Tools and methods for circular dichroism spectroscopy of proteins: a tutorial review.

Circular dichroism (CD) spectroscopy is a widely-used method in biochemistry, structural biology and pharmaceutical chemistry. More than 24 000 papers published in the past decade have included CD characterisations of proteins; many of those studies have also included other complementary chemical, biophysical, and computational chemistry methods. This tutorial review describes the background to the technique of CD spectroscopy and good practice methods for high quality data collection. It specifically focuses on both established and new methods and tools available for experimental design and interpretation, data processing, visualisation, analysis, validation, archiving, and accession, including tools developed to enhance the complementarity of this method with other structural and chemical biology studies.

Circular dichroism spectroscopy of membrane proteins.

Circular dichroism (CD) spectroscopy is a well-established technique for studying the secondary structures, dynamics, folding pathways, and interactions of soluble proteins, and is complementary to the high resolution but generally static structures produced by X-ray crystallography, NMR spectroscopy, and cryo electron microscopy. CD spectroscopy has special relevance for the study of membrane proteins, which are difficult to crystallise and largely ignored in structural genomics projects. However, the requirement for membrane proteins to be embedded in amphipathic environments such as membranes, lipid vesicles, detergent micelles, bicelles, oriented bilayers, or nanodiscs, in order for them to be soluble or dispersed in solution whilst maintaining their structure and function, necessitates the use of different experimental and analytical approaches than those employed for soluble proteins. This review discusses specialised methods for collecting and analysing membrane protein CD data, highlighting where protocols for soluble and membrane proteins diverge.

Structure of the C-terminal domain of the prokaryotic sodium channel orthologue NsvBa.

Crystallographic and electrophysiological studies have recently provided insight into the structure, function, and drug binding of prokaryotic sodium channels. These channels exhibit significant sequence identities, especially in their transmembrane regions, with human voltage-gated sodium channels. However, rather than being single polypeptides with four homologous domains, they are tetramers of single domain polypeptides, with a C-terminal domain (CTD) composed of an inter-subunit four helix coiled coil. The structures of the CTDs differ between orthologues. In NavBh and NavMs, the C-termini form a disordered region adjacent to the final transmembrane helix, followed by a coiled-coil region, as demonstrated by synchrotron radiation circular dichroism (SRCD) and double electron-electron resonance electron paramagnetic resonance spectroscopic measurements. In contrast, in the crystal structure of the NavAe orthologue, the entire C-terminus is comprised of a helical region followed by a coiled coil. In this study, we have examined the CTD of the NsvBa from Bacillus alcalophilus, which unlike other orthologues is predicted by different methods to have different types of structures: either a disordered region adjacent to the transmembrane region, followed by a helical coiled coil, or a fully helical CTD. To discriminate between the two possible structures, we have used SRCD spectroscopy to experimentally determine the secondary structure of the C-terminus of this orthologue and used the results as the basis for modeling the open and closed conformations of the channel.

X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure-function studies of a neuronal calcium-signalling protein.

The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated following a Pavlovian conditioning protocol. Subsequent studies showed that calexcitin regulates the voltage-dependent potassium channel and the calcium-dependent potassium channel as well as causing the release of calcium ions from the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal structure of calexcitin from the squid Loligo pealei showed that the fold is similar to that of another signalling protein, calmodulin, the N- and C-terminal domains of which are known to separate upon calcium binding, allowing interactions with the target protein. Phosphorylation of calexcitin causes it to translocate to the cell membrane, where its effects on membrane excitability are exerted and, accordingly, L. pealei calexcitin contains two protein kinase C phosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimic phosphorylation of the protein were introduced and crystal structures of the corresponding single and double mutants were determined, which suggest that the C-terminal phosphorylation site (Thr188) exerts the greatest effects on the protein structure. Extensive NMR studies were also conducted, which demonstrate that the wild-type protein predominantly adopts a more open conformation in solution than the crystallographic studies have indicated and, accordingly, normal-mode dynamic simulations suggest that it has considerably greater capacity for flexible motion than the X-ray studies had suggested. Like calmodulin, calexcitin consists of four EF-hand motifs, although only the first three EF-hands of calexcitin are involved in binding calcium ions; the C-terminal EF-hand lacks the appropriate amino acids. Hence, calexcitin possesses two functional EF-hands in close proximity in its N-terminal domain and one functional calcium site in its C-terminal domain. There is evidence that the protein has two markedly different affinities for calcium ions, the weaker of which is most likely to be associated with binding of calcium ions to the protein during neuronal excitation. In the current study, site-directed mutagenesis has been used to abolish each of the three calcium-binding sites of calexcitin, and these experiments suggest that it is the single calcium-binding site in the C-terminal domain of the protein which is likely to have a sensory role in the neuron.

