Dr Peter Klappa

Reader in Biochemistry
National Teaching Fellow
Senior Fellow HEA
Master, Rutherford College


Dr Peter Klappa joined the School of Biosciences in 1995. 

Research interests

It has been proposed that the incorrect folding of proteins is the molecular basis of various human diseases, e.g. cystic fibrosis, cancer and Alzheimer's disease. To understand disease-causing alterations in the folding pathway of proteins it is therefore important to investigate how proteins fold in general and why misfolding can occur.
Our interests are mainly focused on protein folding and the role molecular chaperones and folding catalysts play in this process. We are particularly interested in the structure, function and specificity of protein disulphide isomerases (protein folding catalysts that contain thioredoxin-like domains) and peptidyl proly cis-trans isomerases. The techniques we use include peptide synthesis, molecular biology, in-vitro transcription and in-vitro translation in cell free systems, cell culture, chemical cross-linking and immuno-histochemistry 



  • Klappa, P. (2015). Framing Excellence in teaching: Is it the right thing? RI Research Intelligence [Online] 2015. Available at: https://www.bera.ac.uk/wp-content/uploads/2015/12/RI-128-FINAL.pdf?noredirect=1.
    When the ‘Teaching Excellence Framework’
    (TEF) was announced earlier this year,
    I was wondering what it actually meant
    - and I still have lots of questions about
    this Government initiative. First of all,
    I wonder why we want to measure excellence in
    teaching? I agree that with the increase of fees to
    £9k and possibly even higher, there is pressure on
    HEIs to be accountable for the service they deliver.
    While a university’s contribution to research has
    been measured over the years through the various
    ‘Research Assessment Exercises’, followed by
    the latest ‘Research Excellence Framework’, the
    assessment of efficient education has been neglected.
    It is therefore only fitting that we also begin to
    hold universities accountable for the provision of
    excellence in education.
  • Bostan, H. et al. (2012). CMD: A Database to Store the Bonding States of Cysteine Motifs with Secondary Structures. Advances in Bioinformatics [Online] 2012:849830. Available at: http://dx.doi.org/10.1155/2012/849830.
    Computational approaches to the disulphide bonding state and its connectivity pattern prediction are based on various descriptors. One descriptor is the amino acid sequence motifs flanking the cysteine residue motifs. Despite the existence of disulphide bonding information in many databases and applications, there is no complete reference and motif query available at the moment. Cysteine motif database (CMD) is the first online resource that stores all cysteine residues, their flanking motifs with their secondary structure, and propensity values assignment derived from the laboratory data. We extracted more than 3 million cysteine motifs from PDB and UniProt data, annotated with secondary structure assignment, propensity value assignment, and frequency of occurrence and coefficiency of their bonding status. Removal of redundancies generated 15875 unique flanking motifs that are always bonded and 41577 unique patterns that are always nonbonded. Queries are based on the protein ID, FASTA sequence, sequence motif, and secondary structure individually or in batch format using the provided APIs that allow remote users to query our database via third party software and/or high throughput screening/querying. The CMD offers extensive information about the bonded, free cysteine residues, and their motifs that allows in-depth characterization of the sequence motif composition.
  • Ismail, N. et al. (2011). A mutant L-asparaginase II signal peptide improves the secretion of recombinant cyclodextrin glucanotransferase and the viability of Escherichia coli. Biotechnology Letters [Online] 33:999-1005. Available at: https://doi.org/10.1007/s10529-011-0517-8.
    L-Asparaginase II signal peptide was used for the secretion of recombinant cyclodextrin glucanotransferase (CGTase) into the periplasmic space of E. coli. Despite its predominant localisation in the periplasm, CGTase activity was also detected in the extracellular medium, followed by cell lysis. Five mutant signal peptides were constructed to improve the periplasmic levels of CGTase. N1R3 is a mutated signal peptide with the number of positively charged amino acid residues in the n-region increased to a net charge of +5. This mutant peptide produced a 1.7-fold enhancement of CGTase activity in the periplasm and significantly decreased cell lysis to 7.8% of the wild-type level. The formation of intracellular inclusion bodies was also reduced when this mutated signal peptide was used as judged by SDS-PAGE. Therefore, these results provide evidence of a cost-effective means of expression of recombinant proteins in E. coli.
