NMR, X-ray Diffraction, Protein Structure, and RasMol

NMR, X-ray Diffraction, Protein Structure, and RasMol Introