Synthesis and proteasome inhibition of N‐allyl vinyl ester‐based peptides

Inhibition of the proteasome, the multicatalytic protease complex responsible for the turnover of many cellular proteins, represents an attractive target in the development of new drug therapies, proteasome inhibitors being potentially useful tools for the treatment of pathologies such as cancer, as well as inflammatory, immune and neurodegenerative diseases. Based on our previous development of a new class of inhibitors bearing a C‐terminal VE cluster able to interact with catalytic threonine, we report herein the synthesis and activity of new VE‐based peptide analogs bearing an additional allyl pharmacophore unit at the C‐ or N‐terminal position of the pseudotripeptide sequence. In the new series, the structural modification carried out to the prototype determine a decrease of proteasome inhibition. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Mitochondrial dynamics and its responds to proteasome defection during Picea wilsonii pollen tube development

Tip growth of pollen tubes is essential for higher plant sexual reproduction and has been proposed to be highly regulated by the ubiquitin/proteasome pathway (UPP). The dynamics of mitochondria and the functions of the UPP on mitochondrial dynamics during pollen tube development are still poorly understood. In the present study, using real‐time laser scanning and transmission electron microscope, it was revealed that mitochondria in Picea wilsonii, are either ellipsoid or filamentous with various lengths. Time‐lapse images indicated that the two forms of mitochondria interconvert frequently through opposite process of fusion and fission. Examination of mitochondrial morphology during four key stages of in vitro pollen tube development revealed a link between mitochondrial remodeling and the process of pollen tube elongation. We also report that MG132, a specific proteasome inhibitor, not only strongly disturbed the mitochondrial remodeling but also significantly reduced mitochondrial membrane potential during pollen tube development. This finding provides new insight into the function of the proteasome in tip growth of pollen tubes. Copyright © 2010 John Wiley & Sons, Ltd.     

On the terminal homologation of physiologically active peptides as a means of increasing stability in human serum--neurotensin, opiorphin, B27-KK10 epitope, NPY

The terminal homologation by CH₂ insertion into the peptides mentioned in the title is described. This involves replacement of the N‐terminal amino acid residue by a β²‐ and of the C‐terminal amino acid residue by a β³‐homo‐amino acid moiety (β²hXaa and β³hXaa, resp.; Fig. 1). In this way, the structure of the peptide chain from the N‐terminal to the C‐terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1 ) and its C‐terminal fragment NT(8–13) are ligands of the G‐protein‐coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b – 2e , for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5–1.3 vs. 0.6 nM ). At the same time, one of the homologated NT analogs, 2c , survives in human plasma for 7 days at 37° (Fig. 6). An NMR analysis of NT(8–13) (Tables 2 and 4, and Fig. 8) reveals that this N‐terminal NT fragment folds to a turn in CD₃OH. – In the case of the human analgesic opiorphin ( 3a ), a pentapeptide, and of the HIV‐derived B27‐KK10 ( 4a ), a decapeptide, terminal homologation (→ 3b and 4b , resp.) led to a 7‐ and 70‐fold half‐life increase in plasma (Fig. 9). With N‐terminally homologated NPY, 5c , we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c , and 5c , were shown to be agonists (Fig. 7 and 11). A comparison of terminal homologation with other stability‐increasing terminal modifications of peptides is performed (Fig. 5), and possible applications of the neurotensin analogs, described herein, are discussed.     

