Thermal Unfolding Transition Research Articles

Cooperative unfolding of Escherichia coli ribosome recycling factor originating from its domain–domain interaction and its implication for function

Cooperative unfolding of Escherichia coli ribosome recycling factor (RRF) and its implication for function were investigated by comparing the in vitro unfolding and the in vivo activity of wild-type E. coli RRF and its temperature-sensitive mutant RRF V117D. The experiments show that mutation V117D at domain I could perturb the domain II structure as evidenced in the near-UV CD and tyrosine fluorescence spectra though no significant globular conformation change occurred. Both equilibrium unfolding induced by heat or denaturant and kinetic unfolding induced by denaturant obey the two-state transition model, indicating V117D mutation does not perturb the efficient interdomain interaction, which results in cooperative unfolding of the RRF protein. However, the mutation significantly destabilizes the E. coli RRF protein, moving the thermal unfolding transition temperature range from 50–65 to 35–50 °C, which spans the non-permissive temperature for the growth of E. coli LJ14 strain ( frr ts). The in vivo activity assays showed that although V117D mutation results in a temperature sensitive phenotype of E. coli LJ14 strain ( frr ts), over-expression of mutant RRF V117D can eliminate the temperature sensitive phenotype at the non-permissive temperature (42 °C). Taking all the results into consideration, it can be suggested that the mechanism of the temperature sensitive phenotype of the E. coli LJ14 cells is due to inactivation of mutant RRF V117D caused by unfolding at the non-permissive temperatures.

Elimination of the C-cap in ubiquitin—structure, dynamics and thermodynamic consequences

Single amino acid substitutions rarely produce substantial changes in protein structure. Here we show that substitution of the C-cap residue in the α-helix of ubiquitin with proline (34P variant) leads to dramatic structural changes. The resulting conformational perturbation extends over the last two turns of the α-helix and leads to enhanced flexibility for residues 27–37. Thermodynamic analysis of this ubiquitin variant using differential scanning calorimetry reveals that the thermal unfolding transition remains highly cooperative, exhibiting two-state behavior. Similarities with the wild type in the thermodynamic parameters (heat capacity change upon unfolding and m-value) of unfolding monitored by DSC and chemical denaturation suggests that the 34P variant has comparable buried surface area. The hydrophobic core of 34P variant is not packed as well as that of the wild type protein as manifested by a lower enthalpy of unfolding. The increased mobility of the polypeptide chain of this ubiquitin variant allows the transient opening of the hydrophobic core as evidenced by ANS binding. Taken together, these results suggest exceptional robustness of cooperativity in protein structures.

Conformational Stabilities of the Structural Repeats of Erythroid Spectrin and Their Functional Implications

The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (T(m)) ranging from 21 to 72 degrees C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol(-1). Constructs comprising five successive repeats from the beta-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation.

Disulfide Formation and Stability of a Cysteine-Rich Repeat Protein from Helicobacter pylori

Helicobacter pylori cysteine-rich proteins (Hcps) are disulfide-containing repeat proteins. The repeating unit is a 36-residue, disulfide-bridged, helix-loop-helix motif. We use the protein HcpB, which has four repeats and four disulfide bridges arrayed in tandem, as a model to determine the thermodynamic stability of a disulfide-rich repeat protein and to study the formation and the contribution to stability of the disulfide bonds. When the disulfide bonds are intact, the chemical unfolding of HcpB at pH 5 is cooperative and can be described by a two-state reaction. Thermal unfolding is reversible between pH 2 and 5 and irreversible at higher pH 5. Differential scanning calorimetry shows noncooperative structural changes preceding the main thermal unfolding transition. Unfolding of the oxidized protein is not an all-or-none two-state process, and the disulfide bonds prevent complete unfolding of the polypeptide chain. The reduced protein is significantly less stable and does not unfold in a cooperative way. During oxidative refolding of the fully reduced protein, all the possible disulfide intermediates with a correct disulfide bond are formed. Formation of "wrong" (non-native) disulfide bonds could not be demonstrated, indicating that the reduced protein already has some partial repeating structure. There is a major folding intermediate with disulfides in the second, third, and fourth repeat and reduced cysteines in the first repeat. Disulfide formation in the first repeat limits the overall rate of oxidative refolding and contributes about half of the thermodynamic stability to native HcpB, estimated as 27 kJ mol(-1) at 25 degrees C and pH 7. The high contribution to stability of the first repeat may be explained by the repeat acting as a cap to protect the hydrophobic interior of the molecule.

