Protein Engineering Design and Selection - recent issues

Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs

2008-04-14

We have used phage display to isolate a range of human domain antibodies (dAbs) that bind to mouse, rat and/or human serum albumin (SA) and can be expressed at very high levels in bacterial, yeast or mammalian cell culture. In contrast to non-SA-binding dAbs, which have terminal half-lives of less than 45 min, the half-lives of these 12 kDa ‘AlbudAbs’ can match the half-life of SA itself. To demonstrate the use of AlbudAbs for extending the half-lives of therapeutic drugs, we created a fusion of the interleukin-1 receptor antagonist (IL-1ra) with an AlbudAb. Soluble IL-1ra is potent inhibitor of IL-1 signalling that is approved for the treatment of rheumatoid arthritis but has a relatively short in vivo half-life. Here we show that although the AlbudAb/IL-1ra fusion has a similar in vitro potency, its in vivo efficacy can be dramatically improved due to its extended serum half-life. AlbudAbs could potentially be used to generate a range of long half-life versions of many different drugs in order to improve their dosing regimen and/or clinical effect.

Genetic selection for peptide inhibitors of angiogenin

Smith, B. D., Raines, R. T. — 2008-04-14

The improper regulation of angiogenesis is implicit in a variety of diseases, including cancer. Angiogenin is unique among angiogenic factors in having ribonucleolytic activity. Inhibitors of this activity could serve as chemotherapeutics. The ribonucleolytic activity of angiogenin is toxic to the Origami^TM strain of Escherichia coli. Herein, this cytotoxicity was used to identify inhibitors from a random nonapeptide library tethered to the C-terminus of human angiogenin. The selected sequences fell into three classes: (i) extremely hydrophobic, (ii) putative protease (ClpXP) substrates and (iii) slightly anionic. Two peptides corresponding to sequences in the last class were synthesized chemically and found to inhibit the ribonucleolytic activity of human angiogenin in vitro with micromolar values of K_i. Both peptides also inhibit bovine pancreatic ribonuclease, a homolog of angiogenin, though one exhibits selectivity for angiogenin. The affinity and selectivity of these peptides are comparable with the best known inhibitors of angiogenin. Moreover, the strategy used to identify them is general and could be applied to other cytotoxins.

An improved prediction of catalytic residues in enzyme structures

Tang, Y.-R., Sheng, Z.-Y., Chen, Y.-Z., Zhang, Z. — 2008-04-14

The protein databases contain a huge number of function unknown proteins, including many proteins with newly determined 3D structures resulted from the Structural Genomics Projects. To accelerate experiment-based assignment of function, de novo prediction of protein functional sites, like active sites in enzymes, becomes increasingly important. Here, we attempted to improve the prediction of catalytic residues in enzyme structures by seeking and refining different encodings (i.e. residue properties) as well as employing new machine learning algorithms. In particular, considering that catalytic residues can often reveal specific network centrality when representing enzyme structure as a residue contact network, the corresponding measurement (i.e. closeness centrality) was used as one of the most important encodings in our new predictor. Meanwhile, a genetic algorithm integrated neural network (GANN) was also employed. Thanks to the above strategies, our GANN predictor demonstrated a high accuracy of 91.2% in the prediction of catalytic residues based on balanced datasets (i.e. the 1:1 ratio of catalytic to non-catalytic residues). When the GANN method was optimally applied to real enzyme structures, 73.9% of the tested structures had the active site correctly located. Compared with two existing methods, the proposed GANN method also demonstrated a better performance.

