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Carrier proteins are central to the biosynthesis of primary and secondary metabolites in all organisms. Here we describe metabolic labeling and manipulation of native acyl carrier proteins in both type I and II fatty acid synthases. By utilizing natural promiscuity in the CoA biosynthetic pathway in combination with synthetic pantetheine analogues, we demonstrate metabolic labeling of endogenous carrier proteins with reporter tags in Gram-positive and Gram-negative bacteria and in a human carcinoma cell line. The highly specific nature of the post-translational modification that was utilized for tagging allows for simple visualization of labeled carrier proteins, either by direct fluorescence imaging or after chemical conjugation to a fluorescent reporter. In addition, we demonstrate the utility of this approach for the isolation and enrichment of carrier proteins by affinity purification. Finally, we use these techniques to identify a carrier protein from an unsequenced organism, a finding that validates this proteomic approach to natural product biosynthetic enzyme discovery.

Chromogranin A (CgA), the major soluble protein in chromaffin granules, is proteolytically processed to generate biologically active peptides including the catecholamine release inhibitory peptide catestatin. Here we sought to determine whether cysteine protease cathepsin L (CTSL), a novel enzyme for proteolytic processing of neuropeptides, acts like the well-established serine proteases [prohormone convertase (PC)1/3 or PC2] to generate catestatin by proteolytic processing of CgA. We found that endogenous CTSL colocalizes with CgA in the secretory vesicles of primary rat chromaffin cells. Transfection of PC12 cells with an expression plasmid encoding CTSL directed expression of CTSL toward secretory vesicles. Deconvolution fluorescence microscopy suggested greater colocalization of CTSL with CgA than the lysosomal marker LGP110. The overexpression of CTSL in PC12 cells caused cleavage of full-length CgA. CTSL also cleaved CgA in vitro, in time- and dose-dependent fashion, and specificity of the process was documented through E64 (thiol reagent) inhibition. Mass spectrometry on CTSL-digested recombinant CgA identified a catestatin-region peptide, corresponding to CgA360–373. The pool of peptides generated from the CTSL cleavage of CgA inhibited nicotine-induced catecholamine secretion from PC12 cells. CTSL processing in the catestatin region was diminished by naturally occurring catestatin variants, especially Pro370Leu and Gly364Ser. Among the CTSL-generated peptides, a subset matched those found in the catestatin region in vivo. These findings indicate that CgA can be a substrate for the cysteine protease CTSL both in vitro and in cella, and their colocalization within chromaffin granules in cella suggests the likelihood of an enzyme/substrate relationship in vivo.

Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific (15)N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific (15)N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The (15)N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the (15)N-amino acid prevents the scrambling of the (15)N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" (15)N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples.

Proteolytic processing of the amyloid precursor protein by beta- and gamma-secretase generates the amyloid-beta (Abeta) peptides, which are principal drug targets in Alzheimer disease therapeutics. gamma-Secretase has imprecise cleavage specificity and generates the most abundant Abeta40 and Abeta42 species together with longer and shorter peptides such as Abeta38. Several mechanisms could explain the production of multiple Abeta peptides by gamma-secretase, including sequential processing of longer into shorter Abeta peptides. A novel class of gamma-secretase modulators (GSMs) that includes some non-steroidal anti-inflammatory drugs has been shown to selectively lower Abeta42 levels without a change in Abeta40 levels. A signature of GSMs is the concomitant increase in shorter Abeta peptides, such as Abeta38, leading to the suggestion that generation of Abeta42 and Abeta38 peptide species by gamma-secretase is coordinately regulated. However, no evidence for or against such a precursor-product relationship has been provided. We have previously shown that stable overexpression of aggressive presenilin-1 (PS1) mutations associated with early-onset familial Alzheimer disease attenuated the cellular response to GSMs, resulting in greatly diminished Abeta42 reductions as compared with wild type PS1. We have now used this model system to investigate whether Abeta38 production would be similarly affected indicating coupled generation of Abeta42 and Abeta38 peptides. Surprisingly, treatment with the GSM sulindac sulfide increased Abeta38 production to similar levels in four different PS1 mutant cell lines as compared with wild type PS1 cells. This was confirmed with the structurally divergent GSMs ibuprofen and indomethacin. Mass spectrometry analysis and high resolution urea gel electrophoresis further demonstrated that sulindac sulfide did not induce detectable compensatory changes in levels of other Abeta peptide species. These data provide evidence that Abeta42 and Abeta38 species can be independently generated by gamma-secretase and argue against a precursor-product relationship between these peptides.

Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta deposition in Alzheimer's disease. These data also demonstrate the existence and feasibility of 'substrate targeting' by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of 'druggable' targets.

Microorganisms catalyze the formation of naturally occurring Mn oxides, but little is known about the biochemical mechanisms of this important biogeochemical process. We used tandem mass spectrometry to directly analyze the Mn(II)-oxidizing enzyme from marine Bacillus spores, identified as an Mn oxide band with an in-gel activity assay. Nine distinct peptides recovered from the Mn oxide band of two Bacillus species were unique to the multicopper oxidase MnxG, and one peptide was from the small hydrophobic protein MnxF. No other proteins were detected in the Mn oxide band, indicating that MnxG (or a MnxF/G complex) directly catalyzes biogenic Mn oxide formation. The Mn(II) oxidase was partially purified and found to be resistant to many proteases and active even at high concentrations of sodium dodecyl sulfate. Comparative analysis of the genes involved in Mn(II) oxidation from three diverse Bacillus species revealed a complement of conserved Cu-binding regions not present in well-characterized multicopper oxidases. Our results provide the first direct identification of a bacterial enzyme that catalyzes Mn(II) oxidation and suggest that MnxG catalyzes two sequential one-electron oxidations from Mn(II) to Mn(III) and from Mn(III) to Mn(IV), a novel type of reaction for a multicopper oxidase.

Inflammatory myopathies are a group of autoimmune diseases that affect muscles. In humans, the most common inflammatory myopathies are polymyositis, dermatomyositis, and inclusion body myositis. Autoantibodies may be found in humans with inflammatory myopathies, and these play an important role in diagnosis and disease classification. However, these Abs are typically not muscle specific. Spontaneously occurring canine inflammatory myopathies may be good parallel disorders and provide insights into human myositis. In dogs with inflammatory myopathy, muscle-specific autoantibodies have been found, especially in masticatory muscle myositis. We have identified the major Ag recognized by the autoantibodies in canine masticatory muscle myositis. This Ag is a novel member of the myosin binding protein-C family, which we call masticatory myosin binding protein-C (mMyBP-C). mMyBP-C is localized not only within the masticatory muscle fibers, but also at or near their cell surface, perhaps making it accessible as an immunogen. The gene for mMyBP-C also exists in humans, and mMyBP-C could potentially play a role in certain human inflammatory myopathies. Understanding the role of mMyBP-C in this canine inflammatory myopathy may advance our knowledge of mechanisms of autoimmune inflammatory muscle diseases, not only in dogs, but also in humans.

Nuclear factor κB (NF-κB) transcription factors regulate genes responsible for critical cellular processes. IκBα, -β, and -ε bind to NF-κBs and inhibit their transcriptional activity. The NF-κB-binding domains of IκBs contain six ankyrin repeats (ARs), which adopt a β-hairpin/α-helix/loop/α-helix/loop architecture. IκBα appears compactly folded in the IκBα·NF-κB crystal structure, but biophysical studies suggested that IκBα might be flexible even when bound to NF-κB. Amide H/ 2 H exchange in free IκBα suggests that ARs 2–4 are compact, but ARs 1, 5, and 6 are conformationally flexible. Amide H/ 2 H exchange is one of few techniques able to experimentally identify regions that fold upon binding. Comparison of amide H/ 2 H exchange in free and NF-κB-bound IκBα reveals that the β-hairpins in ARs 5 and 6 fold upon binding to NF-κB, but AR 1 remains highly solvent accessible. These regions are implicated in various aspects of NF-κB regulation, such as controlling degradation of IκBα, enabling high-affinity interaction with different NF-κB dimers, and preventing NF-κB from binding to its target DNA. Thus, IκBα conformational flexibility and regions of IκBα folding upon binding to NF-κB are important attributes for its regulation of NF-κB transcriptional activity.

