The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of di

1. The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of dihydrofolate reductase from E. coli Rob Aldina Sarah Hauser Lindsay Vang

2. Background Purpose � to determine the role of domains in defining kinetic and thermodynamic properties of DHFR Method � chain connectivity of the DLD and ABD were altered by permutation Hypothesis � chain cleavage can selectively destabilize the domain in which the N- and C-termini are resident

3. Results Made permutations in one spot in ABD, one in DLD and one at boundary between domains Results showed that a continuous ABD is necessary for a stable thermal intermediate; a continuous DLD is required for a stable urea intermediate Permutation at boundary had a thermal and urea intermediate Observable kinetic folding responses of all 3 permuted proteins were similar to wild type Domains can bind specific ligands, catalyze common chemical reactions or serve to stabilize protein-protein interactions

4. Dihydrofolate Reductase (DHFR) Small, monomeric a/�/a sandwich protein 159 amino acids 18.0 kDa Catalyzes reaction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate as the reducing factor

5. Structure of DHFR DHFR catalyzes reduction of dihydrofolate by means of an NADP(H) cofactor Contains a discontinuous loop domain (DLD), residues 1-37 and 107-159 and an adenosine-binding domain (ABD), residues 38-106 ABD moves as unit relative to the DLD during catalytic cycle, demonstrating its integrity is important for function

6. Rossman Fold Rossman fold (dinucleotide binding domain) is highly conserved in proteins that bind NAD(H) and NADP(H) Consists of parallel sheet formed from 3 extended polypeptide strands First 2 connected by alpha-helix, second 2 connected by helix or less defined structure

7. Rossmann Fold continued Rossman fold of dihydrofolate reductases (1DLR, 8DFR, 1DRH, 3DFR) are different than that of normal folds Parallel and anti-parallel beta strands intercalated by alpha helices are present

8. Gegg Experiment � Fragment Isolation 8 fragments, spanning the sequence and ranging in size from 36-124 amino acids were constructed by chemical cleavage One method is to alter solution conditions to destabilize the native conformation without stabilizing the fully unfolded form Acidic pH or high temperatures are used to populate molten globule species that have substantial secondary structure but little or no tertiary structure Second method is to modify the protein (remove essential ligand or protein engineering)

9. Gegg Experiment, con�t Figure 2 and Figure 3 to show fragments 1-36, 108-159, and 37-107 don�t show properties of folded protein

10. Svensson Approach � Circular Permutation Natural termini are connected by a linker and new termini are introduced at desired positions within the protein Makes it possible to maintain structure while altering connectivity Looked at permutation of DHFR based on host survival in presence of trimethoprim, an inhibitor of DHFR, and saw that it was tolerated at half of 158 possible sites Analysis of spectroscopic features, ability to bind ligand, enzymatic activity, stability to thermal and urea denaturation and kinetics of refolding supports hypothesis that chain connectivity and domain stability are closely linked in DHFR

11. Reagents Urea treated mixed-bed resin before addition of buffers to remove free cyanate and ammonia. Concentration determined by refractive index measurements Standard buffer at pH 7.8: 10 mM potassium phosphate 0.2 mM K2EDTA 1 mM �-mercaptoethanol

12. Protein purification Permutants purified: Cloned protein into pUC19 vector at BamHI site methotrexate affinity chromatography Determined to be >95% pure Concentration of each protein was determined by absorbance at 280 nm Molar extinction coefficient: of 31,100 M-1cm-1 Mass spectrometry data confirmed that for all three permutants, the N-terminal formyl-methionine was not proteolytically cleaved after protein synthesis.

13. Enzyme assay Enzyme used: dihydrofolate reductase Start assay at 15�C with addition of 20 nM of enzyme Activity determined by monitoring the formation of NADPH, which was assumed to be equal to amount of dihydrofolate reductase originally present 340 nm 60 or 180 s 3�5 repetitions.