Thermostability promotes the cooperative function of split adenylate kinases

Nguyen, P. Q., Liu, S., Thompson, J. C., Silberg, J. J. — 2008-04-14

Proteins can often be cleaved to create inactive polypeptides that associate into functional complexes through non-covalent interactions, but little is known about what influences the cooperative function of the ensuing protein fragments. Here, we examine whether protein thermostability affects protein fragment complementation by characterizing the function of split adenylate kinases from the mesophile Bacillus subtilis (AK_Bs) and the hyperthermophile Thermotoga neapolitana (AK_Tn). Complementation studies revealed that the split AK_Tn supported the growth of Escherichia coli with a temperature-sensitive AK, but not the fragmented AK_Bs. However, weak complementation occurred when the AK_Bs fragments were fused to polypeptides that strongly associate, and this was enhanced by a Q16L mutation that thermostabilizes the full-length protein. To examine how the split AK homologs differ in structure and function, their catalytic activity, zinc content, and circular dichroism spectra were characterized. The reconstituted AK_Tn had higher levels of zinc, greater secondary structure, and >10³-fold more activity than the AK_Bs pair, albeit 17-fold less active than full-length AK_Tn. These findings provide evidence that the design of protein fragments that cooperatively function can be improved by choosing proteins with the greatest thermostability for bisection, and they suggest that this arises because hyperthermophilic protein fragments exhibit greater residual structure compared to their mesophilic counterparts.

Amino acid alphabet size in protein evolution experiments: better to search a small library thoroughly or a large library sparsely?

Munoz, E., Deem, M. W. — 2008-04-14

We compare the results obtained from searching a smaller library thoroughly versus searching a more diverse, larger library sparsely. We study protein evolution with reduced amino acid alphabets, by simulating directed evolution experiments at three different alphabet sizes: 20, 5 and 2. We employ a physical model for evolution, the generalized NK model, that has proved successful in modeling protein evolution, antibody evolution and T-cell selection. We find that antibodies with higher affinity are found by searching a library with a larger alphabet sparsely than by searching a smaller library thoroughly, even with well-designed reduced libraries. We also find ranked amino acid usage frequencies in agreement with observations of the CDR-H3 variable region of human antibodies.

Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase

2008-04-14

Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermostable and six thermolabile variants of the TAB5 AP. All of the isolated variants were characterised by their residual activity after heat treatment, Michaelis–Menten kinetics, activation energy and microcalorimetric parameters of unfolding. In addition, they were modelled into the structure of the TAB5 AP. Mutations which affected the cold-adapted properties of the enzyme were all located close to the active site. The destabilised variants H135E and H135E/G149D had 2- and 3-fold higher k_cat, respectively, than the wild-type enzyme. Wild-type AP has a complex heat-induced unfolding pattern while the mutated enzymes loose local unfolding transitions and have large shifts of the T_m values. Comparison of the wild-type and mutated TAB5 APs demonstrates that there is a delicate balance between the enzyme activity and stability and that it is possible to improve the activity and thermostability simultaneously as demonstrated in the case of the H135E/G149D variant compared to H135E.

Diverging catalytic capacities and selectivity profiles with haloalkane substrates of chimeric alpha class glutathione transferases

Kurtovic, S., Shokeer, A., Mannervik, B. — 2008-04-14

Six homologous Alpha class glutathione transferases of human, bovine, and rat origins were hybridized by means of DNA shuffling. The chimeric mutants were compared with the parental enzymes in their activities with several alkyl iodides. In order to facilitate a multivariate analysis of relationships between substrates and enzyme activities, three descriptors were introduced: ‘specific catalytic capacity’, ‘substrate selectivity’, and ‘unit-scaled substrate selectivity’. In some cases the purified mutants showed higher specific activity with a certain alkyl iodide than any of the parental enzymes. However, the overriding effect of DNA shuffling was the generation of chimeras with altered substrate selectivity profiles and catalytic capacities. The altered substrate selectivity profiles of some mutants could be rationalized by changes of the substrate-binding residues in the active site of the enzyme. However, in four of the isolated mutants all active-site residues were found identical with those of rat GST A2-2, even though their substrate specificity profiles were significantly different. Clearly, amino acid residues distant from first-sphere interactions with the substrate influence the catalytic activity. These results are relevant both to the understanding how functional properties may develop in natural enzyme evolution and in the tailoring of novel functions in protein engineering.