Fatty acid, polyketide, and nonribosomal peptide biosynthetic enzymes perform structural modifications upon small molecules that remain tethered to a carrier protein. This manuscript details the design and analysis of cross-linking substrates that are selective for acyl carrier proteins and their cognate condensing enzymes. These inactivators are engineered through a covalent linkage to fatty acid acyl carrier protein via post-translational modification to contain a reactive probe that traps the active site cysteine residue of ketosynthase domains. These proteomic tools are applied to Escherichia coli fatty acid synthase enzymes, where KASI and KASII selectively cross-link ACP-bound epoxide and chloroacrylate moieties. These mechanism-based, protein-protein fusion reagents also demonstrated cross-linking of KASI to type II polyketide ACPs, while nonribosomal peptide carrier proteins showed no reactivity. Similar investigations into protein-protein interactions, proximity effects, and substrate specificities will be required to complete the mechanistic understanding of these pathways.

When iron-starved, the Mn(II)-oxidizing bacteria Pseudomonas putida strains GB-1 and MnB1 produce pyoverdines (PVDGB-1 and PVDMnB1), siderophores that both influence iron uptake and inhibit manganese(II) oxidation by these strains. To explore the properties and genetics of a PVD that can affect manganese oxidation, LC-MS/MS, and various siderotyping techniques were used to identify the peptides of PVDGB-1 and PVDMnB1 as being (for both PVDs): chromophore-Asp-Lys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn, resembling a structure previously reported for P. putida CFML 90-51, which does not oxidize Mn. All three strains also produced an azotobactin and a sulfonated PVD, each with the peptide sequence above, but with unknown regulatory or metabolic effects. Bioinformatic analysis of the sequenced genome of P. putida GB-1 suggested that a particular non-ribosomal peptide synthetase (NRPS), coded by the operon PputGB1_4083-4086, could produce the peptide backbone of PVDGB-1. To verify this prediction, plasmid integration disruption of PputGB1_4083 was performed and the resulting mutant failed to produce detectable PVD. In silico analysis of the modules in PputGB1_4083-4086 predicted a peptide sequence of Asp-Lys-Asp-Ser-Ala-Thr-Lsy-Orn, which closely matches the peptide determined by MS/MS. To extend these studies to other organisms, various Mn(II)-oxidizing and non-oxidizing isolates of P. putida, P. fluorescens, P. marincola, P. fluorescens-syringae group, P. mendocina-resinovorans group, and P. stutzerii group were screened for PVD synthesis. The PVD producers (12 out of 16 tested strains) were siderotyped and placed into four sets of differing PVD structures, some corresponding to previously characterized PVDs and some to novel PVDs. These results combined with previous studies suggested that the presence of OHAsp or the flexibility of the pyoverdine polypeptide may enable efficient binding of Mn(III).