Circular dichroism spectral data and metadata in the Protein Circular Dichroism Data Bank (PCDDB): a tutorial guide to accession and deposition.

The Protein Circular Dichroism Data Bank (PCDDB) is a web-based resource containing circular dichroism (CD) and synchrotron radiation circular dichroism spectral and associated metadata located at http://pcddb.cryst.bbk.ac.uk. This resource provides a freely available, user-friendly means of accessing validated CD spectra and their associated experimental details and metadata, thereby enabling broad usage of this material and new developments across the structural biology, chemistry, and bioinformatics communities. The resource also enables researchers utilizing CD as an experimental technique to have a means of storing their data at a secure site from which it is easily retrievable, thereby making their results publicly accessible, a current requirement of many grant-funding agencies world-wide, as well as meeting the data-sharing requirements for journal publications. This tutorial provides extensive information on searching, accessing, and downloading procedures for those who wish to utilize the data available in the data bank, and detailed information on deposition procedures for creating and validating entries, including comprehensive explanations of their contents and formats, for those who wish to include their data in the data bank.

Effect of anoreceptive intercourse on anorectal function.

This study is the first published assessment of the effect of anoreceptive intercourse (ARI) on anal sphincter tone and function. Forty anoreceptive (AR) male homosexuals were compared with 18 age matched non-anoreceptive (non-AR) heterosexual males. Subjects were questioned about ARI, defaecation and faecal incontinence. Anal resting pressure, maximum voluntary squeeze pressure, anal mucosal electrosensitivity, perineal descent and rectal sensation were measured in all subjects. Fourteen of the AR subjects but only one of the non-AR subjects had symptoms of frequent anal incontinence (P < 0.05). There was a significant reduction in both maximum anal resting pressure (P < 0.01) and anal mucosal electrosensitivity (P < 0.05) and a significant difference in the anal resting pressure profile (P = 0.02) in the AR subjects compared with the non-AR subjects. There was a significant reduction in maximum squeeze pressure in AR subjects with anal incontinence compared with either AR subjects without anal incontinence (P < 0.01) or non-AR subjects (P < 0.01). There were no significant differences in stoll consistency, frequency of defaecation, perineal descent or rectal sensation between the groups. ARI is associated with reduced resting pressure in the anal canal and an increased risk of anal incontinence. The risk of incontinence is greatest amongst AR subjects with reduced maximum squeeze pressure.

Persistent ulceration of the anal margin in homosexuals with HIV infection.

A study of the outcome of surgical treatment of ulceration of the anal margin occurring in male homosexuals with HIV infection was undertaken. Ten patients with acquired immunodeficiency syndrome and three patients with symptomatic HIV infection were referred to the Department of Surgery with painful anal ulceration which had not responded to medical treatment. The medical treatments given prior to surgical referral included high dose oral acyclovir, intravenous foscarnet and broad spectrum antibiotics. Excision biopsy was performed in 12 patients and in 11 cases was followed by healing of the ulcers within 10 weeks. One patient died 2 weeks postoperatively from Pneumocystis carinii pneumonia without healing. The response to excision biopsy was unexpected but suggests that surgical excision may be beneficial for lesions which have failed to respond to aggressive medical treatment.

The impact of stenting obstructing colorectal tumours in a district general hospital.

INTRODUCTION: The technique of stenting malignant obstructing colorectal lesions is established as an acceptable treatment with a low morbidity and mortality. This paper reviews our experience in stenting malignant colorectal obstruction and compares this group with those who underwent emergency surgery as their primary intervention. METHODS: A retrospectively kept database over four years was reviewed and patients who had undergone either stenting or emergency surgery for a malignant colorectal obstruction were identified. These patients' notes were retrieved and reviewed. RESULTS: During the duration of study, a total of 29 stents were placed in 28 patients, with a mean age of 78 y (range 59-96 years). Patients generally had significant co-existing morbidity, with a median ASA score of 2.5. The timing of stent placement was a mean of 3.4 days (1-9 days) after presentation, including time for relevant investigation and diagnosis. Mean length of hospital stay was 9.8 days (2-36 days). In the emergency operation category, during the period of study, a total of 38 patients had operations for large bowel obstruction, either because the lesion was not suitable for stenting, or the personnel for stenting were not available. These patients ranged in age from 45 to 96 years, with a mean age of 72.4 years. Patients in this group were generally a little fitter than the stented group, with a median ASA grade of 2, and 14/38 patients were ASA1 (the largest group). Despite this Post-operative recovery was slow with these patients, the average length of stay being 16 days (range 8-66 days). CONCLUSIONS: In this study, we report our data on the first four years of stenting malignant bowel obstruction. It is a feasible and acceptable means of treatment, and we have demonstrated comparable morbidity and mortality to that reported in medical literature. The technique may avoid the need for emergency operation with its concomitant risks, lengthy in-patient stay, and high likelihood for a stoma. We would advocate the use of self expanding metal stents where appropriate in the management of large bowel obstruction.