In vitro DNA recombination by L-Shuffling during ribosome display affinity maturation of an anti-Fas antibody increases the population of improved variants

Chodorge, M., Fourage, L., Ravot, G., Jermutus, L., Minter, R. — 2008-04-14

The use of random mutagenesis in concert with protein display technologies to rapidly select high affinity antibody variants is an established methodology. In some cases, DNA recombination has been included in the strategy to enable selection of mutations which act cooperatively to improve antibody function. In this study, the impact of L-Shuffling DNA recombination on the eventual outcome of an in vitro affinity maturation has been experimentally determined. Parallel evolution strategies, with and without a recombination step, were carried out and both methods improved the affinity of an anti-Fas single chain variable fragment (scFv). The recombination step resulted in an increased population of affinity-improved variants. Moreover, the most improved variant, with a 22-fold affinity gain, emerged only from the recombination-based approach. An analysis of mutations preferentially selected in the recombined population demonstrated strong cooperative effects when tested in combination with other mutations but small, or even negative, effects on affinity when tested in isolation. These results underline the ability of combinatorial library approaches to explore very large regions of sequence space to find optimal solutions in antibody evolution studies.

Dissimilarity in the oxidative folding of onconase and ribonuclease A, two structural homologues

Gahl, R. F., Narayan, M., Xu, G., Scheraga, H. A. — 2008-03-20

The oxidative folding of frog onconase (ONC), a member of the ribonuclease A family, was examined and shows markedly different behavior compared to its structural homologue bovine pancreatic ribonuclease A (RNase A) under similar conditions. Application of a reduction pulse (using a small amount of reduced dithiothreitol) during the oxidative regeneration of ONC indicated the survival of the native protein along with three other (structured) species, I₁, I₂ and I₃, with the rest of the unstructured species being converted to fully reduced protein. Mass spectrometry indicates that I₁ has two disulfide bonds, whereas I₂ and I₃ have three disulfide bonds each. A disulfide mapping method, based on cyanylation, was used to identify I₂ and I₃ as des-[30–75] and des-[19–68], respectively. On enzymatic digestion using trypsin, I₁ was identified as des-[19–68, 30–75]. Differences in the intermediates that are generated during the oxidative folding of the two structural homologues, RNase A and ONC, demonstrate that regenerative pathways are not necessarily influenced by tertiary structure. This indicates that the lack of a disulfide bond in ONC, analogous to the (65–72) disulfide bond in RNase A which plays an important role in its oxidative regeneration, does not adversely affect the oxidative folding of ONC.

Development of fructosyl amine oxidase specific to fructosyl valine by site-directed mutagenesis

Miura, S., Ferri, S., Tsugawa, W., Kim, S., Sode, K. — 2008-03-20

Docking models of fructosyl amine oxidase (FAOD) from the marine yeast Pichia N1-1 (N1-1 FAOD) with the substrates fructosyl valine (f-Val) and fructosyl-N-lysine (f-Lys) were produced using three-dimensional protein model as reported previously (Miura et al., 2006, Biotechnol. Lett., 28, 1895-1900). The residues involved in recognition of substrates were proposed, particularly Asn354, which interacts closely with f-Lys, but not with f-Val. Substitution of Asn354 to histidine and lysine simultaneously resulted in an increase in activity of f-val and a decrease in activity of f-Lys and thus, increasing the specificity for f-Val from 13- to 19-fold. In addition to creating two mutant FAODs with great potential for the measurement of glycated hemoglobin, we have provided the first structural model of substrate binding with eukaryotic FAOD, which is expected to contribute to further investigation of FAOD.