  • Winter, J. et al. (2011). Protein disulfide isomerase isomerizes non-native disulfide bonds in human proinsulin independent of its peptide-binding activity. Protein Science [Online] 20:588-596. Available at: http://dx.doi.org/10.1002/pro.592.
    Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI-aba′c) where the b′ domain was partially deleted, thus abolishing peptide binding but maintaining a PDI-like redox potential. PDI-aba′c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI-aba′c isomerizes non-native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates.
  • Hayes, N., Smales, C. and Klappa, P. (2009). Protein disulfide isomerase does not control recombinant IgG4 productivity in mammalian cell lines. Biotechnology and Bioengineering [Online] 105:770-779. Available at: http://dx.doi.org/10.1002/bit.22587.
    Post-translational limitations in the endoplasmic reticulum during recombinant monoclonal antibody production are an important factor in lowering the capacity for synthesis and secretion of correctly folded proteins. Mammalian protein disulfide isomerase (PDI) has previously been shown to have a role in the formation of disulfide bonds in immunoglobulins. Several attempts have been made to improve the rate of recombinant protein production by overexpressing PDI but the results from these studies have been inconclusive. Here we examine the effect of (a) transiently silencing PDI mRNA and (b) increasing the intracellular levels of members of the PDI family (PDI, ERp72, and PDIp) on the mRNA levels, assembly and secretion of an IgG4 isotype. Although transiently silencing PDI in NS0/2N2 cells suggests that PDI is involved in disulfide bond formation of this subclass of antibody, our results show that PDI does not control the overall IgG4 productivity. Furthermore, overexpression of members of the PDI family in a Chinese hamster ovary (CHO) cell line does not improve productivity and hence we conclude that the catalysis of disulfide bond formation is not rate limiting for IgG4 production.
  • Stymest, K. and Klappa, P. (2008). The periplasmic peptidyl prolyl cis-trans isomerases PpiD and SurA have partially overlapping substrate specificities. FEBS Journal [Online] 275:3470-3479. Available at: http://dx.doi.org/10.1111/j.1742-4658.2008.06493.x.
    One of the rate-limiting steps in protein folding has been shown to be the cis-trans isomerization of proline residues, catalysed by a range of peptidyl prolyl cis-trans isomerases (PPIases). In the periplasmic space of Escherichia coli and other Gram-negative bacteria, two PPIases, SurA and PpiD, have been identified, which show high sequence similarity to the catalytic domain of the small PPIase parvulin. This observation raises a question regarding the biological significance of two apparently similar enzymes present in the same cellular compartment: do they interact with different substrates or do they catalyse different reactions? The substrate-binding motif of PpiD has not been characterized so far, and no biochemical data were available on how this folding catalyst recognizes and interacts with substrates. To characterize the interaction between model peptides and the periplasmic PPIase PpiD from E. coli, we employed a chemical crosslinking strategy that has been used previously to elucidate the interaction of substrates with SurA. We found that PpiD interacted with a range of model peptides independently of whether they contained proline residues or not. We further demonstrate here that PpiD and SurA interact with similar model peptides, and therefore must have partially overlapping substrate specificities. However, the binding motif of PpiD appears to be less specific than that of SurA, indicating that the two PPIases might interact with different substrates. We therefore propose that, although PpiD and SurA have partially overlapping substrate specificities, they fulfil different functions in the cell.
  • Hall, R. et al. (2008). External pH influences the transcriptional profile of the carbonic anhydrase, CAH-4b in Caenorhabditis elegans. Molecular and Biochemical Parasitology [Online] 161:140-149. Available at: http://dx.doi.org/10.1016/j.molbiopara.2008.06.013.
