Integrated functional-site feature analysis, with application to peroxiredoxin and other redoxin proteins

In collaboration with Professor Leslie Poole (WFUBMC, Biochemistry) and Professor Freddie R. Salsbury Jr. (WFU, Physics); Funded by the NSF

Sequence genomics projects have produced many methods for predicting protein function based on sequence motifs, pairwise sequence alignment, or multiple sequence alignment clustering, providing information on molecular function but not insight into biological mechanism. Structural genomics projects are beginning to supply the data necessary to make general observations about biological mechanism; however, such efforts are hampered by inadequate automated processes to assemble and to characterize such data across protein families and superfamilies. We are working to cross the gap from molecular function to biological mechanism by developing and using computational sequence, structure, bioinformatics and biophysical methods to characterize the molecular function sites of six superfamilies. We use these methods to classify these superfamilies, and compare our classifications to known biological features and mechanisms, allowing progressive improvement in computational approaches and our understanding of the underlying structure/function relationships. We aim to characterize electrostatics, structure, and sequence features of each functional site, which should correlate with mechanism better than current clustering approaches based on global sequence alignment. Our long term goal is to develop an integrated method for describing functional sites that uniquely combines physics, chemistry, and structural information and to use this approach to characterize the general principles that underlie biological mechanism. This detailed analysis will yield insights into biological mechanisms, yielding hypotheses that can be experimentally tested, and ultimately will enable better methods for identifying functional sites from sequence information, thus allowing more accurate identification and classification of many protein functions. The development of these general concepts will allow for the modification of enzymes (to improve or alter their activity) and the design of enzyme inhibitors (lead compounds), an early step in pharmaceutical drug discovery.

Relevant references:

• Fetrow, J. S. Active site profiling to identify protein functional sites in sequences and structures. Current Protocols in Bioinformatics. 2006 In press.
• Huff, R. G., Bayram, E., Tan, H., Knutson, S.T., Knaggs, M.H., Richon, A.B., Santago II, P., and Fetrow, J.S. Chemical and Structural Diversity in Cyclooxygenase Protein Active Sites. Chemistry and Biodiversity. 2005; 2:1533-1552.
• Baxter, S.M., Rosenblum, J.S., Knutson, S.T., Nelson, M.R., Montimurro, J.S., Di Gennaro, J.A., Speir, J.A., Burbaum, J.J. and Fetrow, J.S. Synergistic computational and experimental proteomics approaches for more accurate detection of active serine hydrolases in yeast. Mol. Cell. Proteomics. 2004 Mar; 3(3):209-25.
• Cammer, S.A., Hoffman, B.T., Speir, J.A., Canady, M., Nelson, M.R., Knutson, S.T., Gallina, M., Baxter, S.M., and Fetrow, J.S. Structure-based active site profiles for genome analysis and sub-family classification. J. Mol. Biol. 2003; 334(3):387-401.
• Fetrow, J.S. and Skolnick, J. Method for prediction of protein function from sequence using the sequence-to-structure-to-function paradigm with application to glutaredoxins/thioredoxins and T1 ribonucleases. J. Mol. Biol. 1998 Sep 4; 281(5):949-968.

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Redox signal transduction and modeling of biological networks

In collaboration with Professors David John, Edward Allen, Stan Thomas and William Turkett (WFU); and Leslie Poole, Larry Daniel and Richard Loeser (WFUBMC); Funded by the NIH

Redox-regulated events are fundamental to many cellular pathways and disease processes, including those that cause cancer. Increasing evidence points to a major role for reactive oxygen species (ROS, small molecules involved in redox events) in signal transduction pathways; however, a number of important questions remain unanswered. Answers to these would pave the way for a systems biology understanding of redox signaling networks and the effect of ROS in normal and diseased cells. Until now, proteins in which ROS cause a covalent modification have been studied one at a time. Tools to identify these pathways on a proteomic scale have not been available; thus, the questions posed were not answerable, and a systems biology approach to understanding redox signaling was impossible.

