Islet amyloid deposition limits the viability of human islet grafts but not porcine islet grafts.

Islet transplantation is a promising treatment for diabetes but long-term success is limited by progressive graft loss. Aggregates of the beta cell peptide islet amyloid polypeptide (IAPP) promote beta cell apoptosis and rapid amyloid formation occurs in transplanted islets. Porcine islets are an attractive alternative islet source as they demonstrate long-term graft survival. We compared the capacity of transplanted human and porcine islets to form amyloid as an explanation for differences in graft survival. Human islets were transplanted into streptozotocin-diabetic immune-deficient mice. Amyloid deposition was detectable at 4 weeks posttransplantation and was associated with islet graft failure. More extensive amyloid deposition was observed after 8 weeks. By contrast, no amyloid was detected in transplanted neonatal or adult porcine islets that had maintained normoglycemia for up to 195 days. To determine whether differences in IAPP sequence between humans and pigs could explain differences in amyloid formation and transplant viability, we sequenced porcine IAPP. Porcine IAPP differs from the human sequence at 10 positions and includes substitutions predicted to reduce its amyloidogenicity. Synthetic porcine IAPP was considerably less amyloidogenic than human IAPP as determined by transmission electron microscopy, circular dichroism, and thioflavin T binding. Viability assays indicated that porcine IAPP is significantly less toxic to INS-1 beta cells than human IAPP. Our findings demonstrate that species differences in IAPP sequence can explain the lack of amyloid formation and improved survival of transplanted porcine islets. These data highlight the potential of porcine islet transplantation as a therapeutic approach for human diabetes.

Toxic oligomers and islet beta cell death: guilty by association or convicted by circumstantial evidence?

Type 2 diabetes is a progressive disease characterised by islet amyloid deposits in the majority of patients. Amyloid formation is considered a significant factor in deterioration of islet function and reduction in beta cell mass, and involves aggregation of monomers of the normally soluble beta cell peptide, human islet amyloid polypeptide (hIAPP) into oligomers, fibrils and, ultimately, mature amyloid deposits. Despite extensive in vitro studies, the process of hIAPP aggregation in vivo is poorly understood, though it is widely reported to promote cytotoxicity. Recently, studies have suggested that only the early stages of fibril assembly, and in particular small hIAPP oligomers, are responsible for beta cell cytotoxicity. This challenges the prior concept that newly formed fibrils and/or mature fibrillar amyloid are cytotoxic. Herein, evidence both for and against the toxic hIAPP oligomer hypothesis is presented; from this, it is apparent that what exactly causes beta cell death when hIAPP aggregates remains debatable. Moreover, substantially more work with more specific reagents and techniques than are currently available will be required to identify conclusively the toxic species resulting from hIAPP aggregation. Keeping an open mind on the nature of the cytotoxic insult has implications for therapeutic developments and clinical care in type 2 diabetes.

Local interactions and the role of the 6-120 disulfide bond in alpha-lactalbumin: implications for formation of the molten globule state.

Molten globule states are partially folded states of proteins which are compact and contain a high degree of secondary structure but which lack many of the fixed tertiary interactions associated with the native state. A set of peptides has been prepared in order to probe the role of local interactions in the vicinity of the Cys(6)-Cys(120) disulfide bond in stabilizing the molten globule state of human alpha-lactalbumin. Peptides derived from the N-terminal and C-terminal regions of human alpha-lactalbumin have been analyzed using nuclear magnetic resonance, circular dichroism, fluorescence spectroscopy and sedimentation equilibrium experiments. A peptide corresponding to the first helical region in the native protein, residues 1-13, is only slightly helical in isolation. Extending the peptide to include residues 14-18 results in a modest increase in helicity. A peptide derived from the C-terminal 12 residues, residues 112-123, is predominantly unstructured. Crosslinking the N- and C-terminal peptides by the native disulfide bond results in almost no increase in structure and there is no evidence for any significant cooperative structure formation over the range of pH 2.2-11.7. These results demonstrate that there is very little enhancement of local structure due to the formation of the Cys(6)-Cys(120) disulfide bond. This is in striking contrast to peptides derived from the region of the Cys(28)-Cys(111) disulfide.

pH-dependent interactions and the stability and folding kinetics of the N-terminal domain of L9. Electrostatic interactions are only weakly formed in the transition state for folding.