14. Equilibrium spectroscopy Far-UV CD and Near-UV CD native proteins in the absence and presence of methotrexate (MTX) maintained at 15�C by a thermoelectric temperature control system for far, and a circulating water bath for near. step size: 1 nm bandwidth: 2 nm averaging time: 4 s protein concentration: 5 �M for far, and 10 �M for near Fluorescence steady-state emission spectra Samples excited at 295 nm Emission monitored from 310 to 450 nm at 1 nm intervals. Protein concentrations: 3 �M for both the urea and thermal titrations. Experimental temperature for the urea unfolding experiments: 15�C. Done for both urea and temperature induced denaturation

15. Thermodynamic Analysis Urea and thermal unfolding dependence of far-UV CD and fluoresence emission spectroscopic signals fit to either two-state model: three-state model: four-state model: N* represents a native-like state with altered packing of one or more of the five tryptophans. Free energy of unfolding in the absence of denaturant was calculated

16. Stopped-flow fluoresence spectroscopy Refolding kinetics were measured using either: Stopped-flow fluorescence instrument Applied Photophysics instrument Excitation wavelength: 295 nm Emission monitored using a 320 nm low wavelength cut-off filter In refolding experiments: unfolded protein diluted 10-fold into buffer final unfolded protein concentrations: 1�3 �M protein and 0.50�0.76 M urea All kinetic studies performed at 15�C using standard buffer conditions

17. Selection of sites for permutation Three positions within DHFR chosen as sites for insertion of new N- and C- termini within the DLD (cpN18) at the boundary between the DLD and ABD (cpP39) within the ABD (cpD69) 10 Folding elements identified and avoided prevent the folding of the protein to a stable, active form when disrupted short, neighboring peptide segments distributed throughout an amino acid sequence Ribbon Diagram depicts the two domains: Red: discontinuous loop domain (DLD) Gray: adenosine- binding domain (ABD) Residues chosen as sites for insertion of new N- and C- termini depicted in gray ball and stick Five tryptophan residues shown in green ball and stick Schematic Illustrates altered connectivity due to permutants White triangle is five glycine linker between the original N- and C- termini Secondary structure labels above WT diagram Placement of five tryptophans indicated by green letters Three positions within DHFR chosen as sites for insertion of new N- and C- termini within the DLD (cpN18) at the boundary between the DLD and ABD (cpP39) within the ABD (cpD69) 10 Folding elements identified and avoided prevent the folding of the protein to a stable, active form when disrupted short, neighboring peptide segments distributed throughout an amino acid sequence Ribbon Diagram depicts the two domains: Red: discontinuous loop domain (DLD) Gray: adenosine- binding domain (ABD) Residues chosen as sites for insertion of new N- and C- termini depicted in gray ball and stick Five tryptophan residues shown in green ball and stick Schematic Illustrates altered connectivity due to permutants White triangle is five glycine linker between the original N- and C- termini Secondary structure labels above WT diagram Placement of five tryptophans indicated by green letters

