Sequence Determinants of a Conformational Switch in a Protein

Transcription

1 Sequence Determinants of a Conformational Switch in a Protein Thomas A. Anderson, Matthew H. J. Cordes, and Robert T. Sauer Kyle Skalenko 4/17/09

2 Introduction Protein folding is guided by the following principles: A.) The physical properties of the amino acids i.e.: F=aromatic/non-polar, L=non-polar, E=polar, etc. B.) Because of these physical properties, the environment the protein is in affects its folding rate and native conformation.

3 Re-look at energy landscape S of the system, G is favorable for protein folding. -Protein goes from disordered ordered specie

4 Project Construction: Arc-st11 is a tagged protein (red region is tag) at C-terminal. St11 = HHHHHHKNQHE. Use Ni-column to purify protein by affinity. NOTE: Arc interacts with DNA with NTD.

5 Goal of Paper: 1.) Study Arc mutant protein properties at position 11 when N is changed to different non-polar and polar (only Y in this group) amino acids: N11X; X=G, A, V, I, L, M, F, and Y amino acids. 2.) See which fold is favored (when change N11), either wild type (WT, aka Arc) beta-sheet or mutant (aka switch Arc) 3 10 helix. 3.) Study proteins within different backgrounds: Backgrounds = WT, L12N, and P8L Make N11X mutation in each background to study protein shape.

6 Arc protein: Binds to DNA as a -Arc repressor of bacteriophage P22 is a transcription repressor. It binds to DNA as a tetramer (as shown in figure on next slide). -N-terminal beta-sheet residues are used to interact with the major grove of DNA. tetramer Lane A= free DNA Lane B= Arc+DNA Lanes C-G= includes a variant, Arc- It1. It includes more residues. For this presentation, only care about lane A and B

7 Arc Repressor Homodimer of Phage P22: WT 1.) Arc is a homodimer (top image). It has 2 beta-sheets that interact with DNA. Residues that make up 1 sheet are P 8 Q 9 F 10 N 11 L 12 R 13 W ) Bold N = position 11. By changing this residue to a different amino acid (see previous slide), can change conformation of sheets to 3 10 helix. 3.) Image to right shows ribbon model (one subunit), van der waals, and close up of beta-sheet at position 11. PDB 1BDV

8 Examination of the Mutant Arc: Switch Arc 1.) Top: NMR of switch arc. 2.) Mutation of N11L causes a conformational change in protein from betasheet to 3 10 helix. PDB 1QTG

9 Both WT and Mutant Together: Hydrophobic core N N Hydrophobic core L W L W

10 Pattern of Arc and Switch Arc WT Arc: Beta- sheet: (residues 9-14) Q 9 F 10 N 11 L 12 R 13 W 14 P--NP---P--NP P--NP pattern; P= polar, NP= non-polar Switch Arc: 3 10 helix: N11L, L12N Q 9 F 10 L 11 N 12 R 13 W 14 P--NP--NP--P----P---NP Arc with only N11L: Ambiguous fold. Equilibrium between Beta- sheet and 3 10 helix. Q 9 F 10 L 11 L 12 R 13 W 14 P---NP--NP--NP P----NP

11 Results: Denaturation Assay: L12N WT -L12N destabilizes WT Arc (beta-sheet) fold and favors helix fold. P8L -P8L stabilizes WT Arc by introducing 2 more H- bonds within beta-sheet. Note the high Tm and high [urea] for denaturation Note: G, A, and V seem to be the least stable (lower Tm).

12 Fluorescence of Arc WT and mutants in L12N background: F and W = higher absorbance Q 9 F 10 L 11 N 12 R 13 W 14 -Both figures show that N11X resemble switch (3 10 helix) mutant. -By changing to X to different non-polar residues, the properties of switch Arc stay the same. => N11-non-polar residue can give 3 10 helix. -UV-CD: Data does not look great, but it is looking at the aromatic amino acids within the region b/c absorbance is at 280nm.

13 Location of P on WT Arc protein structure P8 WT Arc PDB 1ARR

14 P8L background: UV-CD data As you can see, N11X; X=G, A, L, I, M, F, and Y + P8L background gave a WT (beta-sheet) fold. As you can see with Tm, N11X + High Tm for larger aa = better packing in core P8L produces a hyperstable fold because of additional H-bonds in beta-sheet. (see slides 10, 11).

