Incidence of betapapillomaviruses in the tumour and perilesional healthy skin in patients with basal cell carcinoma depending on sex, age, hair colour, tumour subtype, its location and dissemination.

INTRODUCTION: Basal cell carcinoma (BCC) is the most common skin cancer in the Caucasian population. It is believed that infections caused by viruses from the genus betapapillomavirus (ß-HPV) might be associated with the risk of BCC, but the spread of data on the prevalence of the virus in biopsies is significant. AIM: To assess the presence and diversity of ß-HPV in skin samples taken from the tumour and a fragment of healthy skin from the patients with BCC, as well as checking the correlation of factors listed below and presence of ß-HPV infection in the studied patients. MATERIAL AND METHODS: The study was conducted on the skin biopsies from 73 patients with histopathologically confirmed BCC. The following data were collected from patients: sex, age, hair colour and tumour location. Using the polymerase chain reaction (PCR) test, the presence of ß-HPV infection was detected in the tested samples. PCR and reverse hybridization assay were also used to genotype 25 types of ß-HPV. RESULTS: A statistically significant correlation was found between the sex and BCC type, BCC type and tumour location, BCC type and exposure to UV radiation, as well as between the hair colour and tumour location. The correlation between the BCC type and the number of tumours and HPV types detected was also noted. CONCLUSIONS: Preliminary studies suggest that one of the risk factors for development of infiltrating lesions is the presence of a single HPV 93 infection, but further research is needed to confirm these assumptions.

Sequence variation analysis of the E1 and E2 genes of human papillomavirus type 16 in cervical lesions in women from the south of Poland.

The E1 and E2 genes of the human papillomavirus encode the so-called early proteins, their sequences are conserved, and regulatory functions are associated with the viral oncoproteins. The purpose of this study is to determine the HPV16 E1 and E2 mutations appearing in the female population of southern Poland, depending on the severity of cervical pathological changes. We also take into account the number of E1 and E2 mutations detected in the E6 gene variant (350G or 350T). This publication is one of the first in the Central and Eastern Europe to deal with this topic. We identified 4 mutations in the E1 gene and 24 mutations in the E2 gene that have not been described so far. In three cases of squamous cell carcinoma a C3409T mutation occurred, which is widely described as oncogenic. This mutation lies in the 3243-3539 area of the E2 hinge region. Statistical analyses show a possible relationship of mutations in this area with oncogenesis. The discovered dependencies may be important in the context of oncogenesis, however, a study with a larger group of patients is needed in order to confirm this view.

Comparison and evaluation of three different molecular methods for detection of human Betapapillomaviruses in skin biopsies from patients with nonmelanoma skin cancer and precancerous lesions.

Betapapillomaviruses have been linked to the development of nonmelanoma skin cancers. A great diversity of these viruses in skin specimens requires the use of sensitive and reliable detection methods. There are currently no standardized assays for diagnostic purposes. A combination of several molecular methods has great practical significance and gives the opportunity to broaden the spectrum of detected Beta-HPV types. In the present study, different molecular methods for Beta-HPVs detection and genotyping were used: PCRs with different sets of primers, PCR followed by reverse hybridization and direct sequencing of PCR amplimers; all performed in skin biopsies from lesions and perilesional healthy area of 118 patients with NMSC or precancerous lesions. Beta-HPVs were detected in 41% of 261 biopsies examined. The RHA for 25 types of Beta-HPVs showed a significantly higher sensitivity than PCR-based methods and allowed to detect 172 genotypes in 86 samples, including 39 with multiple infections. The most frequently identified types were HPV23, HPV24 and HPV93. HPV5 and HPV8, considered high-risk carcinogen types, were detected only in a small percentage of samples. Direct sequencing confirmed the presence of Beta-HPV genotypes from outside of RHA panel in the analysed biopsies. This allowed detecting thirty-two additional genotypes in 5 samples, that were positive only in RHA with the universal probe, which failed to identify the virus genotypes. Our findings confirmed the need to apply different methods to detect Beta-HPV infections.

15-deoxy-Δ12,14-Prostaglandin J2 inhibits human soluble epoxide hydrolase by a dual orthosteric and allosteric mechanism.

Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors.

Structural basis for the regulation of human 5,10-methylenetetrahydrofolate reductase by phosphorylation and S-adenosylmethionine inhibition.