Light flux density threshold at which protein denaturation is induced by synchrotron radiation circular dichroism beamlines.

New high-flux synchrotron radiation circular dichroism (SRCD) beamlines are providing important information for structural biology, but can potentially cause denaturation of the protein samples under investigation. This effect has been studied at the new CD1 dedicated SRCD beamline at ISA in Denmark, where radiation-induced thermal damage effects were observed, depending not only on the radiation flux but also on the focal spot size of the light. Comparisons with similar studies at other SRCD facilities worldwide has lead to the estimation of a flux density threshold under which SRCD beamlines should be operated when samples are to be exposed to low-wavelength vacuum ultraviolet radiation for extended periods of time.

Day case haemorrhoidectomy.

OBJECTIVE: Day case haemorrhoidectomy in a District General Hospital. We have investigated the uptake and outcome of day case haemorrhoidectomy in a small district general hospital. METHOD: Case note review with completion of standard proforma for all patients undergoing haemorrhoidectomy as day case (same day admission and discharge) or inpatient over a 4-year period. RESULTS: Sixty patients underwent day case closed haemorrhoidectomy, 2 day case stapled haemorrhoidectomy and 1 day case open haemorrhoidectomy, whilst 18 patients were treated as in-patients. One patient required re-admission within 31 days for reactionary haemorrhage after day case surgery. None suffered acute urinary retention. Concomitant medical disease or emergency admission were the only reasons for exclusion from day case haemorrhoidectomy. Of the 18 patients treated as in-patients haemorrhoidectomy two had unplanned readmission. CONCLUSION: Closed haemorrhoidectomy with same day discharge should be offered to all patients that require surgical treatment of haemorrhoids and do not have other contra-indications to day case surgery.

VUV irradiation effects on proteins in high-flux synchrotron radiation circular dichroism spectroscopy.

Synchrotron radiation circular dichroism (SRCD) spectroscopy is emerging as an important new tool in structural molecular biology. Previously we had shown that in lower-flux SRCD instruments, such as UV1 at ISA and beamline 3.1 at the SRS, vacuum ultraviolet (VUV) radiation damage to proteins was not evident after exposure over a period of hours. No effects were detected in either the protein primary or the secondary structures. However, with the development of high-flux beamlines, such as CD12 at the SRS, this issue has been revisited because of changes observed in the SRCD spectra of consecutive scans of protein samples obtained on this high-flux beamline. Experiments have been designed to distinguish between two different possible mechanisms: (i) photoionization causing free radicals or secondary electrons producing degradation of the protein, and (ii) local heating of the sample resulting in protein denaturation. The latter appears to be the principal source of the signal deterioration.

Possible occupational asthma among adults presenting with acute asthma.

BACKGROUND: Little is known about the risk of occupational asthma or its causative agents in South Africa. The objective of this study was to determine the proportion of adult asthmatics whose asthma may be occupational, and the main agents or occupations involved. DESIGN AND SETTING: A descriptive surveillance study of adult patients presenting with acute asthma to the casualty units of two large public hospitals in Cape Town. METHODS: A brief questionnaire was completed by the casualty staff for a sample of 140 adult asthmatic patients. Respondents were classified as having possible occupational asthma on the basis of adult-onset asthma, exposure at time of onset to a known or suspected cause of occupational asthma, and symptoms that improved away from work. RESULTS: Eighteen patients (12.9%, 95% confidence interval (CI) 7.8-19.6%) met the criteria for occupational asthma. The main occupational categories in this group were spray painters (4 patients) and domestic workers (4 patients), with cleaning agents, dyes and paints being the most commonly reported exposures. A total of 36 patients (25.7%, 95% CI 18.7-33.7%) reported work aggravation of their asthma. CONCLUSIONS: A clinically significant proportion of adult-onset asthmatics, men and women, may have occupational causation, while an even larger proportion may have occupational aggravation. Since early intervention favourably influences prognosis in occupational asthma, all practitioners dealing with adult asthmatics should explore occupational factors.

Surgical management of anorectal disease in HIV-positive homosexuals.