18. Structural information from solution studies Far-UV CD shows global secondary structure of the permutated versions of DHFR all permuted proteins have the same general spectral shape as WT-DHFR indicative of mixed /� protein minimum at 218 nm, maximum at 195 nm small decreases in ellipticity relative to WT-DHFR suggest a loosening of the secondary structure. MTX bound to the three permuted proteins observed differences are largely reinforced the core structures of the permuted proteins are very similar to WT-DHFR Tryptophan fluoresence emission spectroscopy (figure not included) used to assess tertiary structure five intrinsic tryptophan residues used as probes all three permuted proteins are similar to WT DHFR wavelength of maximum emission: 350 � 1 nm 60�75% of the intensity of WT-DHFR change in the environment of one or more of the tryptophans must enhance their quenching. Near-UV CD spectroscopy (data not included) cpN18 and cpD69 retain characteristic structure of WT-DHFR Retain chiral environments for one or more of their aromatic side chains substantially reduced amplitudes of peaks for cpP39 introduction of the charged N- and C-termini between the DLD and the ABD disrupted the packing near one or more aromatic side chains responsible for these signals. Far-UV CD shows global secondary structure of the permutated versions of DHFR all permuted proteins have the same general spectral shape as WT-DHFR indicative of mixed /� protein minimum at 218 nm, maximum at 195 nm small decreases in ellipticity relative to WT-DHFR suggest a loosening of the secondary structure. MTX bound to the three permuted proteins observed differences are largely reinforced the core structures of the permuted proteins are very similar to WT-DHFR Tryptophan fluoresence emission spectroscopy (figure not included) used to assess tertiary structure five intrinsic tryptophan residues used as probes all three permuted proteins are similar to WT DHFR wavelength of maximum emission: 350 � 1 nm 60�75% of the intensity of WT-DHFR change in the environment of one or more of the tryptophans must enhance their quenching. Near-UV CD spectroscopy (data not included) cpN18 and cpD69 retain characteristic structure of WT-DHFR Retain chiral environments for one or more of their aromatic side chains substantially reduced amplitudes of peaks for cpP39 introduction of the charged N- and C-termini between the DLD and the ABD disrupted the packing near one or more aromatic side chains responsible for these signals.

19. Enzymatic activity steady-state enzyme activity determined for each permuted protein as a measure of folded, functional protein All three permutants had reduced specific activities relative to WT-DHFR the specific activity negligible for cpN18 70% of the wild-type activity for cpP39 and cpD69 low level of activity for cpN18 demonstrates that the TMP screen used to select for viable permutants is not a stringent assay for enzymatic activity not surprising because cpN18 is disrupted in a loop region known to be involved in the catalytic cycle of DHFR activity of cpN18 can be enhanced slightly to 2% of the wild-type activity by increased concentration of enzyme increased temperature pre-incubation with DHF possible to access a native conformation even when M20 loop is disrupted

20. Urea denaturation studies raw data converted to apparent fraction unfolded plots to allow direct visual comparison of the CD and fluoresence data sigmoidal loss of signal at moderate (2�4 M) urea concentrations small linear changes in the optical signal at low (0�2 M) and high (>4 M) urea concentrations cpP39 has additional change in fluoresence signal in the native baseline region (0�1 M urea) reorganization of the native-like packing of the tryptophans in this permutant Normalized CD and fluoresence unfolding transition curves cpN18, when coupled with the ideal sigmoidal behavior, supports a two-state folding mechanism WT-DHFR, with a discontinuous DLD similar to cpN18, also unfolds by a two-state mechanism in response to urea denaturation presence of a stable intermediate in the urea-induced unfolding of cpP39 and cpD69 is implied by the non-coincidence of their CD and fluoresence transition curves SVD analysis simultaneously incorporates data from the entire CD and FL spectra. only two vectors required to describe the unfolding data for cpN18 three vectors required for both cpP39 and cpD69, confirms presence of an intermediate for cpP39 and cpD69 raw data converted to apparent fraction unfolded plots to allow direct visual comparison of the CD and fluoresence data sigmoidal loss of signal at moderate (2�4 M) urea concentrations small linear changes in the optical signal at low (0�2 M) and high (>4 M) urea concentrations cpP39 has additional change in fluoresence signal in the native baseline region (0�1 M urea) reorganization of the native-like packing of the tryptophans in this permutant Normalized CD and fluoresence unfolding transition curves cpN18, when coupled with the ideal sigmoidal behavior, supports a two-state folding mechanism WT-DHFR, with a discontinuous DLD similar to cpN18, also unfolds by a two-state mechanism in response to urea denaturation presence of a stable intermediate in the urea-induced unfolding of cpP39 and cpD69 is implied by the non-coincidence of their CD and fluoresence transition curves SVD analysis simultaneously incorporates data from the entire CD and FL spectra. only two vectors required to describe the unfolding data for cpN18 three vectors required for both cpP39 and cpD69, confirms presence of an intermediate for cpP39 and cpD69

