Serratiopeptidase: a well-known metalloprotease with a new non-proteolytic activity against S. aureus biofilm.

BACKGROUND: The use of indwelling medical devices is associated with a significant risk of infections by Staphylococcus aureus (S. aureus) which possesses a variety of virulence factors including many toxins and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The virulence factors above described are often related to proteins exposed on the bacterial surface. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases. Previously reports evaluated the anti-infective properties of serratiopeptidase (Spep), an extracellular metalloprotease produced by Serratia marcescens ATCC 21074 (E-15), in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. However, to date its mechanism of action is unknown. METHODS: Spep gene was PCR amplified and cloned into expression vector pET28b(+). The mutant EspepA was constructed from plasmid pET28b-Spep applying the one-step overlap extension PCR strategy. There sulting plasmids were costransformed in EcBL21(DE3) cells with the plasmid pRuW4inh1 harboring the Erwinia chrysanthemi secretion system. Bacterial pellets and supernatants were collected and analyzed by SDS-PAGE and zymography. The unambiguous identification and a detailed structure characterization of both the wild type and the mutant Spep were obtained by mass spectrometric analyses. The resultant supernatants sterilized by filtration were separately used to condition biofilm formation of S. aureus. Quantification was based on crystal violet method. RESULTS: In this work we constructed Spep mutant by substituting the glutamic acid in the catalytic site with a residue of alanine. In this manner we were able to evaluate the anti-biofilm activity of Spep mutant in absence of proteolytic activity. As expected, this mutant did not display protease activity but it retained its anti-biofilm properties, suggesting that this action is independent by enzymatic activity. CONCLUSIONS: New knowledge obtained from data reported in this paper calls attention to a novel mechanism of action of Spep. This protein could be developed as a potential "antipathogenic agent" capable to impair the ability of S. aureus to form biofilm on prostheses, catheters and medical devices, exploiting a mechanism different from the proteolytic activity.

Comparison of the action of different proteases on virulence properties related to the staphylococcal surface.

AIMS: The purpose of this study was to evaluate the antimicrobial efficacy of five different proteases belonging to two different families on Staphylococcus aureus and Staphylococcus epidermidis strains. METHODS AND RESULTS: We used three serine proteases and two metalloproteases in single species biofilm formation assays and in human cell invasion processes. Following each protease incubation with bacterial cells, surface protein patterns were analysed by SDS-PAGE and zymography. Some differently expressed proteins were identified by mass spectrometry. CONCLUSIONS: The effect of tested proteases on biofilm formation was not related to the protease category but was strain-dependent and was related to the biofilm formation capacity of each staphylococcal strain. Some proteases showed a nonspecific and indiscriminate effect on surface proteins, while others induced a discrete and reproducible action on protein profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of the surface-related virulence factors is a promising avenue to overcome persistent infections caused by bacterial biofilms. To this end, we show here that proteases, in particular the metalloprotease serratiopeptidase, can interfere with adhesion and invasion of eukaryotic cells and biofilm formation in staphylococci and their use could represent a viable treatment for the development of novel combination therapies.

A new anti-infective strategy to reduce adhesion-mediated virulence in Staphylococcus aureus affecting surface proteins.

Staphylococcus aureus is a flexible microbial pathogen frequently isolated from community-acquired and nosocomial infections. The use of indwelling medical devices is associated with a significant risk of infection by this bacterium which possesses a variety of virulence factors, including many toxins, and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The present study evaluates the anti-infective properties of serratiopeptidase, a secreted protein of Serratia marcescens, in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. SPEP seems to exert its action by modulating specific proteins. Proteomic studies performed on surface proteins extracted from SPEP-treated S. aureus cultures revealed that a number of proteins are affected by the treatment. Among these we found the adhesin/autolysin Atl, FnBP-A, SecA1, Sbi, EF-Tu, EF-G, and alpha-enolase. EF-Tu, EF-G and alpha-enolase are known to perform a variety of functions, depending on their cytoplasmic or surface localization. All these factors can facilitate bacterial colonization, persistence and invasion of host tissues. Our results suggest that SPEP could be developed as a potential anti-infective agent capable to hinder the entry of S. aureus into human tissues, and also impair the ability of this pathogen to form biofilm on prostheses, catheters and medical devices.