One thousand and ninety human immunodeficiency virus (HIV)-positive homosexual or bisexual males were seen in one hospital for management of HIV disease over a 9-year period. One hundred and fifty-five patients were referred by acquired immunodeficiency syndrome (AIDS) physicians for general surgical management. The most frequent reason for surgical referral (64 patients) was anorectal disease which occurred in 5.9 per cent of all HIV-positive patients. One or more diagnoses were reached in 61 of the 64 patients referred with anorectal disease: warts (38 per cent of diagnoses), anorectal ulceration (26 per cent), perianal sepsis (15 per cent), neoplasia (14 per cent) and haemorrhoidal disease (8 per cent). Anorectal symptoms were relieved in 68 per cent of patients and the median survival of those treated was 17.5 months from the time of surgical referral. Both warts and perianal sepsis were associated with in situ neoplasia, but no case of progression from in situ to invasive anal squamous carcinoma was detected. The aetiology of anorectal ulcers was not clear, but surgical excision of anal ulcers and skin tags can produce healing. Palliation of anorectal symptoms in HIV-positive homosexual patients is possible but some conditions are unusual and surgeons should be familiar with their presentation and management.

Randomized trial of laparoscopic cholecystectomy and mini-cholecystectomy.

Three hundred and ten patients having elective cholecystectomy were randomized to either laparoscopic cholecystectomy or mini-cholecystectomy. There were 155 patients in each group. Conversion to open cholecystectomy was significantly more common with laparoscopic cholecystectomy (13 versus 4 per cent) and complications were significantly more frequent with laparoscopic cholecystectomy (9 versus 3 per cent). If laparoscopic cholecystectomy was successful, hospital stay was significantly shorter than for mini-cholecystectomy (2 versus 3 days respectively), but overall the hospital stay was not significantly different. Postoperative analgesia requirements were reduced and return to normal activities and to work were faster after laparoscopic cholecystectomy. There was no significant cost difference between the two procedures.

Promotion of cell adhesion by single-stranded and triple-helical peptide models of basement membrane collagen alpha 1(IV)531-543. Evidence for conformationally dependent and conformationally independent type IV collagen cell adhesion sites.

Several regions within the triple-helical domain of type IV collagen function as cellular recognition sites. We have demonstrated previously that melanoma cell activities promoted by the alpha 1(IV)1263-1277 sequence are enhanced by triple helicity (Fields, C. G., Mickelson, D. J., Drake, S.L., McCarthy, J.B., and Fields, G.B. (1993) J. Biol. Chem. 268, 14153-14160), whereas Eble et al. reached similar conclusions for alpha 1 beta 1 integrin-mediated fibrosarcoma cell adhesion to [alpha 1(IV)]2 alpha 2(IV)434-472 (Eble, J. A., Golbik, R., Mann, K., and Kühn, K. (1993) EMBO J. 12, 4795-4802). In the present study, we have examined the cell adhesion activities of a third region in type IV collagen. A single-stranded peptide (SSP) incorporating the alpha 1(IV)531-543 sequence promoted the adhesion of melanoma, ovarian carcinoma, and Jurkat cells in a dose-dependent manner, with 40% cell adhesion observed at [SSP] = 1.8, 11.5, and 42.2 microM, respectively. Nearly identical results were obtained for cell adhesion to an all-D-enantiomer of the SSP, suggesting that the cell surface receptor(s) for this site do not discriminate based on chirality. The alpha 1(IV)531-543 sequence maintained its cell adhesion promoting activity when incorporated into a homotrimeric triple-helical polypeptide, although relative levels of adhesion were either slightly enhanced or slightly diminished compared with the SSP. Triple-helical conformation was thus not critical for cellular recognition of the alpha 1(IV)531-543 sequence. Single-site substitution experiments of the SSP showed no overall correlation between the biological effects of substitutions and SSP conformation. The SSP, D-SSP, triple-helical polypeptide, and SSP substitution results suggest that cell recognition of the alpha 1(IV)531-543 sequence is generally independent of substrate conformation. The present and prior studies indicate that "conformationally dependent" and "conformationally independent" cellular recognition sites exist within the triple-helical domain of type IV collagen.

Promotion of fibroblast adhesion by triple-helical peptide models of type I collagen-derived sequences.