Reactive cysteinyl residues at regulatory or catalytic sites within enzymes and transcriptional regulators are central to redox signaling networks. Co-investigator Poole has recently shown that these residues are converted first to cysteine sulfenic acids (Cys-SOH), then either re-reduced directly or further modified to form disulfide bonds or other oxidized species. She has developed reagents that react rapidly with Cys-SOH-containing proteins in the cell, allowing for a more comprehensive identification of proteins involved in cellular redox events than would disulfide bond detection alone. With these tools in hand, we have a unique opportunity to characterize the proteins involved in redox signaling on a proteomic scale and to develop novel computational methods for modeling these networks. We aim 1) to identify the components and biological outcomes of redox signaling networks that are modulated in cells exposed to various stimuli; and 2) to produce mathematical models that will reveal both the topology and dynamics of these pathways and to integrate these models with networks produced by other methods.

Relevant references:

• Allen, E.E., Fetrow, J.S., John, D.J., Pecorella A. and Turkett, W. Re-constructing networks using co-temporal functions. Proceedings of the 44th ACM Southeast Conference, (Marius Silaghi, ed), Melbourne, Florida. March 2006, 417-422.
• Allen, E.E., Fetrow, J.S., Daniel, L.W., Thomas, S.J., John, D.J. Algebraic dependency models of protein signal transduction networks from time-series data. J. Theor. Biol. 2006 Jan 21; 238(2):317-30. [Epub 2005 Jul 5]
• Allen, E.E., Fetrow, J.S., John, D.J., Thomas, S.J. Heuristic dependency conjectures in proteomic signaling pathways. Proceedings of the 43rd ACM Southeast Conference, (Victor A. Clincy, ed.), Kennesaw, Georgia, March 2005, 75-79.

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Experimental and computational analysis of the interaction networks in proteins

In collaboration with Professors Freddie R. Salsbury Jr. (WFU) and Marshall Hale Edgell (UNC-Chapel Hill)

Nonadditive effects (in which the sum of the free energy changes resulting from two single mutations do not equal the measured free energy change for the double mutant) are are common in proteins. They are the basis of a crucial functional feature of proteins, allostery, and are also associated with site pairs that are not involved in allostery. The physical basis for nonadditive effects is poorly understood and our predictive capacity, in either qualitative or quantitative terms, is marginal at best. Current generalizations are based on the analysis of a modest number of site pairs and a small number of mutations at those sites. We will develop new generalizations about the interaction network in proteins by doing thermodynamic cycle measurements with several thousand mutant proteins. This will be accomplished using previously developed high throughput mutagenesis techniques and high precision stability measurements. Another objective of this project is to identify parameters extractable from conformational ensembles generated by molecular dynamics simulations that correlate qualitatively and quantitatively with the equilibrium thermodynamic measurements. Extensive correlations between thermodynamic measurements and computer simulation parameters will be a significant step towards a capacity to predict features of the interaction network.

Relevant references:

• Knaggs, M.H., Salsbury, F.R., Edgell, M.H., Fetrow, J.S. Insights into CheY relaxation and relaxation derived from molecular dynamics simulations. Biophys. J. [Epub ahead of print 2006 Dec 15]
• Fetrow, J.S., Knutson, S.T. and Edgell, M.H. Mutations in α-helical solvent exposed sites of eglin c have long-range effects: evidence from molecular dynamics simulations. Proteins: Struct Funct Bioinform. 2006 May 1; 63(2):356-72. [Epub 2005 Dec 9]

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Integrated database of annotated protein structures

The functional and structural elucidation of proteins of unknown function is an essential challenge that spans all areas of biology. We have recently shown that the integration of de novo structure prediction with multiple sources of weak functional information can result in large numbers of functional annotations, not available via sequence based methods. We are developing a database that will present function derived from de novo structure prediction, with functional interpretation of the structures carried out automatically via the matching of the predicted structures to libraries of active site, functional site and fold descriptors. We are integrating these novel structure-derived data with several other sources of annotation (such as ontologies containing pathway, process or localization information) and systems-biology data (such as proteomics and microarray data) to present a comprehensive meta-database for exploring the function of proteins of unknown function/structure. From the perspective of modular implementation, the system will consist of three major parts: 1) the generation of structure predictions and domain organization annotations; 2) extraction of structure derived function annotation and integration with preexisting process, localization and non-specific functional information; and 3) development of the database system wtih graphical access to the database via connections to other analysis tools.

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Fuzzy Functional Forms (FFFs): identification of functional sites in proteins

In collaboration with Professor Jeffrey Skolnick (SUNY Buffalo)

With the flood of data coming from the sequence and structural genomics projects, new methods are needed to better identify functional sites in proteins. Fuzzy Functional Forms (FFFs) are structural descriptors that can be used to identify functional sites in protein structures. FFFs can be applied to structures produced from experimental data or lower quality structures created by computational methods. FFFs have been shown to identify functional sites more accurately than sequence alignment or sequence motif methods.