The oxidation of heme to biliverdin IXα by heme oxygenase involves regiospecific α-meso-hydroxylation followed by extrusion of the α-meso-carbon as CO. In an earlier study, enzymatic oxidation of the fourmeso-methylmesoheme isomers suggested that the reaction regiospecificity is sensitive to the electronic properties of themeso-methyl group (Torpey, J. W., and Ortiz de Montellano, P. R. (1996) J. Biol. Chem. 271, 26067–26073), although we could not exclude the possibility that the altered reaction regiochemistry was due to perturbation of the porphyrin structure by the meso-substituent. To examine this possibility, we have synthesized the fourmeso-formylmesoporphyrin isomers and have examined their oxidation by heme oxygenase. The meso-formyl andmeso-methyl substituents differ in that the former is electron withdrawing and the latter is electron donating. In contrast to α-meso-methylmesoheme, which is exclusively oxidized at the methyl-substituted position, α-meso-formylmesoheme is exclusively oxidized at a non-formyl-substitutedmeso-carbon. The finding that the methyl and formyl groups channel the reaction regiospecificity in opposite directions establishes that the regiochemistry of the heme oxygenase reaction is primarily under electronic rather than steric control. It also confirms that the oxidation involves electrophilic addition of the oxygen to the porphyrin ring.

The oxidation of heme by heme oxygenase (HO-1) results in highly regiospecific extrusion of the α-meso-carbon as CO and the formation of biliverdin IXα. The first step in the accepted mechanism for this process is α-meso-hydroxylation of the heme. To define further the reaction mechanism, the oxidations of the four isomers of meso-methylmesoheme by human truncated HO-1 supported by NADPH-cytochrome P450 reductase, H2O2, or ascorbate have been examined. Surprisingly, the results establish that (a) HO-1 can oxidize an α-meso-methyl-substituted heme to biliverdin IXα without proceeding via the α-meso-hydroxy intermediate; (b) the normal HO-1 α-regiospecificity is inverted in favor of the substituted γ-position by introduction of a γ-meso methyl group; and (c) the β- and δ-meso-methylmesohemes are oxidized less regiospecifically to mixtures of methyl-substituted and -unsubstituted mesobiliverdins. The results indicate that electronic rather than steric effects primarily control the regiospecificity of heme cleavage by HO-1.

Previous studies have established that reaction of the rat heme-heme oxygenase complex with H2O2 proceeds normally to give verdoheme, whereas reaction of the complex with meta-chloroperbenzoic acid yields a ferryl (FeIV = O) species and a protein radical but no verdoheme. The heme-heme oxygenase complex is shown here to react regiospecifically with ethyl hydroperoxide to give alpha-meso-ethoxyheme. Formation of this product exactly parallels the formation of alpha-meso-hydroxyheme in the normal reaction supported by cytochrome P450 reductase/NADPH or H2O2. These results rule out a nucleophilic mechanism for the alpha-meso-hydroxylation catalyzed by heme oxygenase and indicate that it involves electrophilic (or possibly radical) addition of the distal oxygen of iron-bound peroxide (FeIII-OOH) to the porphyrin ring.

Antimicrobial peptides serve as a first line of innate immune defense against invading organisms such as bacteria and viruses. In this study, we hypothesized that peptides produced by a normal microbial resident of human skin, Staphylococcus epidermidis, might also act as an antimicrobial shield and contribute to normal defense at the epidermal interface. We show by circular dichroism and tryptophan spectroscopy that phenol-soluble modulins (PSMs) gamma and delta produced by S. epidermidis have an alpha-helical character and a strong lipid membrane interaction similar to mammalian AMPs such as LL-37. Both PSMs directly induced lipid vesicle leakage and exerted selective antimicrobial action against skin pathogens such as Staphylococcus aureus. PSMs functionally cooperated with each other and LL-37 to enhance antimicrobial action. Moreover, PSMs reduced Group A Streptococcus (GAS) but not the survival of S. epidermidis on mouse skin. Thus, these data suggest that the production of PSMgamma and PSMdelta by S. epidermidis can benefit cutaneous immune defense by selectively inhibiting the survival of skin pathogens while maintaining the normal skin microbiome.