Inhibition of cytotoxicity and amyloid fibril formation by a D-amino acid peptide that specifically binds to Alzheimer's disease amyloid peptide

2008-03-20

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. The ‘amyloid cascade hypothesis’ assigns the amyloid-beta-peptide (Aβ) a central role in the pathogenesis of AD. Although it is not yet established, whether the resulting Aβ aggregates are the causative agent or just a result of the disease progression, polymerization of Aβ has been identified as a major feature during AD pathogenesis. Inhibition of the Aβ polymer formation, thus, has emerged as a potential therapeutic approach. In this context, we identified peptides consisting of d-enantiomeric amino acid peptides (d-peptides) that bind to Aβ. d-peptides are known to be more protease resistant and less immunogenic than the respective l-enantiomers. Previously, we have shown that a 12mer d-peptide specifically binds to Aβ amyloid plaques in brain tissue sections from former AD patients. In vitro obtained binding affinities to synthetic Aβ revealed a K_d value in the submicromolar range. The aim of the present study was to investigate the influence of this d-peptide to Aβ polymerization and toxicity. Using cell toxicity assays, thioflavin fluorescence, fluorescence correlation spectroscopy and electron microscopy, we found a significant effect of the d-peptide on both. Presence of d-peptides (dpep) reduces the average size of Aβ aggregates, but increases their number. In addition, Aβ cytotoxicity on PC12 cells is reduced in the presence of dpep.

A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry

Kronqvist, N., Lofblom, J., Jonsson, A., Wernerus, H., Stahl, S. — 2008-03-20

Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 10⁹ variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host (~10⁶ variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.

Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties

2008-03-20

A monomeric variant of triosephosphate isomerase (TIM) with a new engineered binding groove has been characterized further. In this variant (ml8bTIM), the phosphate binding loop had been shortened, causing the binding site to be much more extended. Here, it is reported that in the V233A variant of ml8bTIM (A-TIM), three important properties of the wild-type TIM active site have been restored: (i) the structural properties of loop-7, (ii) the binding site of a conserved water molecule between loop-7 and loop-8 and (iii) the binding site of the phosphate moiety. It is shown that the active site of A-TIM can bind TIM transition state analogs and suicide inhibitors competently. It is found that the active site geometry of the A-TIM complexes is less compact and more solvent exposed, as in wild-type TIM. This correlates with the observation that the catalytic efficiency of A-TIM for interconverting the TIM substrates is too low to be detected. It is also shown that the A-TIM active site can bind compounds which do not bind to wild-type TIM and which are completely different from the normal TIM substrate, like a citrate molecule. The binding of this citrate molecule is stabilized by hydrogen bonding interactions with the new binding groove.

A novel random mutagenesis approach using human mutagenic DNA polymerases to generate enzyme variant libraries

2008-03-20

The in vitro MutaGen^TM procedure is a new random mutagenesis method based on the use of low-fidelity DNA polymerases. In the present study, this technique was applied on a 2 kb gene encoding amylosucrase, an attractive enzyme for the industrial synthesis of amylose-like polymers. Mutations were first introduced during a single replicating step performed by mutagenic polymerases pol β and pol . Three large libraries (>10⁵ independent clones) were generated (one with pol β and two with pol ). The sequence analysis of randomly chosen clones confirmed the potential of this strategy for the generation of diversity. Variants generated by pol β were 4–7-fold less mutated than those created with pol , indicating that our approach enables mutation rate control following the DNA polymerase employed for mutagenesis. Moreover, pol β and pol provide different and complementary mutation spectra, allowing a wider sequence space exploration than error-prone PCR protocols employing Taq polymerase. Interestingly, some of the variants generated by pol displayed unusual modifications, including combinations of base substitutions and codon deletions which are rarely generated using other methods. By taking advantage of the mutation bias of naturally highly error-prone DNA polymerases, MutaGen^TM thus appears as a very useful tool for gene and protein randomisation.