    Insight into how organisms adapt to environmental stimuli has become increasingly important in recent years for identifying key virulence factors in many species. The life cycle of many pathogenic nematode species forces the organism to experience environments which would otherwise be considered stressful. One of the conditions often encountered by nematodes is a change in environmental pH. Living in a soil environment Caenorhabditis elegans will naturally encounter fluctuations in external pH. Therefore, C elegans has the potential to provide an insight into how pathogenic nematodes survive and proliferate in these environments. We found that C elegans can maintain over 90% survival in pH conditions ranging from pH 3 to 10. This was unrelated to the non-specific protection provided by the cuticle. Global transcriptional analysis identified many genes, which were differentially regulated by pH. The gene cah-4 encodes two putative alpha carbonic anhydrases (CAH-4a and CAH-4b), one of which was five-fold up regulated in an alkaline environment (CAH-4b). Stopped-flow analysis of CAH-4b using 35 different carbonic anhydrase inhibitors identified complex benzenesulfonamide compounds as the most potent inhibitors (K-i 35-89 nM).
  • Karala, A. et al. (2007). Protein disulfide isomerases from C-elegans are equally efficient at thiol-disulfide exchange in simple peptide-based systems but show differences in reactivity towards protein substrates. Antioxidants and Redox Signaling [Online] 9:1815-1823. Available at: http://dx.doi.org/10.1089/ars.2007.1624.
    Although the formation of disulfide bonds is an essential process in every living organism, only little is known about the mechanisms in multicellular eukaryotic systems. The reason for this uncertainty is that in addition to the well-known key enzyme protein disulfide isomerase (PDI), several PDI-like proteins are present in the ER of metazoans. In total, there are now 18 PDI-family members in the human endoplasmic reticulum, with different domain architectures and active site chemistries. To understand why multicellular organisms express multiple proteins with similarity to the archetypal mammalian PDI, the properties of three PDIs from the nematode C.elegans were investigated. Here the authors demonstrate that PDI-1, PDI-2, and PDI-3 show comparable kinetic properties in catalyzing thiol:disulfide exchange reactions in two simple peptide-based assays. However, the three enzymes exhibited clear differences in their reactivity towards protein substrates. The authors therefore propose that the three PDIs can catalyze similar thiol-disulfide exchange reactions in a substrate, but due to differences in substrate binding, they can direct a folding polypeptide chain onto different folding pathways and hence fulfil distinct and different functions in the organism
  • Klappa, P. et al. (2004). A major fraction of endoplasmic reticulum-located glutathione is present as mixed disulfides with protein. Journal of Biological Chemistry [Online] 279:5257-5262. Available at: http://dx.doi.org/10.1074/jbc.M304951200.
    The tripeptide glutathione is the most abundant thiol/disulfide component of the eukaryotic cell and is known to be present in the endoplasmic reticulum lumen. Accordingly, the thiol/disulfide redox status of the endoplasmic reticulum lumen is defined by the status of glutathione, and it has been assumed that reduced and oxidized glutathione form the principal redox buffer. We have determined the distribution of glutathione between different chemical states in rat liver microsomes by labeling with the thiol-specific label monobromobimane and subsequent separation by reversed phase high performance liquid chromatography. More than half of the microsomal glutathione was found to be present in mixed disulfides with protein, the remainder being distributed between the reduced and oxidized forms of glutathione in the ratio of 3:1. The high proportion of the total population of glutathione that was found to be in mixed disulfides with protein has significant implications for the redox state and buffering capacity of the endoplasmic reticulum and, hence, for the formation of disulfide bonds in vivo.
  • Pirneskoski, A. et al. (2004). Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase. Journal of Biological Chemistry [Online] 279:10374-10381. Available at: http://dx.doi.org/10.1074/jbc.M312193200.
    Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b' domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b' domain and show the existence of an intradomain linker between the b' and a' domains. We have expressed the recombinant b' domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b' domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect.
  • Winter, J. et al. (2002). Catalytic activity and chaperone function of human protein-disulfide isomerase are required for the efficient refolding of proinsulin. Journal of Biological Chemistry [Online] 277:310-317. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11694508.