The role of electrostatic interactions in the stability and the folding of the N-terminal domain of the ribosomal protein L9 (NTL9) was investigated by determining the effects of varying the pH conditions. Urea denaturations and thermal unfolding experiments were used to measure the free energy of folding, DeltaG degrees, at 18 different pH values, ranging from pH 1.1 to pH 10.5. Folding rates were measured at 19 pH values between pH 2.1 and pH 9.5, and unfolding rates were determined at 15 pH values in this range using stopped-flow fluorescence experiments. The protein is maximally stable between pH 5.5 and 7.5 with a value of DeltaG degrees =4.45 kcal mol(-1). The folding rate reaches a maximum at pH 5.5, however the change in folding rates with pH is relatively modest. Over the pH range of 2.1 to 5.5 there is a small increase in folding rates, ln (k(f)) changes from 5.1 to 6.8. However, the change in stability is more dramatic, with a difference of 2.6 kcal mol(-1) between pH 2.0 and pH 5.4. The change in stability is largely due to the smaller barrier for unfolding at low pH values. The natural log of the unfolding rates varies by approximately four units between pH 2.1 and pH 5.5. The stability of the protein decreases above pH 7.5 and again the change is largely due to changes in the unfolding rate. ln (k(f)) varies by less than one unit between pH 5.5 and pH 9.5 while DeltaG degrees decreases by 2.4 kcal mol(-1) over the range of pH 5. 4 to pH 10.0, which corresponds to a change in ln K(eq) of 4.0. These studies show that pH-dependent interactions contribute significantly to the overall stability of the protein but have only a small effect upon the folding kinetics, indicating that electrostatic interactions are weakly formed in the transition state for folding.

On the global architecture of initiation factor IF3: a comparative study of the linker regions from the Escherichia coli protein and the Bacillus stearothermophilus protein.

Initiation factor IF3 is a protein involved in the initiation stage of protein synthesis. It consists of two global domains linked by a 20 residue long, solvent-exposed linker. Recently, the structure of the N and C-terminal domains of the Bacillus stearothermophilus protein have been solved by X-ray crystallography and the structure of the intact Escherichia coli protein has been studied by NMR. These two studies have led to apparently contradictory models for the domain organization of IF3. The NMR study of the E. coli protein indicates that the linker region is flexible, while the studies of the isolated N and C-terminal domains of the B. stearothermophilus protein suggest that the linker forms a rigid helical rod. In order to resolve this discrepancy, a set of peptides corresponding to the linker regions of the B. stearothermophilus and the E. coli protein were synthesized. Circular dichroism and NMR spectroscopy were used to study the helical content as a function of pH, temperature, peptide concentration and ionic strength. Both peptides are monomeric. The estimated helical content of the linker fragment from B. stearothermophilus is 68% at high pH and 1 degree C. The measured helicity decreases to 53% at pH 7.0 and 1 degree C. In contrast, the peptide corresponding to the E. coli IF3 linker region is largely unstructured with a maximum helical content of 15% at high pH and only 8% at pH 7.0, 1 degree C. These results suggest that the different structures observed for the two intact proteins may be due to the different intrinsic stability of the two linker peptides. The helical content of the two linker peptides is, however, much closer when the peptides are compared at the respective temperatures of optimum growth for E. coli and B. stearothermophilus (3% versus 17%). The pH and ionic strength dependence of the helical content of the B. stearothermophilus peptide demonstrates that side-chain/side-chain interactions play an important role in stabilizing the helical structure. In addition, studies with mutant peptides show that the first Asp residue in the linker sequence helps to stabilize the helix via an N- capping interaction.

Submillisecond folding of the peripheral subunit-binding domain.