Revisiting the correlation between proteins' thermoresistance and organisms' thermophilicity

Dehouck, Y., Folch, B., Rooman, M. — 2008-03-20

The possibility to rationally design protein mutants that remain structured and active at high temperatures strongly depends on a better understanding of the mechanisms of protein thermostability. Studies devoted to this issue often rely on the living temperature (T_env) of the host organism rather than on the melting temperature (T_m) of the analyzed protein. To investigate the scale of this approximation, we probed the relationship between T_m and T_env on a dataset of 127 proteins, and found a much weaker correlation than previously expected: the correlation coefficient is equal to 0.59 and the regression line is T_m 42.9°C + 0.62T_env. To illustrate the effect of using T_env rather than T_m to analyze protein thermoresistance, we derive statistical distance potentials, describing Glu–Arg and Asp–Arg salt bridges, from protein structure sets with high or low T_m or T_env. The results show that the more favorable nature of salt bridges, relative to other interactions, at high temperatures is more clear-cut when defining thermoresistance in terms of T_m. The T_env-based sets nevertheless remain informative.

CytoPred: a server for prediction and classification of cytokines

Lata, S., Raghava, G.P.S. — 2008-03-20

Cytokines are messengers of immune system. They are small secreted proteins that mediate and regulate the immune system, inflammation and hematopoiesis. Recent studies have revealed important roles played by the cytokines in adjuvants as therapeutic targets and in cancer therapy. In this paper, an attempt has been made to predict this important class of proteins and classify further them into families and subfamilies. A PSI-BLAST+Support Vector Machine-based hybrid approach is adopted to develop the prediction methods. CytoPred is capable of predicting cytokines with an accuracy of 98.29%. The overall accuracy of classification of cytokines into four families and further classification into seven subfamilies is 99.77 and 97.24%, respectively. It has been shown by comparison that CytoPred performs better than the already existing CTKPred. A user-friendly server CytoPred has been developed and available at http://www.imtech.res.in/raghava/cytopred.

Message from the Editors

Daggett, V., Fersht, A. R. — 2008-02-25

Fluorescence resonance energy transfer analysis of the folding pathway of Engrailed Homeodomain

Huang, F., Settanni, G., Fersht, A. R. — 2008-02-25

The Engrailed Homeodomain folds on the microsecond time scale via an intermediate that is experimentally well characterised using structural Engrailed–Homeodomain mimics. Here, we analysed directly the changes in distance between key residues during the kinetics of unfolding and at equilibrium using fluorescence resonance energy transfer (FRET). Trp was the donor and 5-(((acetylamino)ethyl)amino) naphthalene-1-sulphate, the acceptor, substituted in positions that caused little change in stability. Distances calculated for the native state were in good agreement with those derived from the NMR structure. The distances between the N- and C-termini of Helix I and of Helix III increased, then decreased and finally increased again with increasing GdmCl concentration on equilibrium denaturation. This behaviour implied that there was a folding intermediate on the folding pathway and that this intermediate was populated at low concentrations of GdmCl concentration (~1 M). We analysed the changes in distance during temperature-jump relaxation kinetics, using a qualitative and very conservative procedure that drew conclusions only when changes in fluorescence of mutants containing either the donor or the acceptor alone would not obscure the change in the FRET signal when both donor and acceptor were present. The distance changes obtained under equilibrium and kinetic measurements were self-consistent and also consistent with the known high-resolution structures of the mimics of the folding intermediates. We showed that for analysing distances in disordered ensembles, it is important to use FRET probes with a critical distance close to the average separation in the ensemble. Otherwise, average distances could be over or underestimated.