Combined spectroscopic and calorimetric characterisation of rubredoxin reversible thermal transition

Rubredoxins are small iron proteins containing the simplest type of iron-sulphur centre, consisting of an iron atom coordinated by the thiol groups of four cysteines. Here we report studies on the conformational stability of a new type of rubredoxin from the hyperthermophile Methanocaldococcus jannaschii, having an atypical metal site geometry resulting from a modified iron-binding motif. Absorption and fluorescence spectroscopies were used in combination with differential scanning calorimetry to probe different aspects of the thermal unfolding transition: iron site degradation (absorption at 380 nm), tertiary structure unfolding (Trp emission), exposure of hydrophobic regions (1-anilinonaphalene-8-sulphonate fluorescence enhancement) and iron release. Thermal denaturation was found to be irreversible and caused by decomposition of the metal centre. The protein is hyperstable and between pH 4 and 10 it is only thermally denatured in the presence of a strong chemical denaturant. The study of the heating rate dependence of the melting temperature allowed us to determine the reaction equilibrium thermodynamic parameters. At pH 2 the protein is destabilised owing to the absence of salt bridges and it has a T(m) of 65 degrees C. In these conditions, there is excellent agreement between the parameters determined by the different spectroscopic methods and calorimetry. The highest stability was found to be at pH 8, and a detailed study of the heating rate dependence in the presence of guanidine thiocyanate in this condition allowed the determination of a reversible T(m) of 118 degrees C.

Multistate Folding of the Villin Headpiece Domain

The villin headpiece (HP67) is a 67 residue, monomeric protein derived from the C-terminal domain of villin. Wild-type HP67 (WT HP67) is the smallest fragment of villin that retains strong in vitro actin-binding activity. WT HP67 is made up of two subdomains, which form a tightly packed interface. The C-terminal subdomain of WT HP67, denoted HP35, is rich in helical structure, folds in isolation, and has been widely used as a model system for folding studies. In contrast, very little is known about the folding of the intact villin headpiece domain. Here, NMR, CD and H/2H amide exchange measurements are used to follow the pH, thermal and urea-induced unfolding of WT HP67 and a mutant (HP67 H41Y) in which a buried conserved histidine in the N-terminal subdomain, His41, has been mutated to Tyr. Although most small proteins display two-state equilibrium unfolding, the results presented here demonstrate that unfolding of the villin headpiece is a multistate process. The presence of a folded N-terminal subdomain is shown to stabilize the C-terminal subdomain, increasing the midpoints of the thermal and urea-induced unfolding transitions and increasing protection factors for H/2H exchange. Histidine 41 has been shown to act as a pH-dependent switch in wild-type HP67: the N-terminal subdomain is unfolded when His41 is protonated, while the C-terminal subdomain remains folded irrespective of the protonation state of His41. Mutation of His41 to Tyr eliminates the segmental pH-dependent unfolding of the headpiece. The mutation stabilizes both domains, but folding is still multistate, indicating that His41 is not solely responsible for the unusual equilibrium unfolding behavior of villin headpiece domain.

Effect of modulating unfolded state structure on the folding kinetics of the villin headpiece subdomain.

Equilibrium Fourier transform infrared (FTIR) and temperature-jump (T-jump) IR spectroscopic techniques were used to study the thermodynamics and kinetics of the unfolding and folding of the villin headpiece helical subdomain (HP36), a small three-helix protein. A double phenylalanine mutant (HP36 F47L, F51L) that destabilizes the hydrophobic core of this protein also was studied. The double mutant is less stable than wild type (WT) and has been shown to contain less residual secondary structure and tertiary contacts in its unfolded state. The relaxation kinetics after a T-jump perturbation were studied for both HP36 and HP36 F47L, F51L. Both proteins exhibited biphasic relaxation kinetics in response to a T-jump. The folding times for the WT (3.23 micros at 60.2 degrees C) and double phenylalanine mutant (3.01 micros at 49.9 degrees C) at the approximate midpoints of their thermal unfolding transitions were found to be similar. The folding time for the WT was determined to be 3.34 mus at 49.9 degrees C, similar to the folding time of the double phenylalanine mutant at that temperature. The double phenylalanine mutant, however, unfolds faster with an unfolding time of 3.01 micros compared with 6.97 micros for the WT at 49.9 degrees C.