The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.

Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain.

The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme.

Molecular basis of classic galactosemia from the structure of human galactose 1-phosphate uridylyltransferase.

Classic galactosemia is a potentially lethal disease caused by the dysfunction of galactose 1-phosphate uridylyltransferase (GALT). Over 300 disease-associated GALT mutations have been reported, with the majority being missense changes, although a better understanding of their underlying molecular effects has been hindered by the lack of structural information for the human enzyme. Here, we present the 1.9 Å resolution crystal structure of human GALT (hGALT) ternary complex, revealing a homodimer arrangement that contains a covalent uridylylated intermediate and glucose-1-phosphate in the active site, as well as a structural zinc-binding site, per monomer. hGALT reveals significant structural differences from bacterial GALT homologues in metal ligation and dimer interactions, and therefore is a zbetter model for understanding the molecular consequences of disease mutations. Both uridylylation and zinc binding influence the stability and aggregation tendency of hGALT. This has implications for disease-associated variants where p.Gln188Arg, the most commonly detected, increases the rate of aggregation in the absence of zinc likely due to its reduced ability to form the uridylylated intermediate. As such our structure serves as a template in the future design of pharmacological chaperone therapies and opens new concepts about the roles of metal binding and activity in protein misfolding by disease-associated mutants.

Multiplex real-time PCR to identify a possible reinfection with different strains of human cytomegalovirus in allogeneic hematopoietic stem cell transplant recipients.

Human cytomegalovirus (HCMV) infection remains the leading cause of serious contagious complications after allogeneic hematopoietic stem cell transplantation. These infections in HCMV-seropositive recipients can be due to reactivation or reinfection. Different HCMV strains were identified by determining the genotypes isolated from repeatedly tested patients. The UL55 sequences encoding viral glycoprotein B (gB) have been chosen as the target gene. The region, in which the gB precursor protein is cleaved into two fragments by a cellular endoprotease, is characterized by genetic variability, and based on that HCMV is classified into four major genotypes: gB1, gB2, gB3 and gB4. Multiplex real-time PCR assay enabled both, HCMV gB genotyping, as well as simultaneous quantitative assessment of the detected genotypes. This study was carried out in 30 transplant recipients, from whom 105 isolates of HCMV DNA were genotyped. In 40% of recipients, a mixed infection with two or three genotypes was detected. Genotype gB1 dominated in general, and characteristically for mixed infections, the genotype gB3 or gB4 was always present. Although there were no significant differences in the load for each genotype, in case of multiple infections, the number of copies of gB1 genotype was significantly higher when compared to a single gB1 infection. In patients with mixed genotypes, chronic HCMV infections and graft versus host disease were observed more often, as well as antiviral treatment was less effective. It was assumed that these adverse effects can be related to the presence of gB3 and gB4 genotypes.

Crystal structure of Porphyromonas gingivalis peptidylarginine deiminase: implications for autoimmunity in rheumatoid arthritis.

BACKGROUND: Periodontitis (PD) is a known risk factor for rheumatoid arthritis (RA) and there is increasing evidence that the link between the two diseases is due to citrullination by the unique bacterial peptidylarginine deiminase (PAD) enzyme expressed by periodontal pathogen Pophyromonas gingivalis (PPAD). However, the precise mechanism by which PPAD could generate potentially immunogenic peptides has remained controversial due to lack of information about the structural and catalytic mechanisms of the enzyme. OBJECTIVES: By solving the 3D structure of PPAD we aim to characterise activity and elucidate potential mechanisms involved in breach of tolerance to citrullinated proteins in RA. METHODS: PPAD and a catalytically inactive mutant PPAD(C351A) were crystallised and their 3D structures solved. Key residues identified from 3D structures were examined by mutations. Fibrinogen and α-enolase were incubated with PPAD and P. gingivalis arginine gingipain (RgpB) and citrullinated peptides formed were sequenced and quantified by mass spectrometry. RESULTS: Here, we solve the crystal structure of a truncated, highly active form of PPAD. We confirm catalysis is mediated by the following residues: Asp130, His236, Asp238, Asn297 and Cys351 and show Arg152 and Arg154 may determine the substrate specificity of PPAD for C-terminal arginines. We demonstrate the formation of 37 C-terminally citrullinated peptides from fibrinogen and 11 from α-enolase following incubation with tPPAD and RgpB. CONCLUSIONS: PPAD displays an unequivocal specificity for C-terminal arginine residues and readily citrullinates peptides from key RA autoantigens. The formation of these novel citrullinated peptides may be involved in breach of tolerance to citrullinated proteins in RA.