    Protein-disulfide isomerase (PDI) catalyzes the formation, rearrangement, and breakage of disulfide bonds and is capable of binding peptides and unfolded proteins in a chaperone-like manner. In this study we examined which of these functions are required to facilitate efficient refolding of denatured and reduced proinsulin. In our model system, PDI and also a PDI mutant having only one active site increased the rate of oxidative folding when present in catalytic amounts. PDI variants that are completely devoid of isomerase activity are not able to accelerate proinsulin folding, but can increase the yield of refolding, indicating that they act as a chaperone. Maximum refolding yields, however, are only achieved with wild-type PDI. Using genistein, an inhibitor for the peptide-binding site, the ability of PDI to prevent aggregation of folding proinsulin was significantly suppressed. The present results suggest that PDI is acting both as an isomerase and as a chaperone during folding and disulfide bond formation of proinsulin.
  • Klappa, P. et al. (2001). The pancreas-specific protein disulphide-isomerase PDIp interacts with a hydroxyaryl group in ligands. Biochemical Journal [Online] 354:553-559. Available at: http://dx.doi.org/10.1042/0264-6021:3540553.
    Using a cross-linking approach, we have recently demonstrated that radiolabelled model peptides or misfolded proteins specifically interact in vitro with two members of the protein disulphide- isomerase family, namely PDI and PDIp, in a crude extract from sheep pancreas microsomes. In addition, we have shown that tyrosine and tryptophan residues within a peptide are the recognition motifs for the binding to PDIp. Here we examine non-peptide ligands and present evidence that a hydroxyaryl group is a structural motif for the binding to PDIp; simple constructs containing this group and certain xenobiotics and phytoestrogens, which contain an unmodified hydroxyaryl group, can all efficiently inhibit peptide binding to PDIp. To our knowledge this is the first time that the recognition motif of a molecular chaperone or folding catalyst has been specified as a simple chemical structure.
  • Pirneskoski, A. et al. (2001). Domains b' and a' of protein disulfide isomerase fulfill the minimum requirement for function as a subunit of prolyl 4-hydroxylase. The N-terminal domains a and b enhances this function and can be substituted in part by those of ERp57. Journal of Biological Chemistry [Online] 276:11287-11293. Available at: http://dx.doi.org/10.1074/jbc.M010656200.
    Protein disulfide isomerase (PDI) is a modular polypeptide consisting of four domains, a, b, b', and a', plus an acidic C-terminal extension, c. PDI carries out multiple functions, acting as the beta subunit in the animal prolyl 4-hydroxylases and in the microsomal triglyceride transfer protein and independently acting as a protein folding catalyst. We report here that the minimum sequence requirement for the assembly of an active prolyl 4-hydroxylase alpha(2)beta(2) tetramer in insect cell coexpression experiments is fulfilled by the PDI domain construct b'a' but that the sequential addition of the b and a domains greatly increases the level of enzyme activity obtained. In the assembly of active prolyl 4-hydroxylase tetramers, the a and b domains of PDI, but not b' and a', can in part be substituted by the corresponding domains of ERp57, a PDI isoform that functions naturally in association with the lectins calnexin and calreticulin. The a' domain of PDI could not be substituted by the PDI a domain, suggesting that both b' and a' domains contain regions critical for prolyl 4-hydroxylase assembly. All PDI domain constructs and PDI/ERp57 hybrids that contain the b' domain can bind the 14-amino acid peptide Delta-somatostatin, as measured by cross-linking; however, binding of the misfolded protein "scrambled" RNase required the addition of domains ab or a' of PDI. The human prolyl 4-hydroxylase alpha subunit has at least two isoforms, alpha(I) and alpha(II), which form with the PDI polypeptide the (alpha(I))(2)beta(2) and (alpha(II))(2)beta(2) tetramers. We report here that all the PDI domain constructs and PDI/ERp57 hybrid polypeptides tested were more effectively associated with the alpha(II) subunit than the alpha(I) subunit.
  • Kramer, B. et al. (2001). Functional Roles and Efficiencies of the Thioredoxin Boxes of Calcium-binding Proteins 1 and 2 in Protein Folding. Biochemical Journal 357:83-95.