Folding and unfolding rates have been measured for the peripheral subunit-binding domain, a small three-helix protein. The protein folds very fast, with rates too rapid to be measured using traditional stopped-flow techniques. Folding and unfolding rates were measured as a function of temperature using dynamic NMR lineshape analysis. At the lowest temperature at which there is sufficient broadening to measure rates, 41 degrees C, the folding rate is 16,050 s(-1). Thus, the halftime required for folding is 43 micros. At the same temperature, the unfolding rate is 2800 s(-1). Identical rates were measured using resolved resonances from Val16 in the loop and Val21 at the end of the 310-helix. Folding rates have been correlated with protein topology, and this correlation is consistent with the rapid folding of the peripheral subunit-binding domain. The results presented here show that the peripheral subunit-binding domain is the third fastest folding protein for which rates have been estimated. The folding rate is the fastest that has been directly measured and provides further support for the importance of chain topology as a major determinant of folding rates.

Analysis of amylin cleavage products provides new insights into the amyloidogenic region of human amylin.

Human amylin is the primary component of amyloid deposits found in the pancreatic beta-cells of patients with type 2 diabetes mellitus. Recently, two fragments of amylin have been identified in vivo. One fragment contains residues 17 to 37 of human amylin (AMYLIN17-37) and the other contains residues 24 to 37 (AMYLIN24-37). The secondary structure and amyloid forming ability of each peptide was determined at pH 5.5(+/-0.3) and pH 7.4(+/-0.3). Results at these two values of pH were very similar. Both peptides are predominantly unstructured in solution (CD) but adopt a significant amount of beta-sheet secondary structure upon aggregation (FTIR). Transmission electron microscopy (TEM) confirmed the presence of amyloid fibrils. AMYLIN24-37 was further dissected by studying peptides corresponding to residues 24 to 29 and 30 to 37. The AMYLIN30-37 peptide forms amyloid deposits. Samples of the 24 to 29 fragment which had TFA as the associated counterion formed ordered deposits but samples associated with HCl did not. Residues 20 to 29 are traditionally thought to be the amyloidogenic region of amylin, but this study demonstrates that peptides derived from other regions of amylin are capable of forming amyloid, and hence indicates that these regions of amylin can play a role in amyloid formation.

Peptide models of local and long-range interactions in the molten globule state of human alpha-lactalbumin.

alpha-Lactalbumin, a small calcium-binding protein, forms an equilibrium molten globule state under a variety of conditions. A set of four peptides designed to probe the role of local interactions and the role of potential long-range interactions in stabilizing the molten globule of alpha-lactalbumin has been prepared. The first peptide consists of residues 20 through 36 of human alpha-lactalbumin and includes the entire B-helix. This peptide is unstructured in solution as judged by CD. The second peptide is derived from residues 101 through 120 and contains both the D and 310 helices. When this peptide is crosslinked via the native 28 to 111 disulfide to the B-helix peptide, a dramatic increase in helicity is observed. The crosslinked peptide is monomeric, as judged by analytical ultracentrifugation. The peptide binds 1-anilinonaphthalene-8-sulphonate (ANS) and the fluorescence emission maximum of the construct is consistent with partial solvent exposure of the tryptophan residues. The peptide corresponding to residues 101 to 120 adopts significant non-random structure in aqueous solution at low pH. Two hydrophobic clusters, one involving residues 101 through 104 and the other residues 115 through 119 have been identified and characterized by NMR. The hydrophobic cluster formed by residues 101 through 104 is still present in a smaller peptide containing only residues 101 to 111 of alpha-lactalbumin. The cluster also persists in 6 M urea. A non-native, pH-dependent interaction between the Y103 and H107 side-chains that was previously identified in the acid-denatured molten globule state was examined. This interaction was found to be more prevalent at low pH and may therefore be an example of a local interaction that stabilizes preferentially the acid-induced molten globule state.

On the relationship between protein stability and folding kinetics: a comparative study of the N-terminal domains of RNase HI, E. coli and Bacillus stearothermophilus L9.