Thermostability of Irreversible Unfolding α-Amylases Analyzed by Unfolding Kinetics

For most multidomain proteins the thermal unfolding transitions are accompanied by an irreversible step, often related to aggregation at elevated temperatures. As a consequence the analysis of thermostabilities in terms of equilibrium thermodynamics is not applicable, at least not if the irreversible process is fast with respect the structural unfolding transition. In a comparative study we investigated aggregation effects and unfolding kinetics for five homologous alpha-amylases, all from mesophilic sources but with rather different thermostabilities. The results indicate that for all enzymes the irreversible process is fast and the precedent unfolding transition is the rate-limiting step. In this case the kinetic barrier toward unfolding, as measured by unfolding rates as function of temperature, is the key feature in thermostability. The investigated enzymes exhibit activation energies (E(a)) between 208 and 364 kJmol(-1) and pronounced differences in the corresponding unfolding rates. The most thermostable alpha-amylase from Bacillus licheniformis (apparent transition temperature, T(1/2) approximately 100 degrees C) shows an unfolding rate which is four orders of magnitude smaller as compared with the alpha-amylase from pig pancreas (T(1/2) approximately 65 degrees C). Even with respect to two other alpha-amylases from Bacillus species (T(1/2) approximately 86 degrees C) the difference in unfolding rates is still two orders of magnitude.

Enhanced thermal stability achieved without increased conformational rigidity at physiological temperatures: Spatial propagation of differential flexibility in rubredoxin hybrids

The extreme thermal stability of proteins from hyperthermophilic organisms is widely believed to arise from an increased conformational rigidity in the native state. In apparent contrast to this paradigm, both Pyrococcus furiosus (Pf) rubredoxin, the most thermostable protein characterized to date, and its Clostridium pasteurianum (Cp) mesophile homolog undergo a transient conformational opening of their multi-turn segments, which is more favorable in hyperthermophile proteins below room temperature. Substitution of the hyperthermophile multi-turn sequence into the mesophile protein sequence yields a hybrid, (14-33(Pf)) Cp, that exhibits a 12 degrees increase in its reversible thermal unfolding transition midpoint. Nuclear magnetic resonance (NMR) magnetization transfer-based hydrogen exchange was used to monitor backbone conformational dynamics in the subsecond time regime. Despite the substantially increased thermostability, flexibility throughout the entire main chain of the more thermostable hybrid is equal to or greater than that of the wild type mesophile rubredoxin near its normal growth temperature. In comparison to the identical core residues of the (14-33(Pf)) Cp rubredoxin hybrid, six spatially clustered residues in the parental mesophile protein exhibit a substantially larger temperature dependence of exchange. The exchange behavior of these six residues closely matches that observed in the multi-turn segment, consistent with a more extensive conformational process. These six core residues exhibit a much weaker temperature dependence of exchange in the (14-33(Pf)) Cp hybrid, similar to that observed for the multi-turn segment in its parental Pf rubredoxin. These results suggest that differential temperature dependence of flexibility can underlie variations in thermostability observed for mesophile versus hyperthermophile homologs.

Compatible solute effects on thermostability of glutamine synthetase and aspartate transcarbamoylase from Methanococcus jannaschii

Methanococcus jannaschii accumulates α- and β-glutamate as osmolytes. The effect of these and other solutes on the thermostability of two multisubunit metabolic enzymes from M. jannaschii, aspartate transcarbamoylase catalytic trimer (ATCase C 3) and glutamine synthetase (GS), has been measured and compared to solute effects on bacterial mesophilic counterparts in order to explore if osmolytes accumulated by each organism can preferentially stabilize the proteins to thermal unfolding. For both ATCase enzymes and for the B. subtilis GS, the solutes normally accumulated by the organism were very effective in protecting the enzyme from losing activity at high temperatures, although solute effects on loss of secondary structure did not necessarily correlate with this thermoprotection of activity. The recombinant M. jannaschii GS exhibited quite different behavior. The pure enzyme had a thermal unfolding transition with a midpoint temperature ( T m) less than 60 °C, well under the growth temperature of the organism (85 °C). None of the small molecule solutes tested (including the K +-glutamate isomers accumulated by M. jannaschii) significantly stabilized the protein to incubation at 85 °C. Instead, protein–protein interactions, as illustrated by E. coli GroEL or ribosomal protein L2 stabilization of GS, appeared to be the dominant factor in stabilizing this archaeal enzyme at the growth temperature.