Inhibition of tumor proteasome activity by gold–dithiocarbamato complexes via both redox‐dependent and ‐independent processes

We have previously reported on a gold(III) complex, namely [AuBr₂(DMDT)] (N,N‐dimethyldithiocarbamate) showing potent in vitro and in vivo growth inhibitory activities toward human cancer cells and identifying the cellular proteasome as one of the major targets. However, the importance of the oxidation state of the gold center and the involved mechanism of action has yet to be established. Here we show that both gold(III)? and gold(I)–dithiocarbamato species, namely [AuBr₂(ESDT)] (AUL12) and [Au(ESDT)]₂ (AUL15), could inhibit the chymotrypsin‐like activity of purified 20S proteasome and 26S proteasome in human breast cancer MDA‐MB‐231 cells, resulting in accumulation of ubiquitinated proteins and proteasome target proteins, and induction of cell death, but at significantly different levels. Gold(I)‐ and gold(III)‐compound‐mediated proteasome inhibition and cell death induction were completely reversed by the addition of a reducing agent, dithiothreitol or N‐acetyl‐L ‐cysteine, suggesting the involvement of redox processes. Furthermore, treatment of MDA‐MB‐231 cells with gold(III) compound (AUL12), but not the gold(I) analog (AUL15), resulted in the production of significant levels of reactive oxygen species. Our study provides strong evidence that the cellular proteasome is an important target of both gold(I) and gold(III)–dithiocarbamates, but distinct cellular mechanisms of action are responsible for their different overall effect. J. Cell. Biochem. 109: 162–172, 2010. © 2009 Wiley‐Liss, Inc.     

Anti‐inflammatory and anti‐endotoxin properties of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti‐inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)‐induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti‐inflammatory and anti‐endotoxin activities of Os and Os‐C, peptides derived from the carboxy‐terminal of a tick defensin, were investigated. Both Os and Os‐C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin‐binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os‐C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os‐C showed no scavenging activity. Os and Os‐C inhibited LPS/IFN‐γ induced NO and TNF‐α production in RAW 264.7 cells in a concentration‐dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF‐α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os‐C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os‐C, both peptides have in addition anti‐inflammatory and anti‐endotoxin properties. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Genetic analysis of Agrobacterium tumefaciens unipolar polysaccharide production reveals complex integrated control of the motile‐to‐sessile switch

Many bacteria colonize surfaces and transition to a sessile mode of growth. The plant pathogen Agrobacterium tumefaciens produces a u nip olar p olysaccharide (UPP) adhesin at single cell poles that contact surfaces. Here we report that elevated levels of the intracellular signal cyclic diguanosine monophosphate (c‐di‐GMP) lead to surface‐contact‐independent UPP production and a red colony phenotype due to production of UPP and the exopolysaccharide cellulose, when A. tumefaciens is incubated with the polysaccharide stain Congo Red. Transposon mutations with elevated Congo Red staining identified presumptive UPP‐negative regulators, mutants for which were hyperadherent, producing UPP irrespective of surface contact. Multiple independent mutations were obtained in visN and visR, activators of flagellar motility in A. tumefaciens, now found to inhibit UPP and cellulose production. Expression analysis in a visR mutant and isolation of suppressor mutations, identified three diguanylate cyclases inhibited by VisR. Null mutations for two of these genes decrease attachment and UPP production, but do not alter cellular c‐di‐GMP levels. However, analysis of catalytic site mutants revealed their GGDEF motifs are required to increase UPP production and surface attachment. Mutations in a specific presumptive c‐di‐GMP phosphodiesterase also elevate UPP production and attachment, consistent with c‐di‐GMP activation of surface‐dependent adhesin deployment.     

Rapid degradation of solid‐phase bound peptides by the 20S proteasome

Proteasomes are cellular proteases involved in the degradation of numerous cellular proteins. The 20S proteasome is a cylindrical 28‐mer protein complex composed of two outer heptameric α‐rings forming the entrance for the protein substrate and two inner heptameric β‐rings carrying the catalytic sites. Numerous in vitro studies have provided evidence that the 20S proteasome may degrade peptides of various lengths and even unfolded full‐length polypeptide chains. However, a direct demonstration that the 20S proteasome may also cleave surface‐attached immobilized peptides is lacking so far. To this end, we used a model system by coupling peptides from different source proteins covalently to the surface of glass beads and applied nanoLC/MS analysis to monitor the generation of proteolytic fragments in the presence of the 20S proteasome. Detectable amounts of cleavage products occurred within a few minutes indicating a much higher cleavage rate than observed with the same substrates in solution. Our finding lends support to the idea that proteasomes may directly degrade segments of membrane‐bound proteins protruding into the aqueous phase. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Enhancement of the catalytic efficiency and thermostability of Stenotrophomonas sp. keratinase KerSMD by domain exchange with KerSMF

In this study, we enhanced the catalytic efficiency and thermostability of keratinase KerSMD by replacing its N/C‐terminal domains with those from a homologous protease, KerSMF, to degrade feather waste. Replacement of the N‐terminal domain generated a mutant protein with more than twofold increased catalytic activity towards casein. Replacement of the C‐terminal domain obviously improved keratinolytic activity and increased the k_cat/K_m value on a synthetic peptide, succinyl‐Ala‐Ala‐Pro‐Phe‐p‐nitroanilide, by 54.5%. Replacement of both the N‐ and C‐terminal domains generated a more stable mutant protein, with a T_m value of 64.60 ± 0.65°C and a half‐life of 244.6 ± 2 min at 60°C, while deletion of the C‐terminal domain from KerSMD or KerSMF resulted in mutant proteins exhibiting high activity under mesophilic conditions. These findings indicate that the pre‐peptidase C‐terminal domain and N‐propeptide are not only important for substrate specificity, correct folding and thermostability but also support the ability of the enzyme to convert feather waste into feed additives.     

Crystal structure of the N‐terminal domain of EccA1 ATPase from the ESX‐1 secretion system of Mycobacterium tuberculosis

EccA₁ is an important component of the type VII secretion system (T7SS) that is responsible for transport of virulence factors in pathogenic mycobacteria. EccA₁ has an N‐terminal domain of unknown function and a C‐terminal AAA+ (ATPases associated with various cellular activities) domain. Here we report the crystal structure of the N‐terminal domain of EccA₁ from Mycobacterium tuberculosis, which shows an arrangement of six tetratricopeptide repeats that may mediate interactions of EccA₁ with secreted substrates. Furthermore, the size and shape of the N‐terminal domain suggest its orientation in the context of a hexamer model of full‐length EccA₁. Proteins 2014; 82:159–163. © 2013 Wiley Periodicals, Inc.     

Aging and regulated protein degradation: who has the UPPer hand?

In all cells, protein degradation is a constant, ongoing process that is critical for cell survival and repair. The ubiquitin/proteasome pathway (UPP) is the major proteolytic pathway that degrades intracellular proteins in a regulated manner. It plays critical roles in many cellular processes and diseases. Disruption of the UPP is particularly relevant to pathophysiological conditions that provoke the accumulation of aberrant proteins, such as in aging as well as in a variety of neurodegenerative disorders including Alzheimer's and Parkinson's diseases. For unknown reasons, most of these neurodegenerative disorders that include familial and sporadic cases exhibit a late onset. It is possible that these neurodegenerative conditions exhibit a late onset because proteasome activity decreases with aging. Aging‐dependent impairment in proteolysis mediated by the proteasome may have profound ramifications for cell viability. It can lead to the accumulation of modified, potentially toxic proteins in cells and can cause cell injury or premature cell death by apoptosis or necrosis. While it is accepted that aging affects UPP function, the question is why does aging cause a decline in regulated protein degradation by the UPP? Herein, we review some of the properties of the UPP and mechanisms mediating its age‐dependent impairment. We also discuss the relevance of these findings leading to a model that proposes that UPP dysfunction may be one of the milestones of aging.     