There is currently a great deal of interest in proteins that fold in a single highly cooperative step. Particular attention has been focused on elucidating the factors that govern folding rates of simple proteins. Recently, the topology of the native state has been proposed to be the most important determinant of their folding rates. Here we report a comparative study of the folding of three topologically equivalent proteins that adapt a particularly simple alpha/beta fold. The folding kinetics of the N-terminal domain of RNase HI and the N-terminal domain of the ribosomal protein L9 from Escherichia coli (eNTL9) were compared to the previously characterized N-terminal domain of L9 from Bacillus stearothermophilus (bNTL9). This 6.2 kDa protein, which is one of simplest examples of the ABCalphaD motif, folds via a two-state mechanism on the millisecond to submillisecond time scale. The RNase HI domain and bNTL9 have very similar tertiary structures but there is little similarity in primary sequence. bNTL9 and eNTL9 share the same biological function and a similar primary sequence but differ significantly in stability. Fluorescence-detected stopped-flow experiments showed that the three proteins fold in a two-state fashion. The folding rates in the absence of denaturant were found to be very different, ranging form 21 s(-1) to 790 s(-1) at 10 degrees C. The diverse folding rates appear to reflect large differences in the stability of the proteins. When compared at an isostability point, the folding rates converged to a similar value and there is a strong linear correlation between the log of the folding rate and stability for this set of proteins. These observations are consistent with the idea that stability can play an important role in dictating relative folding rates among topologically equivalent proteins.

Global analysis of the effects of temperature and denaturant on the folding and unfolding kinetics of the N-terminal domain of the protein L9.

The folding and unfolding kinetics of the N-terminal domain of the ribosomal protein L9 have been measured at temperatures between 7 and 85 degrees C and between 0 and 6 M guanidine deuterium chloride. Stopped-flow fluorescence was used to measure rates below 55 degrees C and NMR lineshape analysis was used above 55 degrees C. The amplitudes and rate profiles of the stopped-flow fluorescence experiments are consistent with a two-state folding mechanism, and plots of ln(k) versus guanidine deuterium chloride concentration show the classic v-shape indicative of two-state folding. There is no roll over in the plots when the experiments are repeated in the presence of 400 mM sodium sulfate. Temperature and denaturant effects were fit simultaneously to the simple model k=D exp(-DeltaG*/RT) where DeltaG* represents the change in apparent free energy between the transition state and the folded or unfolded state and D represents the maximum possible folding speed. DeltaG* is assumed to vary linearly with denaturant concentration and the Gibbs-Helmholtz equation is used to model stability changes with temperature. Approximately 60% of the surface area buried upon folding is buried in the transition state as evidenced by changes in the heat capacity and m value between the unfolded state and the transition state. The equilibrium thermodynamic parameters, DeltaCp degrees, m and DeltaG degrees, all agree with the values calculated from the kinetic experiments, providing additional evidence that folding is two-state. The folding rates at 0 M guanidine hydrochloride show a non-Arrhenius temperature dependence typical of globular proteins. When the folding rates are examined along constant DeltaG degrees/T contours they display an Arrhenius temperature dependence with a slope of -8600 K. This indicates that for this system, the non-Arrhenius temperature dependence of folding can be accounted for by the anomalous temperature dependence of the interactions which stabilize proteins.

Conformational analysis of a set of peptides corresponding to the entire primary sequence of the N-terminal domain of the ribosomal protein L9: evidence for stable native-like secondary structure in the unfolded state.