Human ISPD Is a Cytidyltransferase Required for Dystroglycan O-Mannosylation.

A unique, unsolved O-mannosyl glycan on α-dystroglycan is essential for its interaction with protein ligands in the extracellular matrix. Defective O-mannosylation leads to a group of muscular dystrophies, called dystroglycanopathies. Mutations in isoprenoid synthase domain containing (ISPD) represent the second most common cause of these disorders, however, its molecular function remains uncharacterized. The human ISPD (hISPD) crystal structure showed a canonical N-terminal cytidyltransferase domain linked to a C-terminal domain that is absent in cytidyltransferase homologs. Functional studies demonstrated cytosolic localization of hISPD, and cytidyltransferase activity toward pentose phosphates, including ribulose 5-phosphate, ribose 5-phosphate, and ribitol 5-phosphate. Identity of the CDP sugars was confirmed by liquid chromatography quadrupole time-of-flight mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. Our combined results indicate that hISPD is a cytidyltransferase, suggesting the presence of a novel human nucleotide sugar essential for functional α-dystroglycan O-mannosylation in muscle and brain. Thereby, ISPD deficiency can be added to the growing list of tertiary dystroglycanopathies.

Structural Insights into the MMACHC-MMADHC Protein Complex Involved in Vitamin B12 Trafficking.

Conversion of vitamin B12 (cobalamin, Cbl) into the cofactor forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl) is required for the function of two crucial enzymes, mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase, respectively. The intracellular proteins MMACHC and MMADHC play important roles in processing and targeting the Cbl cofactor to its destination enzymes, and recent evidence suggests that they may interact while performing these essential trafficking functions. To better understand the molecular basis of this interaction, we have mapped the crucial protein regions required, indicate that Cbl is likely processed by MMACHC prior to interaction with MMADHC, and identify patient mutations on both proteins that interfere with complex formation, via different mechanisms. We further report the crystal structure of the MMADHC C-terminal region at 2.2 Å resolution, revealing a modified nitroreductase fold with surprising homology to MMACHC despite their poor sequence conservation. Because MMADHC demonstrates no known enzymatic activity, we propose it as the first protein known to repurpose the nitroreductase fold solely for protein-protein interaction. Using small angle x-ray scattering, we reveal the MMACHC-MMADHC complex as a 1:1 heterodimer and provide a structural model of this interaction, where the interaction region overlaps with the MMACHC-Cbl binding site. Together, our findings provide novel structural evidence and mechanistic insight into an essential biological process, whereby an intracellular "trafficking chaperone" highly specific for a trace element cofactor functions via protein-protein interaction, which is disrupted by inherited disease mutations.

Inter-domain communication of human cystathionine ß-synthase: structural basis of S-adenosyl-L-methionine activation.

Cystathionine ß-synthase (CBS) is a key enzyme in sulfur metabolism, and its inherited deficiency causes homocystinuria. Mammalian CBS is modulated by the binding of S-adenosyl-l-methionine (AdoMet) to its regulatory domain, which activates its catalytic domain. To investigate the underlying mechanism, we performed x-ray crystallography, mutagenesis, and mass spectrometry (MS) on human CBS. The 1.7 Å structure of a AdoMet-bound CBS regulatory domain shows one AdoMet molecule per monomer, at the interface between two constituent modules (CBS-1, CBS-2). AdoMet binding is accompanied by a reorientation between the two modules, relative to the AdoMet-free basal state, to form interactions with AdoMet via residues verified by mutagenesis to be important for AdoMet binding (Phe(443), Asp(444), Gln(445), and Asp(538)) and for AdoMet-driven inter-domain communication (Phe(443), Asp(538)). The observed structural change is further supported by ion mobility MS, showing that as-purified CBS exists in two conformational populations, which converged to one in the presence of AdoMet. We therefore propose that AdoMet-induced conformational change alters the interface and arrangement between the catalytic and regulatory domains within the CBS oligomer, thereby increasing the accessibility of the enzyme active site for catalysis.