21. Urea denaturation studies, cont. thermodynamic stability for cpN18, 6.1 kcal/mol, comparable to WT consistent with the disruption of the DLD in these three proteins difference in free energy between the native and unfolded forms for cpD69, 6.9 kcal/mol, comparable to WT Not expected because disruption of the ABD yields three-state unfolding transition. cpP39 is 2 kcal/mol more stable to unfolding than WT-DHFR suggests that the presence of both an intact ABD and DLD can enhance the stability of the protein. relative populations of the intermediate species, as a function of urea, calculated from the thermodynamic parameters obtained in the global fits cpP39: 50% population at 3 M urea cpD69: 60% population at 3 M urea continuous DLD found in cpP39 and cpD69 required for a stable intermediate in the urea denaturation reaction stable intermediate in cpD69 has 40% of the native CD ellipticity at 222 nm in absence of urea intermediate in cpP39 hampered by the presence of the NN* transition. thermodynamic stability for cpN18, 6.1 kcal/mol, comparable to WT consistent with the disruption of the DLD in these three proteins difference in free energy between the native and unfolded forms for cpD69, 6.9 kcal/mol, comparable to WT Not expected because disruption of the ABD yields three-state unfolding transition. cpP39 is 2 kcal/mol more stable to unfolding than WT-DHFR suggests that the presence of both an intact ABD and DLD can enhance the stability of the protein. relative populations of the intermediate species, as a function of urea, calculated from the thermodynamic parameters obtained in the global fits cpP39: 50% population at 3 M urea cpD69: 60% population at 3 M urea continuous DLD found in cpP39 and cpD69 required for a stable intermediate in the urea denaturation reaction stable intermediate in cpD69 has 40% of the native CD ellipticity at 222 nm in absence of urea intermediate in cpP39 hampered by the presence of the NN* transition.

22. Thermal denaturation studies Far-UV CD at 226 nm (closed shapes) 236 nm (open shapes) Temperature- Induced Unfolding transitions tryptophan fluoresence between 310 and 450 nm (data not shown) Thermally induced unfolding profiles highly reversible >85% WT and all three perutants Application of slow temperature gradient Normalized unfolding transition curves cpD69, when coupled with the ideal sigmoidal behavior, supports a two-state folding mechanism presence of a stable intermediate in the urea-induced unfolding of cpN18 and cpD39 is implied by the non-coincidence of their CD and fluoresence transition curves Temperature- Induced Unfolding transitions tryptophan fluoresence between 310 and 450 nm (data not shown) Thermally induced unfolding profiles highly reversible >85% WT and all three perutants Application of slow temperature gradient Normalized unfolding transition curves cpD69, when coupled with the ideal sigmoidal behavior, supports a two-state folding mechanism presence of a stable intermediate in the urea-induced unfolding of cpN18 and cpD39 is implied by the non-coincidence of their CD and fluoresence transition curves

23. Thermal denaturation studies, cont. thermally induced unfolding of cpD6 best described by two-state model cpN18 and cpP39 non-coincident transition curves comparable melting temperatures to WT-DHFR existence of a stable thermal intermediate for WT-DHFR, cpN18 and cpP39, but not for cpD69 suggests that continuous ABD required to stabilize partially folded form thermally induced unfolding of cpD6 best described by two-state model cpN18 and cpP39 non-coincident transition curves comparable melting temperatures to WT-DHFR existence of a stable thermal intermediate for WT-DHFR, cpN18 and cpP39, but not for cpD69 suggests that continuous ABD required to stabilize partially folded form

