Sequence- and structure-based protein function prediction from genomic information.

Existing functional annotation transfer is fraught with inaccuracies that may hinder forward interpretation and mining of genomic data. Hand-curation of the annotation placed into databases is not practical. In lieu of experimental evidence, computational biological approaches offer high-throughput tools to predict function accurately; however, these methods are still notably deficient in defining and describing the complexity of protein function. Enriching genomic sequences obtained from sequencing efforts and expression array methods with protein function information and classification will be an efficient first step for incorporating genomic data into drug discovery programs.

Cooperative ordering in homeodomain-DNA recognition: solution structure and dynamics of the MATa1 homeodomain.

The mating type homeodomain proteins, MATa1 and MATalpha2, combine to form a heterodimer to bind DNA in diploid yeast cells. The a1-alpha2 heterodimer tightly and specifically binds haploid-specific gene operators to repress transcription. On its own, however, the a1 homeodomain does not bind DNA in a sequence-specific manner. To help understand this interaction, we describe the solution structure and backbone dynamics of the free a1 homeodomain. Free a1 in solution is an ensemble of structures having flexible hinges at the two turns in the small protein fold. Conformational changes in the a1 homeodomain upon ternary complex formation are located in the loop between helix 1 and helix 2, where the C-terminal tail of alpha2 binds to form the heterodimer, and at the C-terminus of helix 3, the DNA recognition helix. The observed differences, comparing the free and bound a1 structures, suggest a mechanism linking van der Waals stacking changes to the ordering of a final turn in the DNA-binding helix of a1. The tail of alpha2 induces changes in loop 1 of a1 that push it toward a properly folded DNA binding conformation.

Identification of the Archaeoglobus fulgidus endonuclease III DNA interaction surface using heteronuclear NMR methods.

BACKGROUND: Endonuclease III is the prototype for a family of DNA-repair enzymes that recognize and remove damaged and mismatched bases from DNA via cleavage of the N-glycosidic bond. Crystal structures for endonuclease III, which removes damaged pyrimidines, and MutY, which removes mismatched adenines, show a highly conserved structure. Although there are several models for DNA binding by this family of enzymes, no experimental structures with bound DNA exist for any member of the family. RESULTS: Nuclear magnetic resonance (NMR) spectroscopy chemical-shift perturbation of backbone nuclei (1H, 15N, 13CO) has been used to map the DNA-binding site on Archaeoglobus fulgidus endonuclease III. The experimentally determined interaction surface includes five structural elements: the helix-hairpin-helix (HhH) motif, the iron-sulfur cluster loop (FCL) motif, the pseudo helix-hairpin-helix motif, the helix B-helix C loop, and helix H. The elements form a continuous surface that spans the active site of the enzyme. CONCLUSIONS: The enzyme-DNA interaction surface for endonuclease III contains five elements of the protein structure and suggests that DNA damage recognition may require several specific interactions between the enzyme and the DNA substrate. Because the target DNA used in this study contained a generic apurinic/apyrimidinic (AP) site, the binding interactions we observed for A. fulgidus endonuclease III should apply to all members of the endonuclease III family and several interactions could apply to the endonuclease III/AlkA (3-methyladenine DNA glycosylase) superfamily.

Configuration of the catalytic GIY-YIG domain of intron endonuclease I-TevI: coincidence of computational and molecular findings.

I-TevI is a member of the GIY-YIG family of homing endonucleases. It is folded into two structural and functional domains, an N-terminal catalytic domain and a C-terminal DNA-binding domain, separated by a flexible linker. In this study we have used genetic analyses, computational sequence analysis andNMR spectroscopy to define the configuration of theN-terminal domain and its relationship to the flexible linker. The catalytic domain is an alpha/beta structure contained within the first 92 amino acids of the 245-amino acid protein followed by an unstructured linker. Remarkably, this structured domain corresponds precisely to the GIY-YIG module defined by sequence comparisons of 57 proteins including more than 30 newly reported members of the family. Although much of the unstructured linker is not essential for activity, residues 93-116 are required, raising the possibility that this region may adopt an alternate conformation upon DNA binding. Two invariant residues of the GIY-YIG module, Arg27 and Glu75, located in alpha-helices, have properties of catalytic residues. Furthermore, the GIY-YIG sequence elements for which the module is named form part of a three-stranded antiparallel beta-sheet that is important for I-TevI structure and function.

