine-like” CD spectrum, with a negative band at 220 nm. Chapter 3 describes the synthesis and conformational characterization of three hybrid decapeptides : Boc-Leu-Val-βGly-Val-DPro-Gly- Leu-βGly -Val-Val-OMe (BHB10), Boc-Leu-Val-γAbu-Val-DPro-Gly- Leu-γAbu -Val-Val-OMe (BHC10) and Boc- Leu-Val-δAva-Val-DPro-Gly- Leu-δAva -Val-Val-OMe (BHD10). These peptides were designed to systematically investigate the effect of insertion of additional methylene groups into the strands of a hairpin. The incorporation of additional carbon atoms changes the local polarity of the strands. 500 MHz NMR studies establish that BHB10 and BHD10 adopt predominantly β- hairpin conformations in methanol, with interstrand registry established by observation of long range NOEs. The observation of both DPro 4 (CαH) ↔ Gly 5 (NH) and Gly 5 (NH) ↔ Leu 6 (NH) NOEs provides evidence for a Type II ′β - turn for all the hairpins. In BHC10, no long range NOEs were observed. However, X-ray diffraction studies in single crystals reveal a β- hairpin conformation, nucleated by a DPro-Gly Type II′β-turn. Chapter 4 describes an attempt to incorporate one or two ω amino acid residues in the turn region of a potential hairpin, in order to assess the effect of expansion of the nucleating turn. The DPro-LPro segment has been shown to stabilize β-hairpin conformations in both cyclic (Shankaramma,S.C.; Moehle, K. ; James, S.; Vrijbloed, J.W.; Obrecht,D and Robinson, J.A. Chem Commun. 2003,1842-1843) and acyclic sequences ( Raj Kishore Rai ; S.Raghothama and P. Balaram , unpublished results) . In the present study the following turn segments have been considered: βDPro -αLPro , βLPro -αLPro and βLPro -αDPro. The synthesis and conformational analysis of three octapeptide sequences -Boc-Leu-Phe-Val-βDPro-αLPro-Leu-Phe-Val-OMe (βDPαLP8), Boc-Leu-Phe-Val-βLPro-αLPro-Leu-Phe-Val-OMe (βLPαLP8)and Boc-Leu-Phe-Val-βLPro-αDPro-Leu-Phe-Val-OMe (βLPαDP8) are described. In the βDPro-αLPro peptide, NMR evidence clearly supports a β-hairpin conformation, with a nucleating hybrid βα turn stabilized by a C11 (4 →1) hydrogen bond. In the other two octapeptides, no evidence for folded structures was obtained. These results suggest that nucleating turn formation is facilitated only in the heterochiral βD-αL case. Further expansion of the turn segment in potential hairpins has been investigated by inserting two contiguous β-residues into the center of a host α-peptide sequence. The conformational studies on two synthetic hexapeptides, Boc-Leu-Phe-βDPhe-βLPro-Phe-Leu-OMe (βDFβLP6) and Boc-Leu-Phe-βLPhe-βLPro-Phe-Leu-OMe (βLFβLP6) suggest that the βDPhe-βLPro segment is capable of forming a C12 turn in methanol. Two octapeptide sequences, Boc-Leu-Val-Leu-βDPhe-βLPro-Leu-Phe-Val-OMe (βDFβLP8N) and Boc-Leu-Val-Val-βDPhe-βLPro-Leu-Val-Val-OMe (βDFβLP8V) have also been investigated to probe the possible formation of hairpin structures. In these cases, spectroscopic analysis is hampered by the presence of multiple conformations, because of the tendency of the βDPhe-βLPro bond to exist in both cis and trans conformations. NMR studies on the conformational properties of a hexapeptide Boc-Leu-Val-βDPro-βLPro-Leu-Phe-OMe (βDPβLP6) in CDCl3 reveal that in the major conformer the Val 2(NH) ↔ Leu 5 (NH) NOE is observed, suggesting the presence of a 12-membered hydrogen bonded turn. A ββ - segment can give rise to two types of hydrogen bonded rings , 10 – membered (C10) and 12- membered (C12). In an attempt to generate C10 turns, an N-methylamino acid has been inserted next to a ββ - segment, preventing the formation of the 12 – membered turn. In such a situation formation of a 10-membered turn, with reverse hydrogen bond directionality, may be facilitated. The conformational properties of Boc-Leu-Val-βDPhe-βLPro-(N-Me) Leu- Phe-OMe (βDFβLPNMeL6) has been studied by 500 MHz NMR spectroscopy. The data suggests the formation of a C11 turn at the βLPro- (N-Me) Leu segment in CDCl3-DMSO mixtures, instead of formation of a C10 turn at the βDPhe -βLPro segment. Studies on the peptide Boc-Leu-Phe-Val-βLPro-(N-Me) Leu-Leu-Phe-Val-OMe (βLPNMeL8) also suggest the absence of turn formation and folded structures. In hybrid sequences, an important question to be addressed is whether ω amino acids can be accommodated into helical structures. Two contiguous β- residues have been inserted into a helical sequence. The conformational properties of a 11- residue peptide, Boc-Val-Ala-Phe-Aib-βVal-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA11) are described in Chapter 5. This sequence was based on the parent α- peptide Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe, which adopted a complete helical conformation in crystals (Aravinda, S.; Shamala, N.; Das, C .; Sriranjini, A.; Karle, I.L. and Balaram, P. J. Am. Chem. Soc. 2003, 125, 5308-5315). 500 MHz 1H-NMR studies establish a continuous helix over the entire length of the peptide in CDCl3 solution , as evidenced by diagnostic nuclear Overhauser effects. The molecular conformation in crystals reveals a continuous helical fold, stabilized by seven intramolecular hydrogen bonds. The characterization of two synthetic octapeptides Boc-Val-Ala-βPhe-Aib-Val-Ala-βPhe-Aib-OMe (VAβFU8) (βPhe residues have been incorporated at (i /i+4 positions) and Boc-Val-Ala-βPhe-Aib-βPhe-Ala-Val-Aib-OMe (βFUβF8) (βPhe residues have been incorporated at (i /i+2 positions) is also presented. NMR data suggests the retention of helical conformation in both the peptides. In order to delineate the conformations of hybrid peptides with three contiguous β-residues, two peptides have been synthesized Boc-Phe-Aib-βGly-βLeu-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA10) and Boc-Val-Ala-Phe-Aib-βGly-βLeu-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA12). NMR studies in chloroform support continuous helical conformation in the decapeptide.

A família proteína quinases C (PKC) é composta por dez isoenzimas, as quais são capazes de fosforilar resíduos de serina e treonina. A ativação dessas quinases envolve mudanças conformacionais, como a remoção do pseudo-substrato do sítio ativo e associação dessas enzimas com lipídeos em membranas biológicas. Além disso, três fosforilações são importantes para a maturação/ enovelamento da enzima e não estão associadas com o estado de ativação das cPKCs. Apesar dessas quinases estarem envolvidas em vários processos patológicos, como carcinogênese e doenças cardiovasculares, ainda não se estabeleceu a relação entre estado de ativação das PKCs com essas doenças. Isso se deve, em parte, à ausência de ferramentas que possibilitam a distinção das formas ativas e inativas das PKCs. Na presente tese, baseando-se em mudanças conformacionais sofridas pelas PKCs durante o processo de ativação, dois anticorpos contra cPKCs ativas foram racionalmente desenvolvidos, sendo um anticorpo policlonal (anti-C2Cat) e outro monoclonal (4.8E). O anticorpo anti-C2Cat foi desenvolvido a partir de imunização de coelhos com um peptídeo localizado na região de interação entre os domínios C2 e catalítico na PKC inativa. Já o anticorpo monoclonal 4.8E foi produzido após a imunização de camundongos Balb/ C com extrato de proteínas proveniente de células HEK293T superexpressando formas constitutivamente ativas da PKCβI. A seletividade de anti-C2Cat e 4.8E por cPKCs ativas foi demonstrada por ensaios de ELISA e de imunoprecipitação, sendo que os anticorpos sempre apresentaram maior afinidade por cPKCs ativas purificadas, superexpressas ou mesmo as endógenas. O anticorpo anti-C2Cat foi capaz de monitorar a dinâmica espaço-temporal