Heat capacity-independent determination of differential free energy of stability between structurally homologous proteins

Under the assumption of equivalent heat capacity values, the differential free energy of stability for a pair of proteins midway between their thermal unfolding transition temperatures is shown to be independent of Δ C p up to its cubic term in Δ T m . For model calculations reflecting the nearly 30 °C difference in T m for the adenylate kinases from the arctic bacterium Bacillus globisporus and the thermophilic bacterium Geobacillus stearothermophilus, the resultant error in estimating ΔΔ G by the formula 0.5 [Δ S T m1 (1) + Δ S T m2 (2)] Δ T m is less than 1%. Combined with the analogous thermal unfolding data for the adenylate kinase from Escherichia coli, these three homologous proteins exhibit T m and Δ S T m values consistent with differential entropy and enthalpy contributions of equal magnitude. When entropy–enthalpy compensation holds for the differential free energy of stability, the incremental changes in T m values are shown to be proportionate to the changes in free energy.

Helical and Expanded Conformation of Equine β-Lactoglobulin in the Cold-denatured State

The thermal unfolding transition of equine beta-lactoglobulin (ELG) was investigated by circular dichroism (CD) over a temperature range of -15 degrees C to 85 degrees C. In the presence of 2 M urea, a cooperative unfolding transition was observed both with increasing and decreasing temperature. The CD spectrum indicated that the heat and cold-denatured states of ELG have substantial secondary structures but lack persistent tertiary packing of the side-chains. In order to clarify the relation between the heat or cold-denatured state and the acid-denatured (A) state characterized previously, we have attempted to observe the temperature dependence of the CD spectrum at pH 1.5. The CD spectrum in the heat-denatured state is similar to that in the A state. The CD spectrum in the A state does not change cooperatively with increasing temperature. These results indicate that the heat-denatured state and the A state are the same structural state. On the other hand, the CD intensity at acid pH cooperatively increased with decreasing temperature. The CD spectrum at low temperature and acid pH is consistent with that in the cold-denatured state. Therefore, the cold-denatured state is distinguished from the heat-denatured state or the A state, and ELG assumes a larger amount of non-native alpha-helices in the cold-denatured state. Small angle X-ray scattering and analytical ultracentrifugation have indicated that ELG assumes an expanded chain-like conformation in the cold-denatured state in contrast to the compact globular conformation in the A state. The relation between the molecular size and the helical content in the partially folded states is discussed.

The Fusion Activity of HIV-1 gp41 Depends on Interhelical Interactions

Infection by human immunodeficiency virus type I requires the fusogenic activity of gp41, the transmembrane subunit of the viral envelope protein. Crystallographic studies have revealed that fusion-active gp41 is a "trimer-of-hairpins" in which three central N-terminal helices form a trimeric coiled coil surrounded by three antiparallel C-terminal helices. This structure is stabilized primarily by hydrophobic, interhelical interactions, and several critical contacts are made between residues that form a deep cavity in the N-terminal trimer and the C-helix residues that pack into this cavity. In addition, the trimer-of-hairpins structure has an extensive network of hydrogen bonds within a conserved glutamine-rich layer of poorly understood function. Formation of the trimer-of-hairpins structure is thought to directly force the viral and target membranes together, resulting in membrane fusion and viral entry. We test this hypothesis by constructing four series of gp41 mutants with disrupted interactions between the N- and C-helices. Notably, in the three series containing mutations within the cavity, gp41 activity correlates well with the stability of the N-C interhelical interaction. In contrast, a fourth series of mutants involving the glutamine layer residue Gln-653 show fusion defects even though the stability of the hairpin is close to wild-type. These results provide evidence that gp41 hairpin stability is critical for mediating fusion and suggest a novel role for the glutamine layer in gp41 function.