There is considerable interest in the structure of the denatured state and in the role local interactions play in protein stability and protein folding. Studies of peptide fragments provide one method to assess local conformational preferences which may be present in the denatured state under native-like conditions. A set of peptides corresponding to the individual elements of secondary structure derived from the N-terminal domain of the ribosomal protein L9 have been synthesized. This small 56 residue protein adopts a mixed alpha-beta topology and has been shown to fold rapidly in an apparent two-state fashion. The conformational preferences of each peptide have been analyzed by proton nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy. Peptides corresponding to each of the three beta-stands and to the first alpha-helix are unstructured as judged by CD and NMR. In contrast, a peptide corresponding to the C-terminal helix is remarkably structured. This 17 residue peptide is 53 % helical at pH 5.4, 4 degrees C. Two-dimensional NMR studies demonstrate that the helical structure is distributed approximately uniformly throughout the peptide, although there is some evidence for fraying at the C terminus. Detailed analysis of the NMR spectra indicate that the helix is stabilized, in part, by a native N-capping interaction involving Thr40. A mutant peptide which lacks Thr40 is only 32 % helical. pH and ionic strength-dependent studies suggested that charge charge interactions make only a modest net contribution to the stability of the peptide. The protein contains a trans proline peptide bond located at the first position of the C-terminal helix. NMR analysis of the helical peptide and of a smaller peptide containing the proline residue indicates that only a small amount of cis proline isomer (8 %) is likely to be populated in the unfolded state.

A peptide model for proline isomerism in the unfolded state of staphylococcal nuclease.

Nuclear magnetic resonance spectroscopy has been used to investigate a synthetic peptide (YVYKPNNTHE) corresponding to residues 113 to 122 of staphylococcal nuclease. In the major folded state of the protein this region forms a type VIa beta-turn containing a cis Lys116-Pro117 peptide bond. There is, however, no evidence for any significant population of such a turn in the peptide in aqueous solution and the X-Pro bond is predominantly in the trans configuration. The peptide exhibits several well-resolved minor resonances due to the presence of a small fraction (4 +/- 2%) of the cis-proline isomer. The ratio of cis to trans isomer populations was found to be independent of temperature between 5 degrees C and 70 degrees C, indicating that delta H for the isomerism is close to zero. Using magnetization transfer techniques the rate of trans to cis interconversion was found to be 0.025(+/- 0.013) s-1 at 50 degrees C. The thermodynamics and kinetics of isomerism in the peptide are very similar to those estimated for the Lys116-Pro117 peptide bond in unfolded nuclease, suggesting that the cis-trans equilibrium in the unfolded protein is largely determined by the residues adjacent to Pro117 in the sequence. These results are consistent with previous suggestions that the cis-proline bond is stabilized late in the folding process and that the predominance of the cis form in folded nuclease is due to stabilizing interactions within the protein that give rise to a favorable enthalpy term.

Defining the core structure of the alpha-lactalbumin molten globule state.

Molten globules are partially folded states of proteins which are generally believed to mimic structures formed during the folding process. In order to determine the minimal requirements for the formation of a molten globule state, we have prepared a set of peptide models of the molten globule state of human alpha-lactalbumin (alphaLA). A peptide consisting of residues 1-38 crosslinked, via the native 28-111 disulfide bond, to a peptide corresponding to residues 95-120 forms a partially folded state at pH 2.8 which has all of the characteristics of the molten globule state of alphaLA as judged by near and far UV CD, fluorescence, ANS binding and urea denaturation experiments. The structure of the peptide construct is the same at pH 7.0. Deletion of residues 95-100 from the construct has little effect. Thus, less than half the sequence is required to form a molten globule. Further truncation corresponding to the selective deletion of the A (residues 1-19) or D (residues 101-110) helices or the C-terminal 310 helix (residues 112-120) leads to a significant loss of structure. The loss of structure which results from the deletion of any of these three regions is much greater than that which would be expected based upon the non-cooperative loss of local helical structure. Deletion of residues corresponding to the region of the D helix or C-terminal 310 helix region results in a peptide construct which is largely unfolded and contains no more helical structure than is expected from the sum of the helicity of the two reduced peptides. These experiments have defined the minimum core structure of the alphaLA molten globule state.

Rescuing a destabilized protein fold through backbone cyclization.