Amide inequivalence in the fibrillar assembly of islet amyloid polypeptide

Koo, B. W., Hebda, J. A., Miranker, A. D. — 2008-02-25

Amyloid fibers are aggregated, yet highly ordered, β-sheet-rich assemblies of misfolded proteins. Order is established in such systems following profiles indicative of nucleation-dependent assembly. Nucleation dependence suggests that specific interactions, such as long-range contacts and/or strand registration, are critical to establishing initial fiber structure. Here, we show that amino acids at selected positions participate in key interactions that modulate the pathway of amyloid fiber formation by the hormone, islet amyloid polypeptide (IAPP). Specifically, we investigated the role of amide side-chain interactions in the process of IAPP assembly. We mutated five of the asparagine side chains in IAPP and assessed their effects on the kinetics of assembly. We find that the asparagine amide side chains strongly dictate the ability of IAPP to form fibers. In particular, the elimination of two specific asparagines results in near and total loss of amyloid, respectively. Interestingly, the two asparagines are located in a recently identified domain with -helical bias. These sensitivities are unusual for IAPP, as IAPP is generally tolerant to mutation. Here, we demonstrate this mutational tolerance by assessing 10 alterations at five distinct sites. In all cases, the constructs form fibers on timescales perturbed by less than a factor of two compared with wild-type protein. These findings indicate the presence of key specific interactions that are the determinants of IAPP amyloid formation.

The folding pathway of an engineered circularly permuted PDZ domain

Ivarsson, Y., Travaglini-Allocatelli, C., Morea, V., Brunori, M., Gianni, S. — 2008-02-25

To understand the role of sequence connectivity in the folding pathway of a multi-state protein, we have analysed the folding kinetics of an engineered circularly permuted PDZ domain. This variant has been designed with the specific aim of posing two of the strands participating in the stabilisation of an early folding nucleus as contiguous elements in the primary structure. Folding of the circularly permuted PDZ2 has been explored by a variety of different experimental approaches including stopped-flow and continuous-flow kinetics, as well as ligand-induced folding experiments. Data reveal that although circular permutation introduces a significant destabilisation of the native state, a folding intermediate is stabilised and accumulated prior folding. Furthermore, quantitative analysis of the observed kinetics indicates an acceleration of the early folding events by more than two orders of magnitude. The results support the importance of sequence connectivity both in the mechanism and the speed of protein folding.

A strategy for generating polyglutamine 'length libraries' in model host proteins

Tobelmann, M. D., Kerby, R. L., Murphy, R. M. — 2008-02-25

Huntington’s disease is one of nine known neurodegenerative diseases in which a disease-specific protein contains an unusually long polyglutamine (polyQ) stretch. The proteins associated with each disease are unrelated in sequence, size, structure, function or location of the mutation. In all cases, there is an apparent critical number of glutamines below which individuals do not develop disease. Expansion of the polyQ domain is closely associated with misfolding and aggregation of the protein. It is not yet well understood how the length of the polyQ tract, and its location within a given protein, is related to misfolding and to disease. In this work we developed a strategy for generating length libraries of polyQ-containing proteins, with the polyQ inserted at an arbitrary location. This strategy facilitates systematic, detailed study of the relationship among polyQ length, context and misfolding.

Characterization of folding the four-helix bundle protein Rop by real-time NMR

van Nuland, N. A.J., Dobson, C. M., Regan, L. — 2008-02-25

Rop is a four-helix bundle protein composed of two identical helix-loop-helix monomers. Protein folding monitored by stopped-flow fluorescence or CD exhibits biphasic kinetics when folding to low final denaturant concentrations. As the final concentration of denaturant is increased, the amplitude of the fast phase decreases, until at the highest concentrations the kinetics appear monophasic. We propose that the fast phase represents the formation of an intermediate. Here, we use real-time NMR to detect the formation of this intermediate and to characterize its structural features.

The Trp-cage: optimizing the stability of a globular miniprotein

2008-02-25

The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42°C) can be significantly stabilized by mutations; melting points as high as 64°C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (±0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (S_U = 11 ± 2 J/mol K) was measured at the solvent-exposed P17 site. Pro–Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3–3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4–6 kJ/mol) and a buried H-bonded Ser side chain (10 kJ/mol).