da ativação das cPKCs em linhagens de neuroblastoma (Neuro-2A e SK-N-SH) estimuladas com PMA, morfina, ATP ou glutamato por diferentes tempos. Ainda, um maior conteúdo de cPKCs ativas foi detectado por anti-C2Cat na linhagem de câncer de mama MDA-MB-231 (triplo- negativa) do que em células MCF-7 (ER+). Em acordo com esses dados, anti-C2Cat identificou uma maior ativação de cPKCs em tumores mais agressivos de câncer de mama (subtipo triplo-negativo) do que em tumores menos agressivos (ER+, subtipo luminal). Os anticorpos conformacionais anti-C2Cat e 4.8E foram aplicados para elucidar vias de sinalização que levam à carcinogênese em células MDA-MB-231, por meio da realização de ensaios de co-imunoprecipitação, seguida pela identificação das proteínas por espectrometria de massas. Usando essa abordagem, os resultados sugerem que as cPKCs ativas possam estar envolvidas com a tradução de proteínas envolvidas na migração celular, como actina. Em conjunto, os resultado obtidos na presente tese demonstram duas formas racionais de desenvolver anticorpos contra cPKCs ativas, sendo que algumas aplicações para estas ferramentas foram demonstradas. Estratégias baseadas em mudanças conformacionais, similares às apresentadas aqui, poderão ser utilizadas para a produção racional de anticorpos contra outras quinases ou proteínas / The protein kinase C family (PKC) is composed of ten isoenzymes, which are capable of phosphorylating serine and threonine amino acid residues. PKC activation involves conformational changes, such as removing the pseudo-substrate from the active site and binding of the enzyme to lipids in biological membranes. In addition, PKC undergoes three phosphorylations that are important for the maturation/ folding of the enzyme and are not linked with activation status. Despite the fact that these kinases are involved in various pathological processes, such as carcinogenesis and cardiovascular disease, a relationship between PKC activation status with these diseases has not yet been established. This is partly due to the lack of tools to detect active PKC in tissue samples. In this thesis, based on conformational changes suffered by PKC during its activation, two antibodies against active cPKCs were rationally developed; a polyclonal antibody (anti-C2Cat) and a monoclonal (4.8E). Anti-C2Cat was produced after immunization of rabbits with a peptide located at the interface between the C2 and catalytic domains of cPKCs in an inactive PKC. The monoclonal antibody 4.8E was produced after immunization of Balb/C mice with total lysates from HEK293T cells overexpressing constitutively active forms of PKCβI. The anti-C2Cat and 4.8E specificity by active cPKCs was demonstrated by ELISA and immunoprecipitation assays, where the antibodies always showed higher affinity to active cPKCs. Anti-C2Cat was able to detect the temporal and spatial dynamics of cPKC activation upon receptor (morphine, ATP or glutamate) or phorbol ester stimulation in neuroblastoma lines (Neuro-2A and SK-N-SH). Futhermore, anti-C2Cat is able to detect active PKC in human tissues. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Also, both antibodies were applied to study signaling pathways that lead to carcinogenesis in MDA-MB-231 cells by performing co-immunoprecipitation and mass spectrometry. Using this approach, the results suggest that active cPKCs may be involved in translation of proteins involved in cell migration, such as actin. Taken together, the results obtained in this thesis showed two rational ways to develop antibodies against active cPKCs and some applications for these tools were demonstrated. Strategies based on conformational changes, similar to those presented herein may be used for rational production of antibodies against other kinases and proteins.