Identification of the prion protein allotypes which accumulate in the brain of sporadic and familial Creutzfeldt-Jakob disease patients.

A characteristic feature of Creutzfeldt-Jakob disease (CJD) is the accumulation in the brain of the amyloid protease-resistant protein PrPres. PrPres derives from a host-encoded, protease-sensitive isoform, PrPsen. Mutations of this protein are linked to familial variants of the disease, and the presence of a methionine or valine residue at the polymorphic position 129 may be critical in sporadic CJD cases. We found that in the brain of patients heterozygous for the mutation in which isoleucine is substituted for valine at codon 210 (Val21Olle), the PrPres is formed by both the wild-type and mutant PrPsen. We also found that in a sporadic CJD patient, who was heterozygous (Met/Val) at position 129, PrPres is also formed by both allotypes. These data associate transmissible spongiform encephalopathies with other amyloidosis, although the nature of the transmissible agent remains unsettled.

Different carbon sources affect lifespan and protein redox state during Saccharomyces cerevisiae chronological ageing.

In this study, a proteomic approach that combines selective labelling of proteins containing reduced cysteine residues with two-dimensional electrophoresis/mass spectrometry was used to evaluate the redox state of protein cysteines during chronological ageing in Saccharomyces cerevisiae. The procedure was developed on the grounds that biotin-conjugated iodoacetamide (BIAM) specifically reacts with reduced cysteine residues. BIAM-labelled proteins can then be selectively isolated by streptavidin affinity capture. We compared cells grown on 2% glucose in the exponential phase and during chronological ageing and we found that many proteins undergo cysteine oxidation. The target proteins include enzymes involved in glucose metabolism. Both caloric restriction and growth on glycerol resulted in a decrease in the oxidative modification. Furthermore, in these conditions a reduced production of ROS and a more negative glutathione half cell redox potential were observed.

Primary Sjögren's syndrome: Extraglandular manifestations and hydroxychloroquine therapy.

The use of hydroxychloroquine (HCQ) in Primary Sjögren's Syndrome (pSS) has been assessed in different studies over the last years, with conflicting results regarding its efficacy in sicca syndrome and extraglandular manifestations (EGM). The goal of this study was to compare the incidence rate of EGM in pSS patients with and without HCQ therapy.We performed a multicenter retrospective study, including patients with pSS (European classification criteria) with at least 1 year of follow-up. Subjects with concomitant fibromyalgia, autoimmune hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis were excluded. Demographics and pSS characteristics were recorded. The EGM were defined by EULAR-SS disease activity index (ESSDAI). Patients were divided into two groups according to their use or not of HCQ therapy. We evaluated the use of HCQ and its relationship to EGM. HCQ therapy was defined as the continuous use of the drug for at least 3 months. A descriptive analysis of demographics and pSS characteristics was performed. We compared the incidence of EGM between groups defined by HCQ therapy using chi2 test or Fisher's exact test. A total of 221 patients were included (97.3% women), mean age, 55.7 years (SD 14). Mean age at diagnosis, 48.8 years (SD 15); median disease duration, 60 months (IQR 35-84). One hundred and seventy patients (77%) received HCQ. About half of the patients had at least one EGM during the course of the disease, 20% of them developed an EGM before the onset of the sicca syndrome and 26% simultaneously with dryness symptom. Overall, EGM were less frequent in those on HCQ therapy (36.5% vs 63.5%, p < 0.001). Considering each EGM individually, the following manifestations were more frequent in the non-treated group: arthritis (p < 0.001), fatigue (p < 0.001), purpura (p = 0.01), Raynaud phenomenon (p = 0.003), and hypergammaglobulinemia (p = 0.006). Immunosuppressive treatment was indicated on 28 patients (12.7%), 13 of which were receiving also HCQ. The first reason for those treatments was the presence of arthritis in 12/28 patients (42.8%), and the drug used in all the cases was methotrexate. Only three patients required immunosuppressive therapy with cyclophosphamide, due to the presence of glomerulonephritis, vasculitis, and interstitial lung disease. None of the patients received biologic therapy. The lower incidence of EGM was observed in patients on HCQ therapy supports its efficacy in pSS. However, further large scale prospective studies are needed to confirm these findings.