Stabilities and activities of the N‐ and C‐domains of FKBP22 from a psychrotrophic bacterium overproduced in Escherichia coli

FKBP22 from a psychrotrophic bacterium Shewanella sp. SIB1, is a dimeric protein with peptidyl prolyl cis-trans isomerase (PPIase) activity. According to homology modeling, it consists of an N-terminal domain, which is involved in dimerization of the protein, and a C-terminal catalytic domain. A long alpha3 helix spans these domains. An N-domain with the entire alpha3 helix (N-domain+) and a C-domain with the entire alpha3 helix (C-domain+) were overproduced in Escherichia coli in a His-tagged form, purified, and their biochemical properties were compared with those of the intact protein. C-domain+ was shown to be a monomer and enzymatically active. Its optimum temperature for activity (10 degrees C) was identical to that of the intact protein. Determination of the PPIase activity using peptide and protein substrates suggests that dimerization is required to make the protein fully active for the protein substrate or that the N-domain is involved in substrate-binding. The differential scanning calorimetry studies revealed two distinct heat absorption peaks at 32.5 degrees C and 46.6 degrees C for the intact protein, and single heat absorption peaks at 44.7 degrees C for N-domain+ and 35.6 degrees C for C-domain+. These results indicate that the thermal unfolding transitions of the intact protein at lower and higher temperatures represent those of C- and N-domains, respectively. Because the unfolding temperature of C-domain+ is much higher than its optimum temperature for activity, SIB1 FKBP22 may adapt to low temperatures by increasing a local flexibility around the active site. This study revealed the relationship between the stability and the activity of a psychrotrophic FKBP22.

Solution structure of choline binding protein A, the major adhesin of Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) remains a significant health threat worldwide, especially to the young and old. While some of the biomolecules involved in pneumococcal pathogenesis are known and understood in mechanistic terms, little is known about the molecular details of bacterium/host interactions. We report here the solution structure of the 'repeated' adhesion domains (domains R1 and R2) of the principal pneumococcal adhesin, choline binding protein A (CbpA). Further, we provide insights into the mechanism by which CbpA binds its human receptor, polymeric immunoglobulin receptor (pIgR). The R domains, comprised of 12 imperfect copies of the leucine zipper heptad motif, adopt a unique 3-alpha-helix, raft-like structure. Each pair of alpha-helices is antiparallel and conserved residues in the loop between Helices 1 and 2 exhibit a novel 'tyrosine fork' structure that is involved in binding pIgR. This and other structural features that we show are conserved in most pneumococcal strains appear to generally play an important role in bacterial adhesion to pIgR. Interestingly, pneumococcus is the only bacterium known to adhere to and invade human cells by binding to pIgR.

Thermodynamics of α-lactalbumin– dl-α-dipalmitoylphosphatidylcholine interactions and effect of the antioxidant nicotinamide on these interactions

Differential scanning calorimetry has been used to understand the thermodynamics of the interactions of dl-α-dipalmitoylphosphatidylcholine (DPPC) with α-lactalbumin and the effect of the antioxidant nicotinamide on these interactions. Nicotinamide decreases the thermal transition temperature of both the lipid and the protein at high concentrations. The thermal unfolding transitions of the protein were two state and calorimetrically reversible. There was no significant change in the shape and thermodynamic parameters accompanying the lipid endotherms, suggesting that nicotinamide did not penetrate the lipid bilayer. The thermal unfoldings of α-lactalbumin in the presence of DPPC as cosolute also adhered to two-state reversible mechanism. The changes in the thermodynamic parameters accompanying the thermal transitions were small, indicating no significant interaction of α-lactalbumin with DPPC. The changes in the thermodynamic parameters indicate that the lipid bilayer organization, as well as the partitioning of the extrinsic protein α-lactalbumin into the bilayer, is not affected in the entire studied concentration range of the lipid. It is observed that the presence of increasing concentration of nicotinamide (as high as 1.0 mol dm −3) in the lipid–protein mixture does not affect its partitioning into the lipid bilayer, although nicotinamide preferentially interacts with α-lactalbumin. The change in the effect of nicotinamide on lipid transition temperature in the mixture and literature report suggests that nicotinamide may be forming a hydrogen-bonded complex with the protein through its amide functionality. The surface tension data of aqueous nicotinamide in combination with the thermal denaturation results of protein in presence of nicotinamide confirmed that surface tension effect does not have any significant contribution to the effect of nicotinamide on protein.