Relevant references:

• Fetrow, J.S., Siew, N., and Skolnick, J. Structure-based functional motif identifies a potential disulfide oxidoreductase active site in the serine-threonine protein phosphatase-1 subfamily. FASEB J. 1999; Oct;13(13):1866-1874.
• Zhang, L., Godzik, A., Skolnick, J., Fetrow, J. Functional analysis of Escherichia coli proteins for members of the a/b hydrolase family. Fold Des. 1998; 3(6):535-548.
• Fetrow, J., Godzik, A. and Skolnick, J. Functional analysis of the Escherichia coli genome using the sequence-to-structure-to-function paradigm: Identification of proteins exhibiting the glutaredoxin/thioredoxin disulfide oxidoreductase activity. J. Mol. Biol. 1998 Oct 2; 282(4):703-711.
• Fetrow, J.S. and Skolnick, J. Method for prediction of protein function from sequence using the sequence-to-structure-to-function paradigm with application to glutaredoxins/thioredoxins and T1 ribonucleases. J. Mol. Biol. 1998 Sep 4; 281(5):949-968.

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Classification and dynamics simulations of omega loops and and other protein loops

The regular secondary structures, alpha helices and beta strands, are easily recognizable in protein structures. The non-regular secondary structures, such as the various types of loops and turns, are less easily recognized, but no less important in the structure and function of proteins. Omega loops, a type of non-regular secondary structure first described in 1988, are segments of non-regular secondary protein structure that are six or more residues in length and are shaped so that the loop ends are close in three-dimensional space. Omega loops constitute approximately 20-23% of protein structure and have been recognized as playing a variety of roles in protein function. Additional loop types, including S-loops and strap loops, were described in the 1990s. These structures are almost always found at the protein surface and it is generally assumed that these non-regular structures are more flexible than other parts of the protein. However, in 1995, it was suggested that loops could be classified based on their roles in function, folding and stability. Recently, trigger loops were proposed to play a specific role in protein function. We hypothesize that loops playing these different roles will exhibit different dynamic characteristics. We are testing this hypothesis by performing simulations on proteins containing loops of various types that have been well-studied experimentally.

Relevant references:

• Fetrow, J.S., Schaak, D.L., Dreher, U., Wiland, D.J., and Boose, T.L. Mutagenesis of histidine 26 demonstrates the importance of loop-loop and loop-protein attachments for the function of iso-1-cytochrome c. Protein Sci. 1998; 27(4):994-1005.
• Fetrow, J.S., Horner, S.R., Oehrl,W., Schaak, D.L., Boose, T.L., and Burton, R.E. Analysis of the structure and stability of omega loop A replacements in yeast iso-1-cytochrome c. Protein Sci. 1997 Jan; 6(1):197-210.
• Mulligan-Pullyblank, P. Spitzer, J.S., Gilden, B.M., and Fetrow, J.S. Loop replacement and random mutagenesis of omega loop D, residues 70-84, in iso-1-cytochrome c. J. Biol. Chem. 1996 Apr 12; 271(15):8633-8645.
• Fetrow, J.S. Omega Loops: Nonregular secondary structures significant in protein function and stability. FASEB J. 1995 Jun; 9(9):708-717.
• Murphy, M.E.P., Fetrow, J.S., Burton, R.E. and Brayer, G.D. The structure and function of omega loop A replacements in cytochrome c. Protein Sci. 1993; 2(9):1429-1440.
• Fetrow, J.S., Cardillo, T.S., and Sherman, F. Deletions and replacements of omega loops in yeast iso-1-cytochrome c. Proteins. 1989; 6(4):372-381.
• Leszczynski (Fetrow), J.F. and Rose, G.D. Loops in globular proteins: Identification of a novel category of secondary structure. Science. 1986 Nov 14; 234(4778):849-55.