15 Ambiguous fold: N11L only mutation Arc with only N11L: Ambiguous fold. Equilibrium between Beta- sheet and 3 10 helix. Q 9 F 10 L 11 L 12 R 13 W 14 P---NP--NP--NP P----NP The results for this type of mutant is interesting. The mutant can fold into WT Beta-sheet or Switch Arc (3 10 -helix) by being in equilibrium between the 2 folds.

16 Ambiguous fold: N11X UV-CD data As you can see, both WT, switch (N11L) and mutants follow same pattern. The UV-CD data looks very interesting: Both switch and WT form a barrier (roughly) of fold conformation: Mutants equilibrate between the 2 folds: => Shows that mutants listed at top (N11 I, V, Y, M, F, A) form WT fold and N11L, G form switch fold. But for each mutant, they Favored one fold over the other.

17 Ambiguous fold: N11X UV-CD data cont. The paper shows probability of fold conformation for each mutant: Favor WT In equilibrium between WT and helix Favor helix The only difference between these 2 amino acids (I and L) is the position of 1C!! *Shows how amino acid property affects fold, etc.

18 N11X cont. The two fold conformations are in equilibrium with each other. Rate: > 1000 sec -1 -Could this protein selfregulate itself to bind to DNA (ligand) when in close proximity to it? (idea from equilibrium). Arc + DNA Arc-DNA complex Answer: Yes, see next slide.

19 DNA binding Fluorescence data: -Slide 21 states that protein switches between both conformations=> N11L could have its own primitive allosteric system by interchanging between active WT form and inactive helix form.

20 Evolutionary Bridge w.r.t N11L N11L is an evolutionary intermediate. the evolutionary bridge. because it encodes different folds (see data). This intermediate demonstrates that the structural evolution of some proteins can occur continuously by accumulation of substitution mutations. => protein folds do not need to be distinct islands, i.e. fold 1 vs. fold 2, but be linked by these bridges where multiple folds can coexist.

21 Conclusions This paper offered multiple experiments that classified N11X mutants by folds (either WT or switch). The paper can help us understand the dynamics of protein folding, how residues lead to different folds, and stability of proteins in certain environments. Seeing how 1 residue change Polar to Non-polar (N11X) can result in a dramatic structural change within the protein. This can offer information about how aa properties affect fold of protein.

22 References: Anderson, T. A., Cordes, M. H. J, Sauer, R. T. Sequence determinants of a conformational switch in a protein. PNAS, vol. 102, no. 51, , Brown, B. M., Bowie, J. U., Sauer, R. T. Arc repressor is tetrameric when bound to operator DNA. Biochemistry, vol. 29, no. 51, , Cordes, M. H. J., Burton, R. E., Walsh, N. P., McKnight, C. J., Sauer, R. An evolutionary bridge to a new protein fold. Nature Structural Biology, vol. 7, no. 12, , Protein Data Bank (see slides for PDB numbers).

23 Summary of L12N data: Given fluorescence, UV-CD, and HSQC NMR data, it shows that N11X; X=non-polar aa will give a helix structure. Switch Arc: 3 10 helix: N11L, L12N Q 9 F 10 L 11 N 12 R 13 W 14 P--NP--NP--P----P---NP Larger non-polar aa stabilized hydrophobic core (due to better packing) more than G, A, and V (see denaturation assay (CD-urea curves) and Tm.

24 Background: Heteronuclear Single Quantum Correlation (HSQC) NMR: A commonly used technique to determine the position of a proton and a heteronuclear atom ( 15 N or 13 C). For this paper, they are detecting amide H by using isotopic 15 N. Note: cannot detect P proton 2D-spectra observed. Y-axis= 15 N and X-axis= amide H. If know H, can figure out position of N O R and vice versa. + H 3 N R N H COO - Heteronucleus proton

25 Results: Heteronuclear Single Quantum Correlation (HSQC) NMR with L12N background: -The data shows 4 N11X mutants in L12N background. -The red circles are characteristic of helix. -As you can see, each nonpolar aa gives a similar spectrum = proteins have similar folds => Proteins with N11non-polar substitution will give a helix when in L12N background.