Differences in the expression of human papillomavirus type 16 (HPV-16) E6 oncogene mRNA in SiHa cell line inoculated with CMV, HSV or ureaplasmas.

One of the factors associated with an increased risk of HPV-related malignant transformation may be bacterial and/or viral infections. The aim of our study was to examine whether the presence of infectious agents commonly detected in the genitourinary tract such as herpesviruses (HSV, CMV), and ureaplasmas (Ureaplasma urealyticum, Ureaplasma parvum) may lead to alterations in the expression of the HPV-16 E6 oncogene. Quantitative RT-PCR analysis was used to assess the level of HPV-16 E6 mRNA expression in SiHa cells. The presence of HSV-1 or HSV-2 in SiHa cells caused a 1.5-fold increase in HPV-16 E6 mRNA expression as compared with non-inoculated SiHa cells. Ureaplasma urealyticum presence but not Ureaplasma parvum stimulated the expression of HPV-16 E6 resulting in a nearly five-fold (4.8) up-regulated E6 mRNA level in SiHa cells. Our study is the first to suggest that infection of Ureaplasma urealyticum in an urogenital tract could increase the risk of cervical cancer by overexpression of the HPV E6 oncogene.

Enzymatic and structural characterization of rTSγ provides insights into the function of rTSß.

In humans, the gene encoding a reverse thymidylate synthase (rTS) is transcribed in the reverse direction of the gene encoding thymidylate synthase (TS) that is involved in DNA biosynthesis. Three isoforms are found: α, ß, and γ, with the transcript of the α-isoform overlapping with that of TS. rTSß has been of interest since the discovery of its overexpression in methotrexate and 5-fluorouracil resistant cell lines. Despite more than 20 years of study, none of the rTS isoforms have been biochemically or structurally characterized. In this study, we identified rTSγ as an l-fuconate dehydratase and determined its high-resolution crystal structure. Our data provide an explanation for the observed difference in enzymatic activities between rTSß and rTSγ, enabling more informed proposals for the possible function of rTSß in chemotherapeutic resistance.

Identification of Der p 23, a peritrophin-like protein, as a new major Dermatophagoides pteronyssinus allergen associated with the peritrophic matrix of mite fecal pellets.

The house dust mite (HDM) Dermatophagoides pteronyssinus is one of most important allergen sources and a major elicitor of allergic asthma. We screened a D. pteronyssinus expression cDNA library with IgE Abs from HDM allergic patients. A cDNA coding for a new major allergen was isolated, which showed sequence homology to peritrophins, which contain chitin-binding domains and are part of the peritrophic matrix lining the gut of arthropods. The mature Der p 23 allergen was expressed in Escherichia coli as an 8-kDa protein without its hydrophobic leader sequence and purified to homogeneity. It reacted with IgE Abs from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. Thus, Der p 23 represents a new major D. pteronyssinus allergen. Furthermore, rDer p 23 exhibited high allergenic activity as demonstrated by upregulation of CD203c expression on basophils from D. pteronyssinus allergic patients. Immunogold electron microscopy localized the allergen in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets. Thus, we identified a new major D. pteronyssinus allergen as peritrophin-like protein. The high allergenic activity of Der p 23 and its frequent recognition as respiratory allergen may be explained by the fact that it becomes airborne and respirable through its association with mite feces. Der p 23 may be an essential component for diagnosis and specific immunotherapy of HDM allergy.

The AEROPATH project targeting Pseudomonas aeruginosa: crystallographic studies for assessment of potential targets in early-stage drug discovery.

Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.

[UL55 genotype diversity of cytomegalovirus strains isolated from newborns and infants hospitalized in southern Poland]. / Róznorodnosc genotypowa UL55 szczepów wirusa cytomegalii izolowanych od noworodków i niemowlat hospitalizowanych w Polsce poludniowej.