Different disease-causing mutations in transthyretin trigger the same conformational conversion

Steward, R. E., Armen, R. S., Daggett, V. — 2008-02-25

Transthyretin (TTR)-containing amyloid fibrils are deposited in cardiac tissue as a natural consequence of aging. A large number of inherited mutations lead to amyloid diseases by accelerating TTR deposition in other organs. Amyloid formation is preceded by a disruption of the quaternary structure of TTR and conformational changes in the monomer. To study conformational changes preceding the formation of amyloid, we performed molecular dynamics simulations of the wild-type monomer, amyloidogenic variants (V30M, L55P, V122I) and a protective variant (T119M) at neutral and low pH. At low pH, the D strand dissociated from the β-sheet to expose the A strand, consistent with experimental studies. In amyloidogenic variants and in the wild-type at low pH, there was a conformational change in the β-sheets into -sheet via peptide bond flips that was not observed at neutral pH in the wild-type monomer. The same residues participated in conversion in each amyloidogenic variant simulation, originating in the G strand between residues 106 and 109, with accelerated conversion at low pH. The T119M protective variant changed the local conformation of the H strand and suppressed the conversion observed in amyloidogenic variants.

Conservation of mechanism, variation of rate: folding kinetics of three homologous four-helix bundle proteins

Dalal, S., Canet, D., Kaiser, S. E., Dobson, C. M., Regan, L. — 2008-02-25

The amino acid sequence of a protein determines both its final folded structure and the folding mechanism by which this structure is attained. The differences in folding behaviour between homologous proteins provide direct insights into the factors that influence both thermodynamic and kinetic properties. Here, we present a comprehensive thermodynamic and kinetic analysis of three homologous homodimeric four-helix bundle proteins. Previous studies with one member of this family, Rop, revealed that both its folding and unfolding behaviour were interesting and unusual: Rop folds (k⁰_f = 29 s^–1) and unfolds (k⁰_u = 6 x 10^–7 s^–1) extremely slowly for a protein of its size that contains neither prolines nor disulphides in its folded structure. The homologues we discuss have significantly different stabilities and rates of folding and unfolding. However, the rate of protein folding directly correlates with stability for these homologous proteins: proteins with higher stability fold faster. Moreover, in spite of possessing differing thermodynamic and kinetic properties, the proteins all share a similar folding and unfolding mechanism. We discuss the properties of these naturally occurring Rop homologues in relation to previously characterized designed variants of Rop.

Demonstration by burst-phase analysis of a robust folding intermediate in the FF domain

Jemth, P., Johnson, C. M., Gianni, S., Fersht, A. R. — 2008-02-25

The role of intermediates in the folding reaction of single-domain proteins is a controversial issue. It was previously shown by different methods that an on-pathway intermediate is populated in the presence of sodium sulphate during the folding of the FF domain from HYPA/FBP11. Here we demonstrate using analysis of the amplitudes of kinetic traces that this burst-phase folding intermediate is present at different salt concentration and at various pH, and is also found in roughly 30 site-directed mutants. The intermediate appears robust to changing conditions and thus fulfils an important criterion for a productive molecular species on the folding reaction pathway.

Characterisation of transition state structures for protein folding using 'high', 'medium' and 'low' {Phi}-values

Geierhaas, C. D., Salvatella, X., Clarke, J., Vendruscolo, M. — 2008-02-25

It has been suggested that -values, which allow structural information about transition states (TSs) for protein folding to be obtained, are most reliably interpreted when divided into three classes (high, medium and low). High -values indicate almost completely folded regions in the TS, intermediate -values regions with a detectable amount of structure and low -values indicate mostly unstructured regions. To explore the extent to which this classification can be used to characterise in detail the structure of TSs for protein folding, we used -values divided into these classes as restraints in molecular dynamics simulations. This type of procedure is related to that used in NMR spectroscopy to define the structure of native proteins from the measurement of inter-proton distances derived from nuclear Overhauser effects. We illustrate this approach by determining the TS ensembles of five proteins and by showing that the results are similar to those obtained by using as restraints the actual numerical -values measured experimentally. Our results indicate that the simultaneous consideration of a set of low-resolution -values can provide sufficient information for characterising the architecture of a TS for folding of a protein.