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / Jianhan Chen / Intrinsically disordered proteins (IDPs) are functional proteins that lack stable tertiary structures under physiological conditions. IDPs are key components of regulatory networks that dictate various aspects of cellular decision-making, and are over-represented in major disease pathways. For example, about 30% of eukaryotic proteins contain intrinsic disordered regions, and over 70% of cancer-associated proteins have been identified as IDPs. The highly heterogeneous nature of IDPs has presented significant challenge for experimental characterization using NMR, X-ray crystallography, or FRET. These challenges represent a unique opportunity for molecular mod- eling to make critical contributions. In this study, computer simulations at multiple scales were utilized to characterize the structural properties of unbound IDPs as well as to obtain a mechanistic understanding of IDP interactions. These studies of IDPs also reveal significant limitations in the current simulation methodology. In particular, successful simulations of biomolecules not only require accurate molecular models, but also depend on the ability to sufficiently sample the com- plex conformational space. By designing a realistic yet computationally tractable coarse-grained protein model, we demonstrated that the popular temperature replica exchange enhanced sampling is ineffective in driving faster reversible folding transitions for proteins. The second original contribution of this dissertation is the development of novel simulation methods for enhanced sampling of protein conformations, specifically, replica exchange with guided-annealing (RE-GA) method and multiscale enhanced sampling (MSES) method. We expect these methods to be highly useful in generating converged conformational ensembles.

Doctor of Philosophy / Department of Chemical Engineering / Peter H. Pfromm / One area of considerable importance in modern biotechnology is the preparation of highly active and selective enzyme based biocatalysts for applications in organic solvents. A major challenge is posed by the tendency of enzymes to cluster when suspended in organic solvents. Because the clusters obstruct the transport of substrates to the active site of the enzyme, the observed activity is often severely reduced. Over the past two decades, many strategies have been proposed to mitigate this problem. We have tackled this major hurdle by devising an immobilization strategy that utilizes fumed silica as carrier for the enzyme molecules. Fumed silica is a non-porous nanoparticulated fractal aggregate with unique absorptive properties. The enzyme/fumed silica preparation is formed in two steps. The buffered enzyme molecules are physically adsorbed on the fumed silica and then lyophilized. This protocol was shown to be successful with two enzymes of industrial relevance, Candida antarctica Lipase B (CALB) and subtilisin Carlsberg. The maximum observed catalytic activity in hexane reached or even exceeded commercial immobilizates and nonbuffer salt based preparations. The results demonstrated that catalytic activity has an intricate relationship with the nominal surface coverage (%SC) of the support by the enzyme molecules. s. Carlsberg exhibited an ever increasing activity as more surface area was provided per enzyme molecule. The activity leveled off when a sparse surface population was reached. CALB showed a maximum in catalytic activity at an intermediate surface coverage with steep decreases at both lower and higher surface coverage. It was shown that this maximum results from the presence of three distinct surface loading regimes after lyophilization: 1. a low surface coverage where opportunities for multi-attachment to the surface likely lead to detrimental conformational changes, 2. an intermediate surface coverage where interactions with neighboring proteins and the surface help to maintain a higher population of catalytically competent enzyme molecules, and 3. a multi-layer coverage where mass transfer limitations lead to a decrease in the apparent catalytic activity. Conformational stability analyses with both fluorescence and CD spectroscopy showed evidence that these regimes are most likely formed during the adsorption step of our protocol. A low conformational stability region was detected at low surface coverage while adsorbates with highly stable enzyme ensembles were observed at high surface coverage. Secondary structural analysis of the lyophilized nanobiocatalysts with FTIR confirmed a substantial decrease in the alpha-helical components at low surface coverage. In summary, the work presented here traces the phenomenological observation of the catalytic behavior of a nanobiocatalyst to molecular-level: enzyme-enzyme and enzyme-support interactions, which are specific to the intricate properties of the enzyme molecules.