Studies on cytomegalovirus (HCMV) infections more often draw attention to the differences in tropism, pathogenicity and virulence of the virus depending on its genotype. The aim of this study was to assess the individual gB genotypes which are encoded in UL55 region of HCMV genome in a population of newborns and infants from Southern Poland. Genotypic analysis was carried out on 53 children (16 newborns and 37 neonates) with confirmed HCMV infection. The children were tested several times. A total of 101 samples, mainly urine, less blood, swabs from the upper respiratory tract, in justified cases, the cerebrospinal fluid were used in our study. Both genotyping and quantitative assessment of HCMV were performed using real time-PCR (rt-PCR). For identification of four major gB genotypes in one reaction, a modification of multiplex rt-PCR was used. Studies confirmed the presence of all major genotypes: gB1, gB2, gB3 and gB4 in the examined groups of children. Only in one case, the genotype could not be determined, perhaps it belonged to subtypes outside the detectable majority ofgB genotypes. Genotype gB1 (63.5%) which was slightly more frequent in infants than in neonates, dominated in our studies. The other genotypes occurred at a rate: gB2 - 15.4%, gB3 - 21.2%, gB4 - 28.8%, respectively. Mixed infections, caused by two genotypes were found in 16 (31%) children, mainly in older infants. There were no statistically significant differences in viral load when comparing a group of newborns with infants and single vs. mixed infection, as well as individual genotypes. The observed differences in the proportional occurrence of different gB genotypes in the two study groups of children may suggest various preferences of particular HCMV genotypes in congenital and acquired infections. Moreover, by monitoring of HCMV infection and determination the genotypes in consecutive samples, it could be identified infection acquired during hospitalization in three children.

Structure of PA4019, a putative aromatic acid decarboxylase from Pseudomonas aeruginosa.

The ubiX gene (PA4019) of Pseudomonas aeruginosa has been annotated as encoding a putative 3-octaprenyl-4-hydroxybenzoate decarboxylase from the ubiquinone-biosynthesis pathway. Based on a transposon mutagenesis screen, this gene was also implicated as being essential for the survival of this organism. The crystal structure of recombinant UbiX determined to 1.5 Å resolution showed that the protein belongs to the superfamily of homo-oligomeric flavine-containing cysteine decarboxylases. The enzyme assembles into a dodecamer with 23 point symmetry. The subunit displays a typical Rossmann fold and contains one FMN molecule bound at the interface between two subunits.

Lymphotropic herpesvirus DNA detection in patients with active CMV infection - a possible role in the course of CMV infection after hematopoietic stem cell transplantation.

BACKGROUND: The natural history of cytomegalovirus (CMV) infection and disease in transplant recipients prompts researchers to look for other factors contributing to this infection. The ubiquity of lymphotropic herpesviruses (EBV, HHV-6, and HHV-7) and the possibility of their activation during immunosuppression may suggest their participation in progression of CMV infection in patients after hematopoietic stem cell transplantation (HSCT). MATERIAL/METHODS: The presence of CMV, EBV, HHV-6 and HHV-7 was confirmed through detection of viral DNA isolated from leukocytes. Allo-HSCT recipients (n=55) were examined repeatedly within the average period of 14±7.3 months post-transplant. RESULTS: CMV DNA was detected in 24% of samples, while EBV, HHV-6 and HHV-7 were detected in 20%, 15% and 14% of samples, respectively. Based on the presence of CMV infection at particular time-points (months) after transplantation, the recipients were divided into 3 groups: Group I (N=15) with persistent infection, Group II (N=20) with transient infection, and Group III (N=20) without CMV infection. In Group I, the mean CMV load was significantly higher than in Group II, and the clinical condition of Group I patients was poorer. All these patients manifested clinical symptoms, and all had episodes of GvHD. All Group I patients developed multiple infections; EBV in 80%, HHV-6 in 47% and HHV-7 in 87% of patients. In the remaining groups, with the exception of HHV-6 in group II, the frequency of infected patients was lower. In addition, CMV presence was often preceded by another herpesvirus. CONCLUSIONS: The results suggest that other herpesviruses, mainly HHV-7, could predispose CMV to cause chronic infection.

Comparison of fluorescence and light scattering based methods to assess formation and stability of protein-protein complexes.

Thermal shift methods such as differential scanning fluorimetry and differential static light scattering are widely used to identify stabilizing conditions for proteins that might promote crystallization. Here we report a comparison of the two methods when applied to optimization of buffer conditions for protein-protein complexes. Most of the protein complexes under study were amenable to analysis using these two techniques. Protein complexes behave towards thermal denaturation in a manner similar to single proteins, showing a more or less sharp transition consistent with a two-state model of unfolding. A comparison of the melting and aggregation temperatures for single components and the reconstituted complexes can provide additional evidence for complex formation and can be used to identify buffer conditions in which protein-protein complex formation is favored.