    The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys) respectively and, like PDI, may be involved in the folding of nascent proteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is significantly more active than the second trx box. Furthermore, only about 65% re-activation of denatured reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions was significantly reduced in the presence of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-independent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b' domain, a property it shares with PDIp, the pancreas-specific PDI homologue, and although PDI can bind malfolded proteins such as 'scrambled' ribonuclease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a' domain of PDI; (2) CaBP2 lacks the general peptide-binding activity attributed to the b' domain of PDI; (3) interaction of CaBP2 with substrate (RNase AIII) is different from that of PDI and substrate; and (4) both CaBP2 and CaBP1 may promote oxidative folding by different kinetic pathways.
  • Webb, H. et al. (2001). Interaction of the periplasmic peptidylprolyl cis-trans isomerase SurA with model peptides. The N-terminal region of SurA id essential and sufficient for peptide binding. Journal of Biological Chemistry [Online] 276:45622-45627. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11546789.
    One of the rate-limiting steps in protein folding has been shown to be the cis-trans isomerization of proline residues, which is catalyzed by a range of peptidylprolyl cis-trans isomerases. To characterize the interaction between model peptides and the periplasmic peptidylprolyl cis-trans isomerase SurA from E. coli, we employed a chemical cross-linking strategy that has been used previously to elucidate the interaction of substrates with other folding catalysts. The interaction between purified SurA and model peptides was significant in that it showed saturation and was abolished by denaturation of SurA; however the interaction was independent of the presence of proline residues in the model peptides. From results obtained by limited proteolysis we conclude that an N-terminal fragment of SurA, comprising 150 amino acids that do not contain the active sites involved in the peptidylprolyl cis-trans isomerization, is essential for the binding of peptides by SurA. This was confirmed by probing the interaction of the model peptide with the recombinant N-terminal fragment, expressed in Escherichia coli. Hence we propose that, similar to protein disulfide isomerase and other folding catalysts, SurA exhibits a modular architecture composed of a substrate binding domain and distinct catalytically active domains.
  • Ruddock, L., Freedman, R. and Klappa, P. (2000). Specificity in substrate binding by protein folding catalysts: Tyrosine and tryptophan residues are the recognition motifs for the binding of peptides to the pancreas-specific protein disulfide isomerase PDIp. Protein Science 9:758-764.
    Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding.
  • Klappa, P. et al. (2000). Mutations that destabilize the a ' domain of human protein-disulfide isomerase indirectly affect peptide binding. Journal of Biological Chemistry [Online] 275:13213-13218. Available at: http://dx.doi.org/10.1074/jbc.275.18.13213.
    Protein-disulfide isomerase (PDI) is a catalyst of folding of disulfide-bonded proteins and also a multifunctional polypeptide that acts as the beta-subunit in the prolyl 4-hydroxylase alpha(2)beta(2)-tetramer (P4H) and the microsomal triglyceride transfer protein alpha beta-dimer. The principal peptide-binding site of PDI is located in the b' domain, but all domains contribute to the binding of misfolded proteins, Mutations in the C-terminal part of the a' domain have significant effects on the assembly of the P4H tetramer and other functions of PDI, In this study we have addressed the question of whether these mutations in the C-terminal part of the a' domain, which affect P4H assembly, also affect peptide binding to PDI. We observed a strong correlation between P4H assembly competence and peptide binding; mutants of PDI that failed to form a functional P4H tetramer were also inactive in peptide binding. However, there was also a correlation between inactivity in these assays and indicators of conformational disruption, such as protease sensitivity. Peptide binding activity could be restored in inactive, protease-sensitive mutants by selective proteolytic removal of the mutated a' domain. Hence we propose that structural changes in the a' domain indirectly affect peptide binding to the b' domain.
  • Ruddock, L. and Klappa, P. (1999). Oxidative stress: Protein folding with a novel redox switch. Current Biology [Online] 9:R400-R402. Available at: http://dx.doi.org/10.1016/S0960-9822(99)80253-X.
    A novel cellular response to oxidative stress has been discovered, in which the activity of a molecular chaperone, Hsp33, is modulated by the environmental redox potential. This provides a rapid first defence mechanism against the potentially very harmful toxic effects of oxidative stress.
  • Klappa, P. et al. (1998). The b ' domain provides the principal peptide-binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins. Embo Journal [Online] 17:927-935. Available at: http://dx.doi.org/10.1093/emboj/17.4.927.