Limited proteolysis in the investigation of beta2-microglobulin amyloidogenic and fibrillar states.

Amyloid fibrils of patients treated with regular haemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment shows a higher propensity to self-aggregate and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology and the conformational analysis of native beta2-m and the truncated DeltaN6beta2-m species both in the soluble and in the fibrillar forms were investigated by the limited proteolysis/mass spectrometry strategy. The conformation in solution of a further truncated mutant DeltaN3beta2-m lacking three residues at the N-terminus was also examined. This approach appeared particularly suited to investigate the regions that are solvent-exposed, or flexible enough to be accessible to protein-protein interactions and to describe the conformation of transient intermediates. Moreover, proteolysis experiments can also be tailored to investigate amyloid fibrils by discriminating the protein regions constituting the unaccessible core of the fibrils and those still flexible and exposed to the solvent. Although native beta2-m and DeltaN3beta2-m shared essentially the same conformation, significative structural differences exist between the native and the DeltaN6beta2-m proteins in solution with major differences located at the end moiety of strand V and subsequent loop with strand VI and at both the N- and C-termini of the proteins. On the contrary, an identical distribution of preferential proteolytic sites was observed in both proteins in the fibrillar state, which was nearly superimposible to that observed for the soluble form of DeltaN6beta2-m. These data revealed that synthetic fibrils essentially consists of an unaccessible core comprising residues 20-87 of the beta2-m protein with exposed and flexible N- and C-terminal ends. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to take place in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these results, a molecular mechanism for fibril formation has been proposed.

Partial purification and MALDI-TOF MS analysis of UN1, a tumor antigen membrane glycoprotein.

UN1 is a membrane glycoprotein that is expressed in immature human thymocytes, a subpopulation of peripheral T lymphocytes, the HPB acute lymphoblastic leukemia (ALL) T-cell line and fetal thymus. We previously reported the isolation of a monoclonal antibody (UN1 mAb) recognizing the UN1 protein that was classified as "unclustered" at the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. UN1 was highly expressed in breast cancer tissues and was undetected in non-proliferative lesions and in normal breast tissues, indicating a role for UN1 in the development of a tumorigenic phenotype of breast cancer cells. In this study, we report a partial purification of the UN1 protein from HPB-ALL T cells by anion-exchange chromatography followed by immunoprecipitation with the UN1 mAb and MALDI-TOF MS analysis. This analysis should assist in identifying the amino acid sequence of UN1.

Hemoglobin Ozieri: a new alpha-chain variant (alpha 71(E20)Ala-->Val). Characterization using FAB- and electrospray-mass spectrometric techniques.

A new silent hemoglobin variant, Hb Ozieri (alpha 71(E20)Ala-->Val), was observed in five apparently unrelated newborn babies during a screening for hemoglobinopathies on the island of Sardinia. This asymptomatic variant was detected by means of isoelectric focusing (IEF), isolated using IEF in an immobilized ultranarrow pH-gradient and characterized at the structural level using FAB- and electrospray-mass spectrometric techniques. A Val for Ala substitution was unambiguously detected at position 71 of the alpha-globin chain. This substitution indicates that a C to T transition occurred in the GCG codon for Ala which contains one of the 35 unmethylated CpG dinucleotides of the alpha-globin gene. This observation brings the number of variants due to a mutation in the alpha-globin gene CpGs (the third instance of a silent mutation) to 13 and raises the possibility that unmethylated CpGs might be hotspots for mutations as the methylated ones.

Analysis of human serum albumin variants by mass spectrometric procedures.

A new strategy for the structural characterisation of human albumin variants has been developed which makes extensive use of mass spectrometric methodologies. The rationale behind the method is to provide a rapid and effective screening of the entire albumin structure. The first step in this strategy consists in the attempt to determine the accurate molecular mass of the intact variant by electrospray mass spectrometry often providing a first indication on the presence of the variant. An HPLC procedure has been developed io isolate all the seven fragments generated by CNBr hydrolysis of HSA in a single chromatographic step. A rapid screening of the entire albumin structure is achieved by the ESMS analysis of the peptide fragments and the protein region(s) carrying the structural abnormality is identified by its anomalous mass value(s). Mass mapping of the corresponding CNBr peptide, either by Fast Atom Bombardment Mass Spectrometry (FABMS) or by Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDIMS), leads to the definition of the site and the nature of the variation. This combined strategy was applied to the structural characterisation of three HSA genetic variants and provided to be an effective procedure for the rapid assessment of their structural modifications showing considerable advantages over the classical approach.