A novel method to determine thermal transition curves of disulfide-containing proteins and their structured folding intermediates

The stability of a protein or of its folding intermediates is frequently characterized by its resistance to chemical and/or thermal denaturation. The folding/unfolding process is generally followed by spectroscopic methods such as absorbance, fluorescence, circular dichroism spectroscopy, etc. Here, we demonstrate a new method, by using HPLC, for determining the thermal unfolding transitions of disulfide-containing proteins and their structured folding intermediates. The thermal transitions of a model protein, ribonuclease A (RNase A), and a recently found unfolding intermediate of onconase (ONC), des [30–75], have been estimated by this method. Finally, the advantages of this method over traditional techniques are discussed by providing specific examples.

Thermodynamics of denaturation of complexes of barnase and binase with barstar

Differential scanning calorimetry was used to study the thermodynamics of denaturation of protein complexes for which the free energy stabilizing the complexes varied between −8 and −16 kcal/mol. The proteins studied were the ribonucleases barnase and binase, their inhibitor barstar and mutants thereof, and complexes between the two. The results are in good agreement with the model developed by Brandts and Lin for studying the thermodynamics of denaturation for tight complexes between two proteins which undergo two-state thermal unfolding transitions.

Detection of an Intermediate during Unfolding of Bacterial Cell Division Protein FtsZ: LOSS OF FUNCTIONAL PROPERTIES PRECEDES THE GLOBAL UNFOLDING OF FtsZ

Using environment-sensitive fluorescence of 1-anilinonaphthalene-8-sulfonic acid, polarization of fluorescein 5'-isothiocyanate-labeled FtsZ, and far-UV circular dichroism spectroscopy, the chemical unfolding of FtsZ was found to proceed through two steps. The first step of the urea-induced unfolding produced an intermediate, which then unfolded at higher concentrations of urea. The intermediate state contains native-like secondary structure and much less tertiary structure compared with the native state. It is distinct from the native state as well as from the unfolded state. Similar to urea-induced unfolding of FtsZ, thermal unfolding of FtsZ also occurs in two steps. The midpoints for the first and second thermal unfolding transitions were found to be 38 +/- 4 and 77 +/- 5 degrees C, respectively. Further, the functional properties of FtsZ are extremely sensitive to urea, guanidium chloride, and sodium dodecyl sulfate. For example, 50% inhibition of the FtsZ assembly and GTP hydrolysis occurred at 0.1 and 0.2 m of urea, respectively. FtsZ lost its functional properties before any significant perturbation in the secondary or tertiary structure was detected by using several fluorescence techniques and far UV-CD indicating preferential local unfolding of the functional region(s). In addition, the unfolded FtsZ regains its ability to polymerize fully upon removal of urea. The data taken together suggest that FtsZ unfolds reversibly through a multistep process, and local responses that inhibit functional properties precede the global transition of FtsZ to the unfolded state.

Contribution to stability and folding of a buried polar residue at the CARM1 methylation site of the KIX domain of CBP.

The transcriptional coactivator and acetyltransferase CREB Binding Protein (CBP) is comprised of several autonomously folded and functionally independent domains. The KIX domain mediates interactions between CBP and numerous transcriptional activators. The folded region of KIX has all the structural features of a globular protein, including three alpha-helices, two short 3(10) helices, and a well-packed hydrophobic core. KIX contains a buried cation-pi interaction between the positively charged guanidinium group of Arg 600 and the aromatic ring of Tyr 640. Arg 600 is a site for regulatory methylation by CARM1/PRMT4, which negates the CREB-binding function of the KIX domain. The role of the Arg 600-Tyr 640 buried polar interaction in specifying and stabilizing the structure of KIX was investigated by comparing the folding of wild-type KIX with the single point mutants Y640F and R600M. The Y640F mutant disrupts a hydrogen bond involving the Tyr 640 OH and the backbone of V595 but still allows for the cation-pi interaction while the R600M mutant disrupts the cation-pi interaction. Both wild type KIX and Y640F exhibit properties expected of native like, globular proteins such as a single oligomerization state (monomer), cooperative thermal and urea-induced unfolding transitions, and a well-packed core. In contrast, the R600M mutant has properties reminiscent of a molten globule state, including a tendency to aggregate, noncooperative thermal unfolding transition, and a loosely packed core. Thus, the buried cation-pi interaction is critical for specifying the unique cooperatively folded structure of KIX.