Assignment of 15N chemical shifts and 15N relaxation measurements for oxidized and reduced iso-1-cytochrome c.

A protocol for complete isotopic labeling of iso-1-cytochrome c from the eukaryote Saccharomyces cerevisiae is reported. Assignments are reported for the vast majority of the 15N amide resonances in both oxidized and reduced states. 15N heteronuclear relaxation experiments were collected to study the picosecond-nanosecond backbone dynamics of this protein. Relaxation rates were computed and fit to spectral density functions by a model-free analysis. Backbone amides in the overlapping loop B/C region are the most flexible on the picosecond-nanosecond time scale in both forms of the protein. The results show that, on average, the protein backbone is slightly more dynamic in the oxidized than the reduced state, though not significantly so. Exchange terms, which suggest significant motion on a time scale at least an order of magnitude slower than the overall correlation time of 5.2 ns, were required for only two residues in the reduced state and 27 residues in the oxidized state. When analyzed on a per-residue basis, the lower order parameters found in the oxidized state were scattered throughout the protein, with a few continuous segments found in loop C and the C-terminal helix, suggesting greater flexibility of these regions in the oxidized state. The results provide dynamic interpretations for previously presented structural and functional data, including redox-dependent changes that occur in the protein. The way is now paved for extensive dynamic analysis of variant cytochromes c.

Hydrogen exchange behavior of [U-15N]-labeled oxidized and reduced iso-1-cytochrome c.

Heteronuclear NMR spectroscopy was used to measure the hydrogen-deuterium exchange rates of backbone amide hydrogens in both oxidized and reduced [U-15N]iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. The exchange data confirm previously reported data [Marmorino et al. (1993) Protein Sci. 2, 1966-1974], resolve several inconsistencies, and provide more thorough coverage of exchange rates throughout the cytochrome c protein in both oxidation states. Combining the data previously collected on unlabeled C102T with the current data collected on [U-15N]C102T, exchange rates for 53 protons in the oxidized state and 52 protons in the reduced state can now be reported. Most significantly, hydrogen exchange measurements on [U-15N]iso-1-cytochrome c allowed the observation of exchange behavior of the secondary structures, such as large loops, that are not extensively hydrogen-bonded. For the helices, the most slowly exchanging protons are found in the middle of the helix, with more rapidly exchanging protons at the helix ends. The observation for the Omega-loops in cytochrome c is just the opposite. In the loops, the ends contain the most slowly exchanging protons and the loop middles allow more rapid exchange. This is found to be true in cytochrome c loops, even though the loop ends are not attached to any regular secondary structures. Some of the exchange data are strikingly inconsistent with data collected on the C102S variant at a different pH, which suggests pH-dependent dynamic differences in the protein structure. This new hydrogen exchange data for loop residues could have implications for the substructure model of eukaryotic cytochrome c folding. Isotopic labeling of variant forms of cytochrome c can now be used to answer many questions about the structure and folding of this model protein.

High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae a1 homeodomain expressed in Escherichia coli.

The Saccharomyces cerevisiae a1 homeodomain is expressed as a soluble protein in Escherichia coli when cultured in minimal medium. Nuclear magnetic resonance (NMR) spectra of previously prepared a1 homeodomain samples contained a subset of doubled and broadened resonances. Mass spectroscopic and NMR analysis demonstrates that the heterogeneity is largely due to a lysine misincorporation at the arginine (Arg) 115 site. Arg 115 is coded by the 5'-AGA-3' sequence, which is quite rare in E. coli genes. Lower level mistranslation at three other rare arginine codons also occurs. The percentage of lysine for arginine misincorporation in a1 homeodomain production is dependent on media composition. The dnaY gene, which encodes the rare 5'-AGA-3' tRNA(ARG), was co-expressed in E. coli with the a1-encoding plasmid to produce a homogeneous recombinant a1 homeodomain. Co-expression of the dnaY gene completely blocks mistranslation of arginine to lysine during a1 overexpression in minimal media, and homogeneous protein is produced.

Heterodimerization of the yeast homeodomain transcriptional regulators alpha 2 and a1: secondary structure determination of the a1 homeodomain and changes produced by alpha 2 interactions.

The homeodomain proteins, a1 and alpha 2, act cooperatively to regulate cell-type specific genes in yeast. The basis of this cooperativity is an interaction between the two proteins, forming a heterodimer that binds DNA tightly and specifically. A fragment containing the homeodomain of a1, a1(66-126), has been studied by NMR spectroscopy to gain secondary structure information and to characterize the changes in a1 upon heterodimerization with alpha 2. Heteronuclear (1H-15N) NMR methods were used to assign backbone resonances of the 61 amino acid fragment. The a1(66-126) secondary structure was determined using NOE connectivities, 3JHN alpha coupling constants and hydrogen exchange kinetic data. NMR data identify three helical segments separated by a loop and a tight turn that are the characteristic structural elements of homeodomain proteins. The a1 fragment was titrated with alpha 2(128-210), the homeodomain-containing fragment of alpha 2, to study changes in a1(66-126) spectra produced by alpha 2 binding. The a1(66-126) protein was labeled with 15N and selectively observed using isotope-edited NMR experiments. NMR spectra of bound a1(66-126) indicate that residues in helix 1, helix 2, and the loop connecting them are directly involved in the binding of the alpha 2 fragment. Relatively minor effects on the resonances from residues in helix 3, the putative DNA-binding helix, were noted upon alpha 2 binding. We have thus located a region of the a1 homeodomain important for specific protein recognition.

Conformational properties of DNA hairpins with TTT and TTTT loops.

The DNA hairpins d[CGATCG-Tn-CGATCG] (n = 3, 4) have been studied by NMR in order to gain information on hairpin conformation and flexibility. Resonance assignments were made using a combination of DQF-COSY, DQF-COSY[31P], NOESY, and 1H-31P-COSY. These data also provide approximate coupling constant information which points out exceptionally flexible regions of the phosphate backbone. The data for both hairpins reveal substantial flexibility within the loop segments. For n = 4, NOESY data alone are insufficient to distinguish between two loop-folding motifs, although coupling constant data favor a conformation in which Tb is folded toward the minor groove and is highly exposed to solvent. This is in agreement with chemical shift data and susceptibility to modification by KMnO4. The phosphate backbone between Tc and Td is exceptionally flexible, undergoing a facile exchange between (beta t,gamma+) and (beta+,gamma t) conformers. A similar flexible phosphate is observed between Tc and C7 when n = 3. Differences in stem conformation and dynamics in both hairpins are restricted to the two base pairs adjacent to the stem-loop junction. The C7pG8 stem phosphate appears to flip easily between (zeta-,alpha-) and (zeta-,alpha t) conformers when n = 4 but not when n = 3. Hairpin loop size thus affects the conformational flexibility of the adjacent stem segment.

Sequence-dependent oligonucleotide-target duplex stabilities: rules from empirical studies with a set of twenty-mers.

We were interested in developing a better method to predict the thermal stability of specific oligonucleotide-target duplexes. Recognizing that the base sequence can have important effects, we investigated the use of a simple parameter based on nearest-neighbor stacking interactions, the mean stacking temperature. We took values for doublet stabilities from the literature and used a computer program to calculate mean stacking temperatures for all oligonucleotides of specified length and G + C content in the M13 phage genome. As expected, the program predicted a fairly broad range of stabilities for different sequences of equal G + C content. We selected 20-mer sequences representing the highest and lowest mean stacking temperatures at 25%, 50% and 75% G + C and synthesized them for use as probes against M13 DNA immobilized on filters. By hybridizing and washing at different temperatures, we demonstrated that mean stacking temperatures correlate well with observed stabilities. Relative stabilities of the six oligos were predicted correctly in every case. We used conditions appropriate to oligonucleotide probing and polymerase chain reaction and we were able to derive simple linear equations relating the empirical data and mean stacking temperature for both. These observations should be useful in planning experiments with oligonucleotides.