The dissection of the activities mediated by type I collagen requires an approach by which the influence of triple-helical conformation can be evaluated. The alpha 1 beta 1 and alpha 2 beta 1 integrin binding sites within type I collagen are dependent upon triple-helical conformation and contained within residues 14-822 from alpha 1(I). Seven alpha 1(I)-derived triple-helical peptides (THPs) were synthesized based on charge clustering (alpha 1(I)256-270, alpha 1(I)385-396, alpha 1(I)406-417, alpha 1(I)415-423, alpha 1(I)448-456, alpha 1(I)496-507, and alpha 1(I)526-537). Three additional THPs were synthesized (alpha 1(I)85-96, alpha 1(I)433-441, and alpha 1(I)772-786) based on previously described or proposed activities (Kleinman, H. K., McGoodwin, E.B., Martin, G. R., Klebe, R. J., Fietzek, P. P., and Wooley, D. E. (1978) J. Biol. Chem. 253, 5642-5646; Staatz, W. D., Foik, K. F., Zutter, M. M., Adams, S. P., Rodriquez, B. A., and Santoro, S. A. (1991) J. Biol. Chem. 266, 7363-7367; San Antonio, J. D., Lander, A. D., Karnovsky, M. J., and Slayter, H. S. (1994) J. Cell Biol. 125, 1179-1188). Of the ten THPs, alpha 1(I)772-786 THP had the greatest activity, with half-maximal normal dermal fibroblast adhesion occurring at a peptide concentration of 1.6 microM. Triple-helicity was essential for activity of this sequence, as the non-triple-helical peptide analog (alpha 1(I)772-786 SSP) exhibited considerably lower levels of cell adhesion promotion even at peptide concentrations as high as 100 microM. Within the sequence itself, residues 784-786 (Gly-Leu-Hyp) were important for cellular recognition, as the alpha 1(I)772-783 THP had greatly reduced cell adhesion activity compared with alpha 1(I)772-786 THP. Preliminary studies indicate that the beta 1 integrin subunit mediates fibroblast adhesion to alpha 1(I)772-786 THP. The identification of fibroblast integrin binding sites within type I collagen may have important implications for understanding collagen metabolism.

CDtool-an integrated software package for circular dichroism spectroscopic data processing, analysis, and archiving.

CDtool is a software package written to facilitate circular dichroism (CD) spectroscopic studies on both conventional lab-based instruments and synchrotron beamlines. It takes format-independent input data from any type of CD instrument, enables a wide range of standard and advanced processing methods, and, in a single user-friendly graphics-based package, takes raw data through the entire processing procedure and, importantly, uses data-mining techniques to retain in the final output all the information associated with the processing. It permits the facile comparison of data obtained from different instruments without the need for reformatting and displays it in graphical formats suitable for publication. It also includes the ability to automatically archive the processed data. This latter feature may be especially useful in light of recent funding institution directives with regard to data sharing and archiving and requirements for "good practice" and "traceability" within the pharmaceutical industry. In addition, CDtool includes a means of interfacing with protein data bank coordinate files and calculating secondary structures from them using alternate definitions and algorithms. This feature, along with a function that permits the facile production of new reference databases, enables the creation of specialized databases for secondary structural analyses of specific types of proteins. Thus the CDtool software not only enables rapid data processing and analyses but also includes many enhanced features not available in other CD data processing/analysis packages.

A peptide model of basement membrane collagen alpha 1 (IV) 531-543 binds the alpha 3 beta 1 integrin.

Tumor cell adhesion to the triple-helical domain of basement membrane (type IV) collagen occurs at several different regions. Cellular recognition of the sequence spanning alpha 1(IV)531-543 has been proposed to be independent of triple-helical conformation (Miles, A. J., Skubitz, A. P. N., Furcht, L. T., and Fields, G. B. (1994) J. Biol. Chem. 269, 30939-30945). In the present study, integrin interactions with a peptide analog of the alpha 1(IV)-531-543 sequence have been analyzed. Tumor cell adhesion (melanoma, ovarian carcinoma) to the alpha 1(IV)531-543 chemically synthesized peptide was inhibited by a monoclonal antibody against the alpha 3 integrin subunit, and to a lesser extent by monoclonal antibodies against the beta 1 and alpha 2 integrin subunits. An anti-alpha 5 monoclonal antibody and normal mouse IgG were ineffective as inhibitors of tumor cell adhesion to the peptide. Two cell surface proteins of 120 and 150 kDa bound to an alpha 1(IV)531-543 peptide affinity column and were eluted with 20 mM EDTA. When the eluted proteins were incubated with monoclonal antibodies against either the alpha 3 or beta 1 integrin subunit, proteins corresponding in molecular weight to alpha 3 and beta 1 integrin subunits were precipitated. No proteins were immunoprecipated with monoclonal antibodies against the alpha 2 or alpha 5 integrin subunits. Thus, the alpha 3 beta 1 integrin from two tumor cell types has been shown to bind directly to the alpha 1 (IV)531-543 peptide. The alpha 1(IV)531-543 peptide is the first collagen-like sequence that has been shown to bind the alpha 3 beta 1 integrin.