24. Kinetic folding mechanism sub-millisecond, stopped-flow burst phase characterized by simple exponential increase in fluorescence intensity non-monotonic change in intensity between the unfolded and native forms reflects formation of a hyperfluorescent intermediate that results from the burial of Trp74 in a native-like hydrophobic core in the ABD subsequent folding reaction well described by four phases of decreasing fluorescence intensity for WT products all capable of binding MTX four parallel folding channels leading from the hyperfluorescent intermediate to native or native-like species refolding of the permutants reactions under strongly refolding conditions best described by three exponential all three permutated proteins display characteristic hyperfluorescent phase followed by a decrease in the intensity as the folding reaction proceeds cpN18 and cpD69 require two exponentials to describe the hyperfluorescent phase parallel folding channels arise before the formation of the corresponding intermediates and the burial of Trp74 fewer number of phases with decreasing intensity observed for the permutants smaller number of folding channels. lack of hysteresis observed in enzyme activity assay and reduction in the number of folding channels is consistent with the loss of an inactive native conformersub-millisecond, stopped-flow burst phase characterized by simple exponential increase in fluorescence intensity non-monotonic change in intensity between the unfolded and native forms reflects formation of a hyperfluorescent intermediate that results from the burial of Trp74 in a native-like hydrophobic core in the ABD subsequent folding reaction well described by four phases of decreasing fluorescence intensity for WT products all capable of binding MTX four parallel folding channels leading from the hyperfluorescent intermediate to native or native-like species refolding of the permutants reactions under strongly refolding conditions best described by three exponential all three permutated proteins display characteristic hyperfluorescent phase followed by a decrease in the intensity as the folding reaction proceeds cpN18 and cpD69 require two exponentials to describe the hyperfluorescent phase parallel folding channels arise before the formation of the corresponding intermediates and the burial of Trp74 fewer number of phases with decreasing intensity observed for the permutants smaller number of folding channels. lack of hysteresis observed in enzyme activity assay and reduction in the number of folding channels is consistent with the loss of an inactive native conformer

25. Discussion Quantitative analysis of the thermodynamic and kinetic folding properties of the three permutated versions of E.coli DHFR has revealed a correlation between chain connectivity and the roles of the two functional domains in folding. Rational for this is based on an expected increase in chain entropy, and subsequent decrease in free energy of the unfolded state. This is experienced at a domain level.

26. The stable urea-induced folding intermediate Stable urea-induced intermediate was observed in only the cpP39 and cpD69 variants. Absence of this intermediate in WT-DHFR and cpN18 suggests that a continuous DLD is necessary to stabilize this species. These results contradict previous study (Arai et al., 2003) Results of that study are suspect due to small discrepancies between the two transition curves and relatively small absorbance changes during unfolding. This suggests that the stable intermediate may have been present but not detected. Large changes in tryptophan FL for unfolding of DHFR and distinctive response of emission of intermediate properties may have enhanced detectin of partially folded species. Large changes in tryptophan FL for unfolding of DHFR and distinctive response of emission of intermediate properties may have enhanced detectin of partially folded species.

27. The stable thermally induced intermediate A thermal intermediate was observed in cpN18, cpP39, and wild-type-like cysteine-free AS-DHFR and not in cpD69. This suggests a continuous ABD is required for the stability of this intermediate. Some discrepancy in the relative stability of ABD and DLD domains remains despite several studies. The creation of new termini introduces formal positive and negative changes at the N- and C- termini. The result of these new charges may in fact be stabilizing, but could also contribute to the loss of a thermal intermediate for cpD69. Chain connectivity, however, is still likely the principle factor influencing the stabilities of ABD and DLD. Revelation of mutants by different unfolding techniques suggests that there are variations in the relative contributions of the interactions stabilizing these domains.

28. Implications for the kinetic folding mechanism of DHFR Similar observed kinetic folding responses (those occurring after 5ms) for all the permutants and WT-DHFR. Suggests that if ABD or DLD plays a role in folding it must occur in the sub-millisecond time range. This is supported by data from a previous quench-flow hydrogen exchange NMR study (Jones and Mattews, 1995).

29. Conclusions The results on cpN18, cpP39, and cpD69 DHFR demonstrate that there is a crucial role for stable sub-domains in protein folding. Specific placement of new termini does incur an effect on the thermodynamic properties, however, there is minimal effect on the global folding, the post-millisecond folding mechanism, or enzymatic activity. Protein folding mechanisms are sufficiently robust that variations in the connectivity of the polypeptide do not perturb the folding of the protein.