A new type of manganese-oxidizing enzyme has been identified in two alphaproteobacteria, “ Aurantimonas manganoxydans ” strain SI85-9A1 and Erythrobacter sp. strain SD-21. These proteins were identified by tandem mass spectrometry of manganese-oxidizing bands visualized by native polyacrylamide gel electrophoresis in-gel activity assays and fast protein liquid chromatography-purified proteins. Proteins of both alphaproteobacteria contain animal heme peroxidase and hemolysin-type calcium binding domains, with the 350-kDa active Mn-oxidizing protein of A. manganoxydans containing stainable heme. The addition of both Ca 2+ ions and H 2 O 2 to the enriched protein from Aurantimonas increased manganese oxidation activity 5.9-fold, and the highest activity recorded was 700 μM min −1 mg −1 . Mn(II) is oxidized to Mn(IV) via an Mn(III) intermediate, which is consistent with known manganese peroxidase activity in fungi. The Mn-oxidizing protein in Erythrobacter sp. strain SD-21 is 225 kDa and contains only one peroxidase domain with strong homology to the first 2,000 amino acids of the peroxidase protein from A. manganoxydans . The heme peroxidase has tentatively been named MopA (manganese-oxidizing peroxidase) and sheds new light on the molecular mechanism of Mn oxidation in prokaryotes.

Proteolytic processing of the amyloid precursor protein by beta- and gamma-secretase generates the amyloid-beta (Abeta) peptides, which are principal drug targets in Alzheimer disease therapeutics. gamma-Secretase has imprecise cleavage specificity and generates the most abundant Abeta40 and Abeta42 species together with longer and shorter peptides such as Abeta38. Several mechanisms could explain the production of multiple Abeta peptides by gamma-secretase, including sequential processing of longer into shorter Abeta peptides. A novel class of gamma-secretase modulators (GSMs) that includes some non-steroidal anti-inflammatory drugs has been shown to selectively lower Abeta42 levels without a change in Abeta40 levels. A signature of GSMs is the concomitant increase in shorter Abeta peptides, such as Abeta38, leading to the suggestion that generation of Abeta42 and Abeta38 peptide species by gamma-secretase is coordinately regulated. However, no evidence for or against such a precursor-product relationship has been provided. We have previously shown that stable overexpression of aggressive presenilin-1 (PS1) mutations associated with early-onset familial Alzheimer disease attenuated the cellular response to GSMs, resulting in greatly diminished Abeta42 reductions as compared with wild type PS1. We have now used this model system to investigate whether Abeta38 production would be similarly affected indicating coupled generation of Abeta42 and Abeta38 peptides. Surprisingly, treatment with the GSM sulindac sulfide increased Abeta38 production to similar levels in four different PS1 mutant cell lines as compared with wild type PS1 cells. This was confirmed with the structurally divergent GSMs ibuprofen and indomethacin. Mass spectrometry analysis and high resolution urea gel electrophoresis further demonstrated that sulindac sulfide did not induce detectable compensatory changes in levels of other Abeta peptide species. These data provide evidence that Abeta42 and Abeta38 species can be independently generated by gamma-secretase and argue against a precursor-product relationship between these peptides.

Microbial Mn(II) oxidation has important biogeochemical consequences in marine, freshwater, and terrestrial environments, but many aspects of the physiology and biochemistry of this process remain obscure. Here, we report genomic insights into Mn(II) oxidation by the marine alphaproteobacterium Aurantimonas sp. strain SI85-9A1, isolated from the oxic/anoxic interface of a stratified fjord. The SI85-9A1 genome harbors the genetic potential for metabolic versatility, with genes for organoheterotrophy, methylotrophy, oxidation of sulfur and carbon monoxide, the ability to grow over a wide range of O 2 concentrations (including microaerobic conditions), and the complete Calvin cycle for carbon fixation. Although no growth could be detected under autotrophic conditions with Mn(II) as the sole electron donor, cultures of SI85-9A1 grown on glycerol are dramatically stimulated by addition of Mn(II), suggesting an energetic benefit from Mn(II) oxidation. A putative Mn(II) oxidase is encoded by duplicated multicopper oxidase genes that have a complex evolutionary history including multiple gene duplication, loss, and ancient horizontal transfer events. The Mn(II) oxidase was most abundant in the extracellular fraction, where it cooccurs with a putative hemolysin-type Ca 2+ -binding peroxidase. Regulatory elements governing the cellular response to Fe and Mn concentration were identified, and 39 targets of these regulators were detected. The putative Mn(II) oxidase genes were not among the predicted targets, indicating that regulation of Mn(II) oxidation is controlled by other factors yet to be identified. Overall, our results provide novel insights into the physiology and biochemistry of Mn(II) oxidation and reveal a genome specialized for life at the oxic/anoxic interface.