β‐amino acid substitution to investigate the recognition of angiotensin II (AngII) by angiotensin converting enzyme 2 (ACE2)

In spite of the important role of angiotensin converting enzyme 2 (ACE2) in the cardiovascular system, little is known about the substrate structural requirements of the AngII–ACE2 interaction. Here we investigate how changes in angiotensin II (AngII) structure affect binding and cleavage by ACE2. A series of C3 β‐amino acid AngII analogs were generated and their secondary structure, ACE2 inhibition, and proteolytic stability assessed by circular dichroism (CD), quenched fluorescence substrate (QFS) assay, and LC‐MS analysis, respectively. The β‐amino acid‐substituted AngII analogs showed differences in secondary structure, ACE2 binding and proteolytic stability. In particular, three different subsets of structure‐activity profiles were observed corresponding to substitutions in the N‐terminus, the central region and the C‐terminal region of AngII. The results show that β‐substitution can dramatically alter the structure of AngII and changes in structure correlated with ACE2 inhibition and/or substrate cleavage. β‐amino acid substitution in the N‐terminal region of AngII caused little change in structure or substrate cleavage, while substitution in the central region of AngII lead to increased β‐turn structure and enhanced substrate cleavage. β‐amino acid substitution in the C‐terminal region significantly diminished both secondary structure and proteolytic processing by ACE2. The β‐AngII analogs with enhanced or decreased proteolytic stability have potential application for therapeutic intervention in cardiovascular disease. Copyright © 2010 John Wiley & Sons, Ltd.     

Curcusone D,a novel ubiquitin–proteasome pathway inhibitor via ROS-induced DUB inhibition,is synergistic with bortezomib against multiple myeloma cell growth

Background

Ubiquitin–proteasome pathway (UPP) plays a very important role in the degradation of proteins. Finding novel UPP inhibitors is a promising strategy for treating multiple myeloma (MM).

Methods

Ub-YFP reporter assays were used as cellular UPP models. MM cell growth, apoptosis and overall death were evaluated with the MTS assay, Annexin V/PI dual-staining flow cytometry, poly (ADP-ribose) polymerase (PARP) cleavage, and PI uptake, respectively. The mechanism of UPP inhibition was analyzed by western blotting for ubiquitin, in vitro and cellular proteasomal and deubiquitinases (DUBs) activity assays. Cellular reactive oxygen species (ROS) were measured with H₂DCFDA.

Results

Curcusone D, identified as a novel UPP inhibitor, causes cell growth inhibition and apoptosis in MM cells. Curcusone D induced the accumulation of poly-ubiquitin-conjugated proteins but could not inhibit proteasomal activity in vitro or in cells. Interestingly, the mono-ubiquitin level and the total cellular DUB activity were significantly downregulated following curcusone D treatment. Furthermore, curcusone D could induce ROS, which were closely correlated with DUB inhibition that could be nearly completely reversed by NAC. Finally, curcusone D and the proteasomal inhibitor bortezomib showed a strong synergistic effect against MM cells.

Conclusions

Curcusone D is novel UPP inhibitor that acts via the ROS-induced inhibition of DUBs to produce strong growth inhibition and apoptosis of MM cells and synergize with bortezomib.

General significance

The anti-MM molecular mechanism study of curcusone D will promote combination therapies with different UPP inhibitors against MM and further support the concept of oxidative stress regulating the activity of DUBs.     

EFFECT OF ELEVATED TEMPERATURE,DARKNESS, AND HYDROGEN PEROXIDE TREATMENT ON OXIDATIVE STRESS AND CELL DEATH IN THE BLOOM‐FORMING TOXIC CYANOBACTERIUM MICROCYSTIS AERUGINOSA1