We describe the physicochemical characterization of various circular and linear forms of the approximately 60 residue N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein. Structural, dynamic, thermodynamic, kinetic and biochemical studies reveal that backbone circularization does not prevent the adoption of the natural folded structure in any of the circular proteins. Both the folding and unfolding rate of the protein increased slightly upon circularization. Circularization did not lead to a significant thermodynamic stabilization of the full-length protein, suggesting that destabilizing enthalpic effects (e.g. strain) negate the expected favorable entropic contribution to overall stability. In contrast, we find circularization results in a dramatic stabilization of a truncated version of the SH3 domain lacking a key glutamate residue. The ability to rescue the destabilized mutant indicates that circularization may be a useful tool in protein engineering programs geared towards generating minimized proteins.

Cooperative folding of a protein mini domain: the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex.

The peripheral subunit-binding domain from the dihydrolipoamide acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus is stably folded, despite its short sequence of only 43 amino acid residues. A 41 residue peptide derived from this domain, psbd41, undergoes a cooperative thermal unfolding transition with a tm of 54 degrees C. This three-helix protein is monomeric as judged by ultracentrifugation and concentration-dependent CD measurements. Peptides corresponding to the individual helices are largely unstructured both alone and in combination, indicating that the unusual stability of this protein does not arise solely from unusually stable alpha-helices. Chemical denaturation by guanidine hydrochloride is also cooperative with a delta GH2O of 3.1 kcal mol-1 at pH 8.0 and 25 degrees C. The chemical denaturation is broad with an m-value of 760 cal mol-1 M-1. psbd41 contains a buried aspartate residue at position 34 that may provide stability and specificity to the fold. A mutant peptide, psbd41Asn was synthesized in which the buried aspartate residue was mutated to asparagine. This peptide still folds cooperatively and it is monomeric, but is much less thermostable than the wild-type with a tm of only 31 degrees C. Chemical denaturations at 4 degrees C give an m-value of 740 cal mol-1 M-1, similar to the wild-type, but the stability delta GH2O is only 1.4 kcal mol-1. Both the wild-type and the mutant unfold at extremes of pH, but at 4 degrees C psbd41Asn is folded over a narrower pH range than the wild-type. Although the mutant unfolds cooperatively by thermal and by chemical denaturation, its NMR spectrum is significantly broader than that of the wild-type and it binds ANS. These results show that Asp34 is vital for the stability and specificity of this structure, the second smallest natural sequence known to fold in the absence of disulfide bonds or metal or ligand-binding sites.

Effects of varying the local propensity to form secondary structure on the stability and folding kinetics of a rapid folding mixed alpha/beta protein: characterization of a truncation mutant of the N-terminal domain of the ribosomal protein L9.

The N-terminal domain of the ribosomal protein L9 forms a split betaalphabeta structure with a long C-terminal helix. The folding transitions of a 56 residue version of this protein have previously been characterized, here we report the results of a study of a truncation mutant corresponding to residues 1-51. The 51 residue protein adopts the same fold as the 56 residue protein as judged by CD and two-dimensional NMR, but it is less stable as judged by chemical and thermal denaturation experiments. Studies with synthetic peptides demonstrate that the C-terminal helix of the 51 residue version has very little propensity to fold in isolation in contrast to the C-terminal helix of the 56 residue variant. The folding rates of the two proteins, as measured by stopped-flow fluorescence, are essentially identical, indicating that formation of local structure in the C-terminal helix is not involved in the rate-limiting step of folding.

An exceptionally stable helix from the ribosomal protein L9: implications for protein folding and stability.

The ribosomal protein L9 has an unusual structure comprising two compact globular domains connected by a 34 residue alpha-helix. The middle 17 residues of the helix are exposed to solvent while the first seven pack against and form part of the N-terminal domain, and the last ten form part of the C-terminal domain. Here we report results which show that a peptide corresponding to the central helix of L9 is monomeric in aqueous solution and >85% helical at 1 degrees C and 68(+/-7)% helical at 25 degrees C. This is considerably more helical than any other protein fragment studied to date. Another peptide corresponding to the middle 17 residues of the helix is monomeric and is 41(+/-4)% helical at 1 degrees C. Because the central helix has high intrinsic stability the globular N and C-terminal domains will likely be stabilized by their interactions with the helix. Therefore, the stability of the two terminal domains should not be completely independent because both domains gain stability from a shared structural element, the central helix. Also, the ability of the central helix to form native-like structure in isolation highlights a potential role for the helix in the early stages of the folding process.