Silent myocardial ischemia during ambulatory electrocardiographic monitoring in patients with effort angina.

The frequency and characteristics of asymptomatic ischemic attacks were investigated in 39 patients with effort angina. During 24 hour Holter monitoring, 32 of the 39 patients displayed one or more episodes of ischemic ST depression. Eight patients had attacks (n = 25) always accompanied by pain; 9 patients had only asymptomatic ischemic episodes (n = 40) and 15 patients had both symptomatic (n = 29) and asymptomatic attacks (n = 76). In the 15 patients exhibiting both symptomatic and asymptomatic attacks, mean duration of symptomatic episodes was longer (probability [p] less than 0.001) and mean maximal ST depression was greater (p less than 0.001). When patients exhibiting only symptomatic episodes were compared with those exhibiting only asymptomatic attacks, differences were not significant. All symptomatic and asymptomatic attacks during Holter monitoring were correlated with the results of stress testing: patients experiencing a delayed response to pain after the onset of St ischemic depression during stress testing had a higher ratio of asymptomatic to symptomatic attacks during Holter monitoring compared with those patients reporting pain before or at the moment of the appearance of ischemic electrocardiographic features during stress testing. It is concluded that: 1) asymptomatic episodes of ischemia are more frequent than symptomatic episodes in patients with effort angina; 2) in the same patient, the severity of ischemia is generally a fundamental factor in determining the presence or absence of pain during an ischemic attack; and 3) differences among patients with respect to predominance of symptomatic or asymptomatic attacks probably depend on individual factors.

Prognostic implications of restrictive left ventricular filling in reperfused anterior acute myocardial infarction.

OBJECTIVES: We sought to assess the relative prognostic role of a restrictive left ventricular (LV) filling pattern after a first anterior acute myocardial infarction (AMI) in patients treated with primary percutaneous transluminal coronary angioplasty (PTCA). BACKGROUND: In thrombolized patients, a short Doppler-derived mitral deceleration time (DT) of early filling is a powerful independent predictor of heart failure and death. However, it is still unknown whether the outcome of patients with AMI with a short DT may be improved by a more aggressive treatment. METHODS: In 104 patients, two-dimensional and Doppler echocardiograms were obtained three days after the index AMI. Coronary angiography was performed in all patients one and six months after PTCA. The patients were classified into two groups according to the DT duration: group 1 (n = 34) with DT < or = 130 ms and group 2 (n = 70) with DT >130 ms. All patients were followed-up for a mean (+/- SD) period of 32 +/- 10 months. RESULTS: During the follow-up period, 14 patients (13%) were admitted to the hospital for congestive heart failure, and 9 patients (9%) died. All cardiac deaths (n = 7) occurred in group 1. The survival rate at mean follow-up was 79% in group 1 and 97.2% in group 2 (p = 0.003). Multivariate Cox analysis showed that only age and restrictive filling were independent predictors of event-free survival. Furthermore, when survival with no cardiovascular events was analyzed, a short DT still emerged as the most powerful independent predictor. CONCLUSIONS: Patients with a restrictive LV filling pattern early after anterior AMI have a poor clinical outcome, even if treated with primary PTCA.

Topology of the calmodulin-melittin complex.

The topology of the Ca2+-calmodulin-melittin ternary complex has been investigated by a combined strategy which integrates limited proteolysis and cross-linking experiments with mass spectrometric methodologies. The rationale behind the methods is that the interface regions of two interacting proteins are accessible to the solvent in the isolated molecules, whereas they become protected following the formation of the complex. Therefore, when limited proteolysis experiments are carried out on both the isolated proteins and the complex, differential peptide maps are obtained from which the interface regions can be inferred. Alternatively, cross-linking reactions performed under strictly controlled conditions lead to the identification of spatially closed amino acid residues in the complex. Mass spectrometry can be employed in both procedures for the definition of the cleavage sites and to identify covalently linked residues. Our results show that melittin interacts with calmodulin by adopting a parallel orientation, i.e. the N and C-terminal halves of the peptide are anchored to the amino and carboxy-terminal domains of the protein, respectively. This orientation is inverted with respect to all the peptide substrates examined so far. A model of the complex was designed and refined on the basis of the experimental results, supporting the above conclusions. This finding reveals a further dimension to the already remarkable capability of calmodulin in binding different protein substrates, providing this protein with the capability of regulating an even larger number of enzymes.