This study assessed the implication of oxidative stress in the mortality of cells of Microcystis aeruginosa Kütz. Cultures grown at 25°C were exposed to 32°C, darkness, and hydrogen peroxide (0.5 mM) for 96 h. The cellular abundance, chl a concentration and content, maximum photochemical efficiency of PSII (F_v/F_m ratio), intracellular oxidative stress (determined with dihydrorhodamine 123 [DHR]), cell mortality (revealed by SYTOX‐labeling of DNA), and activation of caspase 3–like proteins were assessed every 24 h. The presence of DNA degradation in cells of M. aeruginosa was also assessed using a terminal deoxynucletidyl transferase‐mediated dUTP nick end labeling (TUNEL) assay at 96 h. Transferring cultures from 25°C to 32°C was generally beneficial to the cells. The cellular abundance and chl a concentration increased, and the mortality remained low (except for a transient burst at 72 h) as did the oxidative stress. In darkness, cells did not divide, and the F_v/F_m continuously decreased with time. The slow increase in intracellular oxidative stress coincided with the activation of caspase 3–like proteins and a 15% and 17% increase in mortality and TUNEL‐positive cells, respectively. Exposure to hydrogen peroxide had the most detrimental effect on cells as growth ceased and the F_v/F_m declined to near zero in less than 24 h. The 2‐fold increase in oxidative stress matched the activation of caspase 3–like proteins and a 40% and 37% increase in mortality and TUNEL‐positive cells, respectively. These results demonstrate the implication of oxidative stress in the stress response and mortality of M. aeruginosa.     

Distinct solid and solution state self‐assembly pathways of RADA16‐I designer peptide

Solid state NMR measurements on selectively ¹³C‐labeled RADA16‐I peptide (COCH₃–RADARADARADARADA–NH₂) were used to obtain new molecular level information on the conversion of α‐helices to β‐sheets through self‐assembly in the solid state with increasing temperature. Isotopic labeling at the A4 C_β site enabled rapid detection of ¹³C NMR signals. Heating to 344–363 K with simultaneous NMR detection allowed production of samples with systematic variation of α‐helix and β‐strand content. These samples were then probed at room temperature for intermolecular ¹³C–¹³C nuclear dipolar couplings with the PITHIRDS‐CT NMR experiment. The structural transition was also characterized by Fourier transform infrared spectroscopy and wide angle X‐ray diffraction. Independence of PITHIRDS‐CT decay shapes on overall α‐helical and β‐strand content infers that β‐strands are not observed without association with β‐sheets, indicating that β‐sheets are formed at elevated temperatures on a timescale that is fast relative to the NMR experiment. PITHIRDS‐CT NMR data were compared with results of similar measurements on RADA16‐I nanofibers produced by self‐assembly in aqueous salt solution. We report that β‐sheets formed through self‐assembly in the solid state have a structure that differs from those formed through self‐assembly in the solution state. Specifically, solid state RADA16‐I self‐assembly produces in‐register parallel β‐sheets, whereas nanofibers are composed of stacked parallel β‐sheets with registry shifts between adjacent β‐strands in each β‐sheet. These results provide evidence for environment‐dependent self‐assembly mechanisms for RADA16‐I β‐sheets as well as new constraints on solid state self‐assembled structures, which must be avoided to maximize solution solubility and nanofiber yields. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of Aglycon Structure on Saccharide Elongation by Cells

Alkyl N‐acetyl‐β‐D ‐glucosaminide (GlcNAc primers) with different aglycon moieties were synthesized and used to determine the effect of the aglycon structure on cellular saccharide elongation. Dodecyl N‐acetyl‐β‐D ‐glucosaminide (GlcNAc‐C12), tridecan‐7‐yl N‐acetyl‐β‐D ‐glucosaminide (GlcNAc‐2C6), and pentacosan‐13‐yl N‐acetyl‐β‐D ‐glucosaminide (GlcNAc‐2C12) primers were synthesized by glycosylation of dodecan‐1‐ol, tridecan‐7‐ol, and pentacosan‐13‐ol, respectively, with peracetylglucosamine. These primers were introduced to mouse B16 melanoma cells to prepare glycolipids. After 48 h incubation, results showed that GlcNAc‐C12 was elongated to give NeuAc‐Gal‐GlcNAc‐C12. GlcNAc‐2C6 was also elongated to afford Gal‐GlcNAc‐2C6 and NeuAc‐Gal‐GlcNAc‐2C6. On the other hand, GlcNAc‐2C12 primer was not elongated. Significantly, the results demonstrated that the amount of glycosylated product increased 1.5‐times by modifying the aglycon structure of GlcNAc from C₁₂ to 2 C₆ despite having almost the same number of C‐units.     