    Protein disulfide isomerase (PDI) is a very efficient catalyst of folding of many disulfide-bonded proteins, A great deal is known about the catalytic functions of PDI, while little is known about its substrate binding, We recently demonstrated by cross-linking that PDI binds peptides and misfolded proteins, with high affinity but broad specificity. To characterize the substrate-binding site of PDI, we investigated the interactions of various recombinant fragments of human PDI, expressed in Escherichia coil, with different radiolabelled model peptides, We observed that the b' domain of human PDI is essential and sufficient for the binding of small peptides, In the case of larger peptides, specifically a 28 amino acid fragment derived from bovine pancreatic trypsin inhibitor, or misfolded proteins, the b' domain is essential but not sufficient for efficient binding, indicating that contributions from additional domains are required, Hence we propose that the different domains of PDI all contribute to the binding site, with the b' domain forming the essential core.
  • Klappa, P. et al. (1998). A pancreas-specific glycosylated protein disulphide-isomerase binds to misfolded proteins and peptides with an interaction inhibited by oestrogens. European Journal of Biochemistry [Online] 254:63-69. Available at: http://dx.doi.org/10.1046/j.1432-1327.1998.2540063.x.
    Using a cross-linking approach, we have demonstrated that radiolabeled model peptides or misfolded proteins specifically interact in vitro with two different luminal proteins in a crude extract from sheep pancreas microsomes. One of the proteins was identified as protein disulphide-isomerase (PDI), the other one was a related protein (PDIp). We have shown that PDIp was expressed exclusively in the pancreas. Interspecies conservation of PDIp, was confirmed and, unlike other members of the PDI family, PDIp from various sources was found to be a glycoprotein. PDIp interacted with peptides and also a misfolded protein, but not with native proteins, suggesting that it might act as a molecular chaperone. The inital binding process was independent of the presence of Cys residues in the probed peptides. Certain oestrogens strongly inhibited the interaction between peptides and PDIp, with 17 beta-oestradiol being the most patent inhibitor.
  • Klappa, P., Hawkins, H. and Freedman, R. (1997). Interactions between protein disulphide isomerase and peptides. European Journal of Biochemistry [Online] 248:37-42. Available at: http://dx.doi.org/10.1111/j.1432-1033.1997.t01-1-00037.x.
    There is growing evidence that protein disulphide isomerase (PDI) has a common chaperone function in the endoplasmic reticulum. To characterise this function, we investigated the interaction of purified PDI with radiolabelled model peptides, somatostatin and mastoparan, by cross-linking. The interaction between the peptides and PDI was specific, for it showed saturation and was abolished by denaturation of PDI. The interaction between a hydrophobic peptide without cysteine residues was much more sensitive to Triton X-100 than the interaction between PDI and a more hydrophilic peptide wither without cysteine residues. We therefore propose that hydrophobic interactions between protein disulphide isomerase and peptides play an important role in the binding process. The interaction between PDI and the bound peptide therefore is enhanced by the formation of mixed disulphide bonds.
  • Klappa, P., Freedman, R. and Zimmermann, R. (1995). Protein Disulfide-Isomerase and a Lumenal Cyclophin-Type Petidyl-Prolyl Cis-Trans Isomerase are in Transient Contact with Secretroy Proteins During Late Stages of Translocation. European Journal of Biochemistry 232:755-764.
    The transport of a presecretory protein into the mammalian endoplasmic reticulum can be divided into early translocation events which include specific targeting of the presecretory protein to and insertion into the endoplasmic reticulum membrane and late translocation events, comprising signal sequence cleavage, completion of translocation and folding of the secretory protein into a functional conformation. The microsomal membrane proteins Sec61 alpha p and translocating-chain-associating membrane protein were previously identified as being in close contact with a nascent presecretory protein at an early step of translocation. Here, we investigated whether additional microsomal proteins are in contact with translocating chains during or immediately after transit. This was addressed by crosslinking after release of the nascent chain from Sec61 alpha p. We observed two additional membrane proteins interacting with the nascent precursor in the early stages of translocation and three lumenal proteins interacting with the processed polypeptide chain in the late stages of translocation. One of the lumenal proteins was identified as protein disulphide isomerase by immunoprecipitation. Another of the lumenal proteins was suggested to be a lumenal cyclophilin-type peptidyl prolyl cis-trans isomerase by the effect of cyclosporin A. We propose that molecular chaperones, such as protein disulphide isomerase and cyclophilin may represent two of the lumenal proteins which are involved in completion of translocation.