Nine proline-rich peptides ending with a proline-glutamine C terminus in a salivary peptidome were sequenced by matrix-assisted laser desorption ionization time of flight time of flight tandem mass spectrometry. A GPPPQGGRPQ peptide binds gram-positive Propionibacterium acnes and considerably inhibits bacterial growth. The peptide exhibiting innate immunity may be applied for treatment of various P. acnes -associated human diseases.

The plasma level of chromogranin A (CgA) is elevated in genetic hypertension. Conversely, the plasma level of the CgA peptide catestatin is diminished in individuals with established hypertension and those with a genetic risk of this disease. Resequencing of the human CHGA gene identified three naturally occurring variants of catestatin (Gly364Ser, Pro370Leu, and Arg374Gln) that exhibit different potencies in inhibiting catecholamine secretion. Here, we have examined whether there is any differential processing of the three CHGA variants to catestatin by the endoproteolytic enzyme plasmin. Plasmin digestion of the purified CgA proteins generated a stable biologically active 14-amino acid peptide (human CgA(360-373)) from the wild-type, Gly364Ser, and Arg374Gln proteins despite the disruption of the dibasic site (Arg(373)Arg(374)) in the Arg374Gln variant. Unexpectedly, the action of plasmin in generating the catestatin peptide from the Pro370Leu protein was less efficient. The efficiency of cleavage at the dibasic Arg(373) downward arrowArg(374) site in synthetic human CgA(360-380) was 3- to 4-fold less in Pro370Leu CgA, compared with the wild type. Circular dichroism of the synthetic CgA(352-372) suggested a difference in the amount of alpha-helix and beta-sheet between the wild-type and Pro370Leu CgA peptides. Because the Pro(370) residue is in the P4 position, the local secondary structure in the vicinity of the cleavage site may enforce the specificity or accessibility to plasmin. The less efficient proteolytic processing of the Pro370Leu protein by plasmin, coupled with the strong association of this variant with ethnicity, suggests that the Pro370Leu CHGA gene variant may contribute to the differential prevalence of cardiovascular disease across ethnic groups.

One advantage of detecting amide H/2H exchange by mass spectrometry instead of NMR is that the more rapidly exchanging surface amides are still detectable. In this study, we present quench-flow amide H/2H exchange experiments to probe how rapidly the surfaces of two different proteins exchange. We compared the amide H/2H exchange behavior of thrombin, a globular protein, and IkappaBalpha, a nonglobular protein, to explore any differences in the determinants of amide H/2H exchange rates for each class of protein. The rates of exchange of only a few of the surface amides were as rapid as the "intrinsic" exchange rates measured for amides in unstructured peptides. Most of the surface amides exchanged at a slower rate, despite the fact that they were not seen to be hydrogen bonded to another protein group in the crystal structure. To elucidate the influence of the surface environment on amide H/2H exchange, we compared exchange data with the number of amides participating in hydrogen bonds with other protein groups and with the solvent accessible surface area. The best correlation with amide H/2H exchange was found with the total solvent accessible surface area, including side chains. In the case of the globular protein, the correlation was modest, whereas it was well correlated for the nonglobular protein. The nonglobular protein also showed a correlation between amide exchange and hydrogen bonding. These data suggest that other factors, such as complex dynamic behavior and surface burial, may alter the expected exchange rates in globular proteins more than in nonglobular proteins where all of the residues are near the surface.