Early intermediates in the PDI-assisted folding of ribonuclease A.

The oxidative refolding of ribonuclease A has been investigated in several experimental conditions using a variety of redox systems. All these studies agree that the formation of disulfide bonds during the process occurs through a nonrandom mechanism with a preferential coupling of certain cysteine residues. We have previously demonstrated that in the presence of glutathione the refolding process occurs through the reiteration of two sequential reactions: a mixed disulfide with glutathione is produced first which evolves to form an intramolecular S-S bond. In the same experimental conditions, protein disulfide isomerase (PDI) was shown to catalyze formation and reduction of mixed disulfides with glutathione as well as formation of intramolecular S-S bonds. This paper reports the structural characterization of the one-disulfide intermediate population during the oxidative refolding of Ribonuclease A under the presence of PDI and glutathione with the aim of defining the role of the enzyme at the early stages of the reaction. The one-disulfide intermediate population occurring at the early stages of both the uncatalyzed and the PDI-catalyzed refolding was purified and structurally characterized by proteolytic digestion followed by MALDI-MS and LC/ESIMS analyses. In the uncatalyzed refolding, a total of 12 disulfide bonds out of the 28 theoretical possible cysteine couplings was observed, confirming a nonrandom distribution of native and nonnative disulfide bonds. Under the presence of PDI, only two additional nonnative disulfides were detected. Semiquantitative LC/ESIMS analysis of the distribution of the S-S bridged peptides showed that the most abundant species were equally populated in both the uncatalyzed and the catalyzed process. This paper shows the first structural characterization of the one-disulfide intermediate population formed transiently during the refolding of ribonuclease A in quasi-physiological conditions that mimic those present in the ER lumen. At the early stages of the process, three of the four native disulfides are detected, whereas the Cys26-Cys84 pairing is absent. Most of the nonnative disulfide bonds identified are formed by nearest-neighboring cysteines. The presence of PDI does not significantly alter the distribution of S-S bonds, suggesting that the ensemble of single-disulfide species is formed under thermodynamic control.

Conformational changes in the NS3 protease from hepatitis C virus strain Bk monitored by limited proteolysis and mass spectrometry.

Conformational changes occurring within the NS3 protease domain from the hepatitis C virus Bk strain (NS3(1-180)) under different physico-chemical conditions either in the absence or in the presence of its cofactor Pep4A were investigated by limited proteolysis experiments. Because the surface accessibility of the protein is affected by conformational changes, when comparative experiments were carried out on NS3(1-180) either at different glycerol concentrations or in the presence of Pep4A, differential peptide maps were obtained from which protein regions involved in the structural changes could be inferred. The surface topology of isolated NS3(1-180) in solution was essentially consistent with the crystal structure of the protein with the N-terminal segment showing a high conformational flexibility. At higher glycerol concentration, the protease assumed a more compact structure showing a decrease in the accessibility of the N-terminal segment that either was forced to interact with the protein or originate intermolecular interactions with neighboring molecules. Binding of the cofactor Pep4A caused the displacement of the N-terminal arm from the protein moiety, leading this segment to again adopt an open and flexible conformation, thus suggesting that the N-terminus of the protease contributes only marginally to the stability of the complex. The observed conformational changes might be directly correlated with the activation mechanism of the protease by either the cosolvent or the cofactor peptide because they lead to tighter packing of the substrate binding site.

Selective and asymmetric action of trypsin on the dimeric forms of seminal RNase.