  • Klappa, P. (2015). Innovative pedagogies series: Videos for learning and teaching. Higher Education Academy. Available at: https://www.heacademy.ac.uk/system/files/peter_klappa_final2.pdf.
    The expression ‘chalk and talk’ has been used synonymously for boring and old-fashioned teaching style, but
    this approach can have advantages over more ‘modern’ teaching styles, notably the use of slides and
    asynchronous online delivery of lectures (for review see Seth 2010). However, a major disadvantage of this
    teaching style is the transient nature of the notes scribbled on the board: once the notes and explanations
    have been wiped off, they are gone, persisting only in the more or less complete notes of the students. As a
    result students focus their attention predominantly on trying to keep up with copying the notes from the
    board while at the same time listening to the lecturer, which can easily lead to cognitive load (Mayer 2003). It
    is not a surprise that many students find it challenging to engage with the topic while anxiously trying to get a
    complete set of notes and at the same time listening to the lecturer. In my view, students should focus on the
    development of the topic rather than copying the board contents. It was important to me to improve the
    learning experience and to provide accessible, flexible and diverse learning opportunities that improve
    student engagement with their studies.


  • Freedman, R., Klappa, P. and Ruddock, L. (2002). Model peptide substrates and ligands in analysis of action of mammalian protein disulfide-isomerase. Methods in Enzymology [Online] 348:342-354. Available at: http://dx.doi.org/10.1016/S0076-6879(02)48653-3.
    Protein disulfide-isomerase (PDI) polypeptide comprises four distinct but homologous domains can function alone, as homo-oligomers, or as an obligatory component of hetero-oligomeric species, such as prolyl-4-hydroxylase and microsomal triglyceride transfer protein. Thus, PDI is a complex enzyme that catalyzes a complex reaction. Advances in understanding the action of this complex enzyme have come from three directions: (1) the definition of the domain structure of the PDI polypeptide; (2) the definition and validation of “partial reactions” with simple substrates, representing specific elements of the overall reaction catalyzed by PDI on its complex physiological substrates; and (3) the combination of these inputs to express recombinant PDI constructs comprising individual domains, combinations of domains or active-site mutant species, and to characterize them in functional terms. This chapter focuses on the second of these advances, describing peptide substrates for partial reactions of PDI and their use to define the overall catalytic process and to establish the roles within it of individual domains of PDI.
  • Freedman, R., Klappa, P. and Ruddock, L. (2002). Protein disulfide isomerases exploit synergy between catalytic and specific binding domains. EMBO Reports [Online] 3:136-140. Available at: http://dx.doi.org/10.1093/embo-reports/kvf035.
    Protein disulfide isomerases (PDIs) catalyse the formation of native disulfide bonds in protein folding pathways. The key steps involve disulfide formation and isomerization in compact folding intermediates. The high-resolution structures of the a and b domains of PDI are now known, and the overall domain architecture of PDI and its homologues can be inferred. The isolated a and a' domains of PDI are good catalysts of simple thiol-disulfide interchange reactions but require additional domains to be effective as catalysts of the rate-limiting disulfide isomerizations in protein folding pathways. The b' domain of PDI has a specific binding site for peptides and its binding properties differ in specificity between members of the PDI family. A model of PDI function can be deduced in which the domains function synergically: the b' domain binds unstructured regions of polypeptide, while the a and a' domains catalyse the chemical isomerization steps.


  • Klappa, P. (2017). Lectures in a virtual space - live-streaming on Facebook. in: Bilham, T. ed. Reframing space for learning: Empowering Excellence and Innovation in University Teaching and Learning. Palgrave MacMillan.