Deliberate Microbial Infection Research Reveals Limitations to Current Safety Protections of Healthy Human Subjects.

Here we identify approximately 40,000 healthy human volunteers who were intentionally exposed to infectious pathogens in clinical research studies dating from late World War II to the early 2000s. Microbial challenge experiments continue today under contemporary human subject research requirements. In fact, we estimated 4,000 additional volunteers who were experimentally infected between 2010 and the present day. We examine the risks and benefits of these experiments and present areas for improvement in protections of participants with respect to safety. These are the absence of maximum limits to risk and the potential for institutional review boards to include questionable benefits to subjects and society when weighing the risks and benefits of research protocols. The lack of a duty of medical care by physician-investigators to research subjects is likewise of concern. The transparency of microbial challenge experiments and the safety concerns raised in this work may stimulate further dialogue on the risks to participants of human experimentation.

Improving the Proteomic Analysis of Archival Tissue by Using Pressure-Assisted Protein Extraction: A Mechanistic Approach.

Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE tissue. Our laboratory has taken a mechanistic approach to developing improved protein extraction protocols, by first studying the reactions of formaldehyde with proteins and ways to reverse these reactions, then applying this approach to a model system called a "tissue surrogate", which is a gel formed by treating high concentrations of cytoplasmic proteins with formaldehyde, and finally FFPE mouse liver tissue. Our studies indicate that elevated pressure improves the recovery of proteins from FFPE tissue surrogates by hydrating and promoting solubilization of highly aggregated proteins allowing for the subsequent reversal (by hydrolysis) of formaldehyde-induced protein adducts and cross-links. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency and up to a 30-fold increase in the number of non-redundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of non-redundant proteins identified in the FFPE tissue was nearly identical to that of the corresponding frozen tissue.

An ultra-sensitive immunoassay for quantifying biomarkers in breast tumor tissue.

Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) have been validated at the highest level of evidence as clinical biomarkers of prognosis in breast cancer. The American Society of Clinical Oncology recommends using uPA and PAI-1 levels in breast tumors for deciding whether patients with newly diagnosed node-negative breast cancer can forgo adjuvant chemotherapy. The sole validated method for quantifying uPA and PAI-1 levels in breast tumor tissue is a colorimetric ELISA assay that takes 3 days to complete and requires 100-300 mg of fresh or frozen tissue. In this study we describe a new assay method for quantifying PAI-1 levels in human breast tumor tissue. This assay combines pressure-cycling technology to extract PAI-1 from breast tumor tissue with a highly sensitive liposome polymerase chain reaction immunoassay for quantification of PAI-1 in the tissue extract. The new PAI-1 assay method reduced the total assay time to one day and improved assay sensitivity and dynamic range by >100, compared to ELISA.

Bedside to bench: role of muscarinic receptor activation in ultrarapid growth of colorectal cancer in a patient with pheochromocytoma.

An elderly man with long-standing, nonresectable pheochromocytoma had rapid development of rectal adenocarcinoma despite close endoscopic surveillance. We determined that the patient's colorectal cancer overexpressed muscarinic receptor subtype 3, whereas his pheochromocytoma expressed choline acetyltransferase, an enzyme required to produce acetylcholine, which is a muscarinic receptor agonist. These findings suggested that acetylcholine release from the pheochromocytoma stimulated rapid growth of the rectal neoplasm. As proof of principle, we found that culture media conditioned by pheochromocytoma cells stimulates proliferation of a human colon cancer cell line, an effect attenuated by atropine, a muscarinic receptor inhibitor. Our observations provide both clinical and laboratory evidence that muscarinic receptor agonists promote the growth of colorectal neoplasia.

Toward improving the proteomic analysis of formalin-fixed, paraffin-embedded tissue.

Archival formalin-fixed, paraffin-embedded (FFPE) tissue and their associated diagnostic records represent an invaluable source of retrospective proteomic information on diseases for which the clinical outcome and response to treatment are known. However, analysis of archival FFPE tissues by high-throughput proteomic methods has been hindered by the adverse effects of formaldehyde fixation and subsequent tissue histology. This review examines recent methodological advances for extracting proteins from FFPE tissue suitable for proteomic analysis. These methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, allow at least a qualitative analysis of the proteome of FFPE archival tissues. The authors also discuss recent advances in the proteomic analysis of FFPE tissue; including liquid-chromatography tandem mass spectrometry, reverse phase protein microarrays and imaging mass spectrometry.