Dimeric seminal RNase (BS-RNase) is an equilibrium mixture of conformationally different quaternary structures, one characterized by the interchange between subunits of their N-terminal ends (the MXM form); the other with no interchange (the M=M form). Controlled tryptic digestion of each isolated quaternary form generates, as limit digest products, folded and enzymatically active molecules, very resistant to further tryptic degradation. Electrospray mass spectrometric analyses and N-terminal sequence determinations indicate that trypsin can discriminate between the conformationally different quaternary structures of seminal RNase, and exerts a differential and asymmetric action on the two dimeric forms, depending on the original quaternary conformation of each form. The two digestion products from the MXM and the M=M dimeric forms have different structures, which are reminiscent of the original quaternary conformation of the dimers: one with interchange, the other with no interchange, of the N-terminal ends. The surprising resistance of these tryptic products to further tryptic action is explained by the persistence in each digestion product of the original intersubunit interface.

Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: the case of Minibody.

A strategy that combines limited proteolysis experiments and mass spectrometric analysis of the fragments generated has been developed to probe protease-accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64-residue protein consisting of a beta-sheet scaffold based on the heavy-chain variable-domain structure of a mouse immunoglobulin and containing two segments corresponding to the hypervariable H1 and H2 regions. The low solubility of the protein prevented a detailed characterization by NMR and/or X-ray. Different proteases were used under strictly controlled conditions and the cleavage sites were mapped onto the anticipated Minibody model, leading to the identification of the most exposed regions. A single-residue mutant was constructed and characterized, following the same procedure, showing a slightly higher correspondence with the predicted model. This strategy can be used to effectively supplement NMR and X-ray investigations of protein tertiary structure, where these procedures cannot provide definitive data, or to verify and refine protein models.

Conformational analysis of putative regulatory subunit D of the toluene/o-xylene-monooxygenase complex from Pseudomonas stutzeri OX1.

A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for toluene/o-xylene-monooxygenase. The putative regulatory D subunit was expressed in Escherichia coli and purified. Its protein sequence was verified by mass spectrometry mapping and found to be identical to the sequence predicted on the basis of the DNA sequence. The surface topology of subunit D in solution was probed by limited proteolysis carried out under strictly controlled conditions using several proteases as proteolytic probes. The same experiments were carried out on the homologous P2 component of the multicomponent phenol hydroxylase from Pseudomonas putida CF600. The proteolytic fragments released from both proteins in their native state were analyzed by electrospray mass spectrometry, and the preferential cleavage sites were assessed. The results indicated that despite the relatively high similarity between the sequences of the two proteins, some differences in the distribution of preferential proteolytic cleavages were detected, and a much higher conformational flexibility of subunit D was inferred. Moreover, automatic modeling of subunit D was attempted, based on the known three-dimensional structure of P2. Our results indicate that, at least in this case, standard modeling procedures based on automatic alignment on the structure of P2 fail to produce a model consistent with limited proteolysis experimental data. Thus, it is our opinion that reliable techniques such as limited proteolysis can be employed to test three-dimensional models and highlight problems in automatic model building.

Surface topology of Minibody by selective chemical modifications and mass spectrometry.

The surface topology of the Minibody, a small de novo-designed beta-protein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysis of the modified fragments. Under appropriate conditions, the susceptibility of individual residues primarily depends on their surface accessibility so that their relative reactivities can be correlated with their position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramolecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values. The results of this study indicate that, while overall the Minibody model is correct, the beta-sheet formed by the N- and C-terminal segments is most likely distorted. This is also in agreement with previous results that were obtained using a similar approach where mass spectrometry was used to identify Minibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianchi E, Venturini S, Sollazzo M, Tramontano A. Marino G, Pucci P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of Minibody. Protein Sci 5:802-813). The chemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural information to complement simulation techniques. This is especially valuable when, as in the Minibody case, an NMR and/or X-ray structure cannot be obtained due to insufficient solubility of the molecule.

Removal of the N-terminal hexapeptide from human beta2-microglobulin facilitates protein aggregation and fibril formation.

The solution structure and stability of N-terminally truncated beta2-microglobulin (deltaN6beta2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that deltaN6beta2-m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at microM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that deltaN6beta2-m is significantly less protected than its wild-type counterpart. Analysis of deltaN6beta2-m by NMR shows that this loss of protection occurs in beta strands I, III, and part of II. At mM concentration gel filtration analysis shows that deltaN6beta2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of beta2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that deltaN6beta2-m could be a key intermediate of a proteolytic pathway of beta2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of beta2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between beta-strands far removed from this constrain is greatly perturbed.