Global analysis of the thermal and chemical denaturation of the N-terminal domain of the ribosomal protein L9 in H2O and D2O. Determination of the thermodynamic parameters, deltaH(o), deltaS(o), and deltaC(o)p and evaluation of solvent isotope effects.

The stability of the N-terminal domain of the ribosomal protein L9, NTL9, from Bacillus stearothermophilus has been monitored by circular dichroism at various temperatures and chemical denaturant concentrations in H2O and D2O. The basic thermodynamic parameters for the unfolding reaction, deltaH(o), deltaS(o), and deltaC(o)p, were determined by global analysis of temperature and denaturant effects on stability. The data were well fit by a model that assumes stability varies linearly with denaturant concentration and that uses the Gibbs-Helmholtz equation to model changes in stability with temperature. The results obtained from the global analysis are consistent with information obtained from individual thermal and chemical denaturations. NTL9 has a maximum stability of 3.78 +/- 0.25 kcal mol(-1) at 14 degrees C. DeltaH(o)(25 degrees C) for protein unfolding equals 9.9 +/- 0.7 kcal mol(-1) and TdeltaS(o)++(25 degrees C) equals 6.2 +/- 0.6 kcal mol(-1). DeltaC(o)p equals 0.53 +/- 0.06 kcal mol(-1) deg(-1). There is a small increase in stability when D2O is substituted for H2O. Based on the results from global analysis, NTL9 is 1.06 +/- 0.60 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 5.8 +/- 3.6 degrees C in D2O. Based on the results from individual denaturation experiments, NTL9 is 0.68 +/- 0.68 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 3.5 +/- 2.1 degrees C in D2O. Within experimental error there are no changes in deltaH(o) (25 degrees C) when D2O is substituted for H2O.

Amide proton exchange measurements as a probe of the stability and dynamics of the N-terminal domain of the ribosomal protein L9: comparison with the intact protein.

Amide H/D exchange rates have been measured for the N-terminal domain of the ribosomal protein L9, residues 1-56. The rates were measured at pD 3.91, 5.03, and 5.37. At pD 5.37, 18 amides exchange slowly enough to give reliable rate measurements. At pD 3.91, seven additional residues could be followed. The exchange is shown to occur by the EX2 mechanism for all conditions studied. The rates for the N-terminal domain are very similar to those previously measured for the corresponding region in the full-length protein (Lillemoen J et al., 1997, J Mol Biol 268:482-493). In particular, the rates for the residues that we have shown to exchange via global unfolding in the N-terminal domain agree within the experimental error with the rates measured by Hoffman and coworkers, suggesting that the structure of the domain is preserved in isolation and that the stability of the isolated domain is comparable to the stability of this domain in intact L9.

The failure of simple empirical relationships to predict the viscosity of mixed aqueous solutions of guanidine hydrochloride and glucose has important implications for the study of protein folding.

Viscosities of aqueous solutions of guanidine hydrochloride (GuHCl) were measured in the presence of varying amounts of glucose. At high concentrations of glucose or GuHCl, the measured viscosities showed significant deviation from the values computed using a method proposed by Tanford (1966, J Biol Chem 241:3228-3232). This method was originally derived to allow the calculation of the effects of buffer or low concentrations of salts and other additives on the density and viscosity of aqueous solutions of urea or GuHCl. Recently it has been used to estimate the viscosity of denaturant solutions that contain high concentrations of viscogens. Our results show that the extrapolation of this approach to solutions of highly concentrated viscous co-solutes leads to significant errors. The implications for experimental studies of the viscosity dependence of conformational transitions in proteins is discussed.