Structure–activity relationship of a novel pentapeptide with cancer cell growth‐inhibitory activity

We previously reported that yamamarin, a pentapeptide with an amidated C‐terminus (DILRG‐NH₂) isolated from larvae of the silkmoth, and its palmitoylated analog (C16‐DILRG‐NH₂) suppressed proliferation of rat hepatoma (liver cancer) cells. In this study, we investigated the structure–activity relationship of yamamarin by in vitro assay and spectroscopic methods (CD and NMR) for various analogs. The in vitro assay results demonstrated that the chemical structure of the C‐terminal part (‐RG‐NH₂) of yamamarin is essential for its activity. The CD and NMR results indicated that yamamarin and its analog adopt predominantly a random coil conformation. Moreover, a comparison of NMR spectra of DILRG‐NH₂ and C16‐DILRG‐NH₂ revealed that the N‐terminal palmitoyl group of C16‐DILRG‐NH₂ did not affect the conformation of the C‐terminal part, which is essential for activity. Together, these results should assist in the design of more sophisticated anticancer drugs. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Pollen S–locus F–box proteins of Petunia involved in S–RNase‐based self‐incompatibility are themselves subject to ubiquitin‐mediated degradation

Many flowering plants show self‐incompatibility, an intra‐specific reproductive barrier by which pistils reject self‐pollen to prevent inbreeding and accept non‐self pollen to promote out‐crossing. In Petunia, the polymorphic S–locus determines self/non‐self recognition. The locus contains a gene encoding an S–RNase, which controls pistil specificity, and multiple S‐locus F‐box (SLF) genes that collectively control pollen specificity. Each SLF is a component of an SCF (Skp1/Cullin/F‐box) complex that is responsible for mediating degradation of non‐self S‐RNase(s), with which the SLF interacts, via the ubiquitin–26S proteasome pathway. A complete set of SLFs is required to detoxify all non‐self S‐RNases to allow cross‐compatible pollination. Here, we show that SLF1 of Petunia inflata is itself subject to degradation via the ubiquitin–26S proteasome pathway, and identify an 18 amino acid sequence in the C‐terminal region of S₂‐SLF1 (SLF1 of S₂ haplotype) that contains a degradation motif. Seven of the 18 amino acids are conserved among all 17 SLF proteins of S₂ haplotype and S₃ haplotype involved in pollen specificity, suggesting that all SLF proteins are probably subject to similar degradation. Deleting the 18 amino acid sequence from S₂‐SLF1 stabilized the protein but abolished its function in self‐incompatibility, suggesting that dynamic cycling of SLF proteins is an integral part of their function in self‐incompatibility.     

Noncollagenous region of the streptococcal collagen‐like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

Proper folding of the (Gly‐Xaa‐Yaa)_n sequence of animal collagens requires adjacent N‐ or C‐terminal noncollagenous trimerization domains which often contain coiled‐coil or beta sheet structure. Collagen‐like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen‐like protein from Streptococcus pyogenes has an N‐terminal globular domain, designated V_sp, adjacent to its triple‐helix domain. The V_sp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V_sp domain alone is shown to form trimers with a significant α‐helix content and to have a thermal stability of T_m = 45°C. Examination of a new construct shows that the V_sp domain facilitates efficient in vitro refolding only when it is located N‐terminal to the triple‐helix domain but not when C‐terminal to the triple‐helix domain. Fusion of the V_sp domain N‐terminal to a heterologous (Gly‐Xaa‐Yaa)_n sequence from Clostridium perfringens led to correct folding and refolding of this triple‐helix, which was unable to fold into a triple‐helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly‐Xaa‐Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple‐helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.