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Motion and dynamics in yeast iso-1-cytochrome c

Proteins are not static structures. Rather, they exhibit many different kinds of motions on a very wide range of time scales. To better understand these motions in a well-studied protein system, we have used NMR and EPR spectroscopy to study protein motion and dynamics in yeast iso-1-cytochrome c. Beginning with the first successful isotopic labeling of cytochrome c, we have studied the psec-nsec dynamics of the protein backbone using both hydrogen exchange and ¹⁵N relaxation measurements. Site-directed spin labeling of the cysteine in the C-terminal helix of iso-1-cytochrome c provides an indication of the flexibility of the C-terminus of the protein.

Relevant references:

• DeWeerd, K., Grigoryants, V., Sun, Y., Fetrow, J.S., Scholes, C.P. EPR-detected folding kinetics of externally located cysteine-directed spin-labeled mutants of iso-1-cytochrome c. Biochemistry. 2001 Dec 25; 40(51):15846-15855.
• Fetrow, J.S. and Baxter, S.M. Assignment of ¹⁵N chemical shifts and ¹⁵N relaxation measurements for oxidized and reduced iso-1-cytochrome c. Biochemistry. 1999 Apr 6; 38(14):4480-4492.
• Baxter, S.M. and Fetrow, J.S. Hydrogen exchange behavior of [U-¹⁵N]-labeled oxidized and reduced iso-1-cytochrome c. Biochemistry. 1999 Apr 6; 38(14):4493-4503.
• Baxter, S.M., Boose, T.L., and Fetrow, J.S. ¹⁵N isotopic labeling and amide hydrogen exchange rates of oxidized iso-1-cytochrome c. J. Am. Chem. Soc. 1998; 119(41):9899-9900.
• Qu, K. Vaughn, J.L., Sienkiewicz, A. Scholes, C.P., and Fetrow, J.S. Kinetics and motional dynamics of spin labeled yeast iso-1-cytochrome c: 1. Stopped-flow EPR as a probe for protein folding/unfolding of the C-terminal helix spin labeled at cysteine 102. Biochemistry. 1998; 36(10):2884-2897.

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Cytochrome c structure/function relationships

To better understand the role that omega loops play in proteins, we have developed methods of directed, random mutagenesis of yeast cytochrome c. Yeast cytochrome c can be analyzed for both function and structure in vivo, which makes it an ideal protein for analysis of structure-function relationships. Using directed, random mutagenesis, we mutagenized several pairs of residues in yeast cytochrome c to identify which residue pairs are consistent with structure and function of this protein in vivo.

Relevant references:

• Fetrow, J.S., Spitzer, J.S., Gilden, B.M., Mellender, S.J., Begley, T., Haas, B., and Boose, T.L. Structure, function, and temperature sensitivity analysis of directed, random mutants of proline 76 and glycine 77 in omega loop D of yeast iso-1-cytochrome c. Biochemistry.1998; 37(8):2477-2487.
• Fetrow, J.S., Schaak, D.L., Dreher, U., Wiland, D.J., and Boose, T.L. Mutagenesis of histidine 26 demonstrates the importance of loop-loop and loop-protein attachments for the function of iso-1-cytochrome c. Protein Sci. 1998; 27(4):994-1005.
• Fumo, G. and Fetrow, J.S. A method of directed random mutagenesis of the yeast chromosome shows that iso-1-cytochrome c heme ligand His18 is essential. Gene. 1995 Oct 16; 164(1):33-39.

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Structural Building Blocks (SBBs) and automatic identification of protein secondary structures

Automated methods for identification of secondary structure are necessary for large-scale analysis of protein structure. We developed a method for identification and classification of protein secondary structure, without previous knowledge of the types of secondary structures. This method utilizes artificial neural networks to classify and cluster segments of protein structure based on their geometry. Clustering of six-residue segments in a large group of protein structures results in the identification of six classes of secondary structures, which we term structural building blocks (SBBs). Two of these are the canonical alpha helix and beta strand structures, while two other SBBs coincide with N- and C-terminal helix capping structures.

Relevant references:

• Fetrow, J.S., Palumbo, M.J., and Berg, G. Patterns, structures, and amino acid frequencies in structural building blocks, a protein secondary structure classification scheme. Proteins. 1997 Feb; 27(2):249-71.
• Zhang, X., Fetrow, J.S., Rennie, W.A., Waltz, D.L., and Berg, G. Automatic derivation of substructures yields novel structural building blocks in globular proteins. (1993) Proceedings: First International Conference on Intelligent Systems for Molecular Biology. p. 438-446. L. Hunter, D. Searls, J. Shavlik, eds.

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