Complete solubilization of formalin-fixed, paraffin-embedded tissue may improve proteomic studies.

Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues.

Pressure-assisted protein extraction: a novel method for recovering proteins from archival tissue for proteomic analysis.

Formaldehyde-fixed, paraffin-embedded (FFPE) tissue repositories represent a valuable resource for the retrospective study of disease progression and response to therapy. However, the proteomic analysis of FFPE tissues has been hampered by formaldehyde-induced protein modifications, which reduce protein extraction efficiency and may lead to protein misidentification. Here, we demonstrate the use of heat augmented with high hydrostatic pressure (40,000 psi) as a novel method for the recovery of intact proteins from FFPE mouse liver. When FFPE mouse liver was extracted using heat and elevated pressure, there was a 4-fold increase in protein extraction efficiency, a 3-fold increase in the extraction of intact proteins, and up to a 30-fold increase in the number of nonredundant proteins identified by mass spectrometry, compared to matched tissue extracted with heat alone. More importantly, the number of nonredundant proteins identified in the FFPE tissue was nearly identical to that of matched fresh-frozen tissue.

Novel mineral contrast agent for magnetic resonance studies of bone implants grown on a chick chorioallantoic membrane.

Magnetic resonance imaging (MRI) studies of tissue engineered constructs prior to implantation clearly demonstrate the utility of the MRI technique for studying the bone formation process. To test the utility of our MRI protocols for explant studies, we present a novel test platform in which osteoblast-seeded scaffolds were implanted on the chorioallantoic membrane of a chick embryo. Scaffolds from the following experimental groups were examined by high-resolution MRI: (a) cell-seeded implanted scaffolds (CIM), (b) unseeded implanted scaffolds (UCIM), (c) cell-seeded scaffolds in static culture (CIV) and (d) unseeded scaffolds in static culture (UCIV). The reduction in water proton transverse relaxation times and the concomitant increase in water proton magnetization transfer ratios for CIM and CIV scaffolds, compared to UCIV scaffolds, were consistent with the formation of a bone-like tissue within the polymer scaffold, which was confirmed by immunohistochemistry and fluorescence microscopy. However, the presence of angiogenic vessels and fibrotic adhesions around UCIM scaffolds can confound MRI findings of bone deposition. Consequently, to improve the specificity of the MRI technique for detecting mineralized deposits within explanted tissue engineered bone constructs, we introduce a novel contrast agent that uses alendronate to target a Food and Drug Administration-approved MRI contrast agent (Gd-DOTA) to bone mineral. Our contrast agent termed GdALN was used to uniquely identify mineralized deposits in representative samples from our four experimental groups. After GdALN treatment, both CIM and CIV scaffolds, containing mineralized deposits, showed marked signal enhancement on longitudinal relaxation time-weighted (T1W) images compared to UCIV scaffolds. Relative to UCIV scaffolds, some enhancement was observed in T1W images of GdALN-treated UCIM scaffolds, subjacent to the dark adhesions at the scaffold surface, possibly from dystrophic mineral formed in the fibrotic adhesions. Notably, residual dark areas on T1W images of CIM and UCIM scaffolds were attributable to blood inside infiltrating vessels. In summary, we present the efficacy of GdALN for sensitizing the MRI technique to the deposition of mineralized deposits in explanted polymeric scaffolds.

The effect of formaldehyde fixation on RNA: optimization of formaldehyde adduct removal.

Formalin-fixed, paraffin-embedded tissues generally provide low yields of extractable RNA that exhibit both covalent modification of nucleic acid bases and strand cleavage. This frustrates efforts to perform retrospective analyses of gene expression using archival tissue specimens. A variety of conditions have been reported to demodify formaldehyde-fixed RNA in different model systems. We studied the reversal of formaldehyde fixation of RNA using a 50 base RNA oligonucleotide and total cellular RNA. Formaldehyde-adducted, native, and hydrolyzed RNA species were identified by their bioanalyzer electrophoretic migration patterns and RT-quantitative PCR. Demodification conditions included temperature, time, buffer, and pH. The reversal of formaldehyde-fixed RNA to native species without apparent RNA hydrolysis was most successfully performed in dilute Tris, phosphate, or similar buffers (pH 8) at 70°C for 30 minutes. Amines were not required for efficient formaldehyde demodification. Formaldehyde-fixed RNA was more labile than native RNA to treatment with heat and buffer, suggesting that antigen retrieval methods for proteins may impede RNA hybridization or RNA extraction. Taken together, the data indicate that reliable conditions may be used to remove formaldehyde adducts from RNA to improve the quality of RNA available for molecular studies.

Tissue microarrays: construction and uses.

Tissue microarrays (TMAs) are produced by taking small punches from a series of paraffin-embedded (donor) tissue blocks and transferring these tissue cores into a positionally encoded array in a recipient paraffin block. Though TMAs are not used for clinical diagnosis, they have several advantages over using conventional whole histological sections for research. Tissue from multiple patients or blocks can be examined on the same slide, and only a very small amount of reagent is required to stain or label an entire array. Multiple sections (100-300) can be cut from a single array block, allowing for hundreds of analyses per microarray. These advantages allow the use of TMAs in high-throughput procedures, such as screening antibodies for diagnostics and validating prognostic markers that are impractical using conventional whole tissue sections. TMAs can be used for immunohistochemistry, immunofluorescence, in situ hybridization, and conventional histochemical staining. Finally, several tissue cores may be taken without -consuming the tissue block, allowing the donor block to be returned to its archive for any additional studies.

Protein mass spectrometry applications on FFPE tissue sections.

Formalin-fixed, paraffin-embedded (FFPE) tissue archives and their associated diagnostic records represent an invaluable source of proteomic information on diseases where the patient outcomes are already known. Over the last few years, advances in methodology have made it possible to recover peptides from FFPE tissues that yield a reasonable representation of the proteins recovered from identical fresh or frozen specimens. These new methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, have developed sufficiently to allow at least a qualitative analysis of the proteome of FFPE archival tissues. This chapter describes the approaches for performing proteomic analysis on FFPE tissues by liquid chromatography and mass spectrometry.

Antigen retrieval causes protein unfolding: evidence for a linear epitope model of recovered immunoreactivity.

Antigen retrieval (AR), in which formalin-fixed paraffin-embedded tissue sections are briefly heated in buffers at high temperature, often greatly improves immunohistochemical staining. An important unresolved question regarding AR is how formalin treatment affects the conformation of protein epitopes and how heating unmasks these epitopes for subsequent antibody binding. The objective of the current study was to use model proteins to determine the effect of formalin treatment on protein conformation and thermal stability in relation to the mechanism of AR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to identify the presence of protein formaldehyde cross-links, and circular dichroism spectropolarimetry was used to determine the effect of formalin treatment and high-temperature incubation on the secondary and tertiary structure of the model proteins. Results revealed that for some proteins, formalin treatment left the native protein conformation unaltered, whereas for others, formalin denatured tertiary structure, yielding a molten globule protein. In either case, heating to temperatures used in AR methods led to irreversible protein unfolding, which supports a linear epitope model of recovered protein immunoreactivity. Consequently, the core mechanism of AR likely centers on the restoration of normal protein chemical composition coupled with improved accessibility to linear epitopes through protein unfolding.

Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates.

BACKGROUND: Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing. PRINCIPAL FINDINGS: In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates. CONCLUSIONS: These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.

Elevated Pressure Improves the Rate of Formalin Penetration while Preserving Tissue Morphology.

Formaldehyde fixation and paraffin-embedding remains the most widely used technique for processing cancer tissue specimens for pathologic examination, the study of tissue morphology, and archival preservation. However, formaldehyde penetration and fixation is a slow process, requiring a minimum of 15 hr for routine processing of pathology samples. Routinely fixed samples often have a well-fixed outer rim, with a poorly-fixed inner core of tissue. In this study, we show that the application of elevated pressure up to 15,000 psi improves the rate of formaldehyde fixation by approximately 5 to 7-fold while preserving the tissue morphology of porcine liver. The tissue also exhibited much more uniform formaldehyde penetration after 30-60 min incubation under elevated pressure than samples fixed for the same length of time at atmospheric pressure.

Modeling formalin fixation and histological processing with ribonuclease A: effects of ethanol dehydration on reversal of formaldehyde cross-links.

Understanding the chemistry of protein modification by formaldehyde fixation and subsequent tissue processing is central to developing improved methods for antigen retrieval in immunohistochemistry and for recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissues for proteomic analysis. Our initial studies of single proteins, such as bovine pancreatic ribonuclease A (RNase A), in 10% buffered formalin solution revealed that upon removal of excess formaldehyde, monomeric RNase A exhibiting normal immunoreactivity could be recovered by heating at 60 degrees C for 30 min at pH 4. We next studied tissue surrogates, which are gelatin-like plugs of fixed proteins that have sufficient physical integrity to be processed using normal tissue histology. Following histological processing, proteins could be extracted from the tissue surrogates by combining heat, detergent, and a protein denaturant. However, gel electrophoresis revealed that the surrogate extracts contained a mixture of monomeric and multimeric proteins. This suggested that during the subsequent steps of tissue processing protein-formaldehyde adducts undergo further modifications that are not observed in aqueous proteins. As a first step toward understanding these additional modifications we have performed a comparative evaluation of RNase A following fixation in buffered formaldehyde alone and after subsequent dehydration in 100% ethanol by combining gel electrophoresis, chemical modification, and circular dichroism spectroscopic studies. Our results reveal that ethanol-induced rearrangement of the conformation of fixed RNase A leads to protein aggregation through the formation of large geometrically compatible hydrophobic beta-sheets that are likely stabilized by formaldehyde cross-links, hydrogen bonds, and van der Waals interactions. It requires substantial energy to reverse the formaldehyde cross-links within these sheets and regenerate protein monomers free of formaldehyde modifications. Accordingly, the ethanol-dehydration step in tissue histology may be important in confounding the successful recovery of proteins from FFPE tissues for immunohistochemical and proteomic analysis.

Elevated hydrostatic pressure promotes protein recovery from formalin-fixed, paraffin-embedded tissue surrogates.

High-throughput proteomic studies on formalin-fixed, paraffin-embedded (FFPE) tissues have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications to proteins. We previously reported a method for the formation of 'tissue surrogates' as a model to study formalin fixation, histochemical processing, and protein retrieval from FFPE tissues. In this study, we demonstrate the use of high hydrostatic pressure as a method for efficient protein recovery from FFPE tissue surrogates. Reversal of formaldehyde-induced protein adducts and crosslinks was observed when lysozyme tissue surrogates were extracted at 45 000 psi and 80-100 degrees C in Tris buffers containing 2% sodium dodecyl sulfate and 0.2 M glycine at pH 4. These conditions also produced peptides resulting from acid-catalyzed aspartic acid cleavage. Additives such as trimethylamine N-oxide or copper (II) chloride decreased the total percentage of these aspartic acid cleavage products, while maintaining efficient reversal of intermolecular crosslinks in the FFPE tissue surrogates. Mass spectrometry analysis of the recovered lysozyme yielded 70% sequence coverage, correctly identified all formaldehyde-reactive amino acids, and demonstrated hydrolysis at all of the expected trypsin cleavage sites. This study demonstrates that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues for proteomic analysis.

'Tissue surrogates' as a model for archival formalin-fixed paraffin-embedded tissues.

High-throughput proteomic studies of archival formalin-fixed paraffin-embedded (FFPE) tissues have the potential to be a powerful tool for examining the clinical course of disease. However, advances in FFPE tissue-based proteomics have been hampered by inefficient methods to extract proteins from archival tissue and by an incomplete knowledge of formaldehyde-induced modifications in proteins. To help address these problems, we have developed a procedure for the formation of 'tissue surrogates' to model FFPE tissues. Cytoplasmic proteins, such as lysozyme or ribonuclease A, at concentrations approaching the protein content in whole cells, are fixed with 10% formalin to form gelatin-like plugs. These plugs have sufficient physical integrity to be processed through graded alcohols, xylene, and embedded in paraffin according to standard histological procedures. In this study, we used tissue surrogates formed from one or two proteins to evaluate extraction protocols for their ability to quantitatively extract proteins from the surrogates. Optimal protein extraction was obtained using a combination of heat, a detergent, and a protein denaturant. The addition of a reducing agent did not improve protein recovery; however, recovery varied significantly with pH. Protein extraction of >80% was observed for pH 4 buffers containing 2% (w/v) sodium dodecyl sulfate (SDS) when heated at 100 degrees C for 20 min, followed by incubation at 60 degrees C for 2 h. SDS-polyacrylamide gel electrophoresis of the extracted proteins revealed that the surrogate extracts contained a mixture of monomeric and multimeric proteins, regardless of the extraction protocol employed. Additionally, protein extracts from surrogates containing carbonic anhydrase:lysozyme (1:2 mol/mol) had disproportionate percentages of lysozyme, indicating that selective protein extraction in complex multiprotein systems may be a concern in proteomic studies of FFPE tissues.

Complex of an active mu-opioid receptor with a cyclic peptide agonist modeled from experimental constraints.

Site-directed mutagenesis and design of Zn(2+)-binding centers have been used to determine a set of specific tertiary interactions between the mu-opioid receptor, a rhodopsin-like G protein-coupled receptor (GPCR), and its cyclic peptide agonist ligand, Tyr(1)-c(S-Et-S)[d-Cys(2)-Phe(3)-d-Pen(4)]NH(2) (JOM6). The binding affinity of the tetrapeptide is strongly dependent on the nature of its first and third residues and on substitutions at positions 213, 216, 237, 300, 315, and 318 of the mu-opioid receptor. His(1) and His(3) analogues of the ligand were able to form metal-binding complexes with the V300C and G213C/T315C receptor mutants, respectively. Direct contact of the Phe(3) residue of JOM6 with Gly(213), Asp(216), Thr(315), and Trp(318) of the receptor was suggested by the binding affinities of His(3)-, Nle(3)-, Leu(3)-, Aci(3)-, Delta(E)Phe(3)-, and Delta(Z)Phe(3)-substituted peptides with the G213C/T315C, D216V, T315C, and W318L mutants. The improved binding affinity of the free carboxylate analogue of JOM6 for binding to the E229D mutant revealed an interaction between the C-terminal group of the peptide and Glu(229) of the receptor. The experimental constraints that were obtained were applied for distance geometry modeling of the mu-receptor in complex with the tetrapeptide agonist ligand, JOM6. The active conformation of the opioid receptor was calculated using the crystal structure of "inactive" rhodopsin and published engineered and intrinsic metal-binding sites and disulfide bonds that allow or facilitate activation of GPCRs. Interhelical H-bonds existing in the mu-receptor were applied as additional distance constraints. The calculated model of the receptor-ligand complex can serve as a prototype of the active state for all rhodopsin-like GPCRs. It displays a strongly shifted transmembrane helix 6 (TM6) and reorientation of the conserved Trp(293) residue in TM6 upon its interaction with the agonist. Importantly, the binding pockets of the active and inactive states are not identical, which implies distinct interaction modes of agonists and antagonists. In the active state, the binding pocket of the mu-receptor is complementary to the previously proposed receptor-bound conformation of JOM6.

Refinement of a homology model of the mu-opioid receptor using distance constraints from intrinsic and engineered zinc-binding sites.

Publication of the rhodopsin X-ray structure has facilitated the development of homology models of other G protein-coupled receptors. However, possible shifts of transmembrane (TM) alpha helices, expected variations in helical distortions, and differences in loop size necessitate experimental verification of these comparative models. To refine a rhodopsin-based homology model of the mu-opioid receptor (MOR), we experimentally determined structural-distance constraints from intrinsic and engineered metal-binding sites in the rat MOR. Investigating the relatively high intrinsic affinity of MOR for Zn(2+) (IC(50) approximately 30microM), we observed that mutation of His(319) (TM7) abolished Zn(2+) inhibition of ligand binding, while mutation of Asp(216) (extracellular loop 2) decreased the effect of Zn(2+), suggesting these residues participate in the intrinsic Zn(2+)-binding center of MOR. To verify the relative orientation of TM5 and TM6 and to examine whether a rhodopsin-like alpha aneurism is present in TM5, we engineered Zn(2+)-binding centers by mutating residues of TM5 and TM6 to Cys or His, making use of the native His(297) in TM6 as an additional Zn(2+)-coordination site. Inhibition of opioid ligand binding by Zn(2+) suggests that residues Ile(234) and Phe(237) in TM5 face the binding-site crevice and form a metal-binding center with His(297) and Val(300) in TM6. This observation is inconsistent with a rhodopsin-like structure, which would locate Ile(234) on the lipid-exposed side of TM5, too distant from other residues making up the Zn(2+)-binding site. Subsequent distance geometry refinement of the MOR model indicates that the rhodopsin-like alpha aneurism is likely absent in TM2 but present in TM5.