The influx of genomic sequence information has led to the concept of structural proteomics, the determination of protein structures on a genome-wide scale. Here we describe an approach to structural proteomics of small proteins using NMR spectroscopy. Over 500 small proteins from several organisms were cloned, expressed, purified, and evaluated by NMR. Although there was variability among proteomes, overall 20% of these proteins were found to be readily amenable to NMR structure determination. NMR sample preparation was centralized in one facility, and a distributive approach was used for NMR data collection and analysis. Twelve structures are reported here as part of this approach, which allowed us to infer putative functions for several conserved hypothetical proteins. S tructural proteomics, which aims to determine the threedimensional (3D) structures of all proteins, has become a major initiative within the biomedical community (see ref. 1 and other articles in the same issue). The large number of protein structures expected from these projects will yield valuable clues to the rules for predicting protein folding and understanding biochemical function. In these early stages of the structural proteomics effort, one of the main goals is to identify the best technologies and the most efficient processes to convert gene sequence into 3D structural information. One of the decisions will be to determine the optimal use of x-ray crystallography and NMR spectroscopy, which are the two techniques that will provide the majority of experimental data for these initiatives.X-ray crystallography currently is perceived as the potential workhorse for structural proteomics, because if provided with a well diffracting crystal it is possible to determine a 3D structure in hours. However, the throughput of structure determination using x-ray crystallography remains unclear, because the ratedetermining step continues to be the production of well diffracting crystals, a process that is unpredictable and can take between hours and months.NMR structure determination is limited currently by size constraints and lengthy data collection and analysis times (often months), and the method is best applied to proteins smaller than 250 amino acids. On the other hand, NMR experiments do not require crystals, and samples appropriate for structure determination can be identified within minutes of the protein being purified. In summary, x-ray crystallography and NMR spectroscopy seem to have complementary deficiencies, and the relative success of these methods in structural proteomics remains to be determined.We have shown previously that NMR spectroscopy can play a significant role in structural proteomics even with its current limitations (2). The initial pilot project, based on a limited number of proteins from the thermophilic archaebacterium Methanobacterium thermoautotrophicum (Mth) suggested that smaller proteins may be more amenable to structure analysis, because in this genome a higher proportion of smaller proteins were soluble compar...

Chaperones and foldases in the endoplasmic reticulum (ER) ensure correct protein folding. Extensive protein-protein interaction maps have defined the organization and function of many cellular complexes, but ER complexes are under-represented. Consequently, chaperone and foldase networks in the ER are largely uncharacterized. Using complementary ER-specific methods, we have mapped interactions between ER-lumenal chaperones and foldases and describe their organization in multiprotein complexes. We identify new functional chaperone modules, including interactions between protein-disulfide isomerases and peptidylprolyl cis-trans-isomerases. We have examined in detail a novel ERp72-cyclophilin B complex that enhances the rate of folding of immunoglobulin G. Deletion analysis and NMR reveal a conserved surface of cyclophilin B that interacts with polyacidic stretches of ERp72 and GRp94. Mutagenesis within this highly charged surface region abrogates interactions with its chaperone partners and reveals a new mechanism of ER protein-protein interaction. This ability of cyclophilin B to interact with different partners using the same molecular surface suggests that ER-chaperone/foldase partnerships may switch depending on the needs of different substrates, illustrating the flexibility of multichap-

The first structure of a 2'-deoxy-2'-fluoro-D-arabinose nucleic acid (2'F-ANA)/RNA duplex is presented. We report the structural characterization by NMR spectroscopy of a small hybrid hairpin, r(GGAC)d(TTCG)2'F-a(GTCC), containing a 2'F-ANA/RNA stem and a four-residue DNA loop. Complete (1)H, (13)C, (19)F, and (31)P resonance assignments, scalar coupling constants, and NOE constraints were obtained from homonuclear and heteronuclear 2D spectra. In the chimeric duplex, the RNA strand adopts a classic A-form structure having C3' endo sugar puckers. The 2'F-ANA strand is neither A-form nor B-form and contains O4' endo sugar puckers. This contrasts strongly with the dynamic sugar conformations previously observed in the DNA strands of DNA/RNA hybrid duplexes. Structural parameters for the duplex, such as minor groove width, x-displacement, and inclination, were intermediate between those of A-form and B-form duplexes and similar to those of DNA/RNA duplexes. These results rationalize the enhanced stability of 2'F-ANA/RNA duplexes and their ability to elicit RNase H activity. The results are relevant for the design of new antisense drugs based on sugar-modified nucleic acids.

The endoplasmic reticulum (ER) is the cell compartment where membrane and secretory proteins fold. The rate-limiting step for the folding of many proteins is the formation of disulfide bonds. As polypeptides are synthesized, their cysteine thiols enter the oxidizing environment of the ER and form covalent intramolecular and intermolecular disulfide links. Although this oxidative folding process occurs spontaneously [1], non-native disulfide-bonded intermediates often occur, acting as kinetic traps along the folding pathway [2,3]. To avoid these, the ER contains a large family of enzymes called protein disulfide isomerases (PDIs) that catalyze both disulfide bond formation and the rearrangement of incorrect disulfide bonds [4][5][6][7].PDI family members are loosely defined by homology to thioredoxin and ER localization. There are at least 17 PDI family proteins in humans, 13 of which contain CXXC active-site motifs, and 9 have been shown to catalyze disulfide-exchange reactions [4,5]. The best studied and most abundant member of the family is PDI, a ubiquitous enzyme found at very high concentrations in the ER. Its concentration has been estimated to be 10 lm in dog pancreatic microsomes [8], the highest of all ER resident proteins. PDI has four thioredoxin-like domains, a-b-b¢-a¢, where the two a domains contain catalytic CGHC motifs, and the two b domains lack the conserved cysteine residues and are noncatalytic. The linkers between the domains are generally short. The longest is a stretch of 19 amino acids between the b¢ and a¢ domains, referred to as the x-linker [9]. Protein disulfide isomerase is the most abundant and best studied of the disulfide isomerases that catalyze disulfide bond formation in the endoplasmic reticulum, yet the specifics of how it binds substrate have been elusive. Protein disulfide isomerase is composed of four thioredoxin-like domains (abb¢a¢). Cross-linking studies with radiolabeled peptides and unfolded proteins have shown that it binds incompletely folded proteins primarily via its third domain, b¢. Here, we determined the solution structure of the second and third domains of human protein disulfide isomerase (b and b¢, respectively) by triple-resonance NMR spectroscopy and molecular modeling. NMR titrations identified a large hydrophobic surface within the b¢ domain that binds unfolded ribonuclease A and the peptides mastoparan and somatostatin. Protein disulfide isomerase-catalyzed refolding of reduced ribonuclease A in vitro was inhibited by these peptides at concentrations equal to their affinity to the bb¢ fragment. Our findings provide a structural basis for previous kinetic and cross-linking studies which have shown that protein disulfide isomerase exhibits a saturable, substratebinding site.Abbreviations ER, endoplasmic reticulum; GST, glutathione S-transferase; HSQC, heteronuclear single-quantum correlation; PDI, protein disulfide isomerase; RDC, residual dipolar coupling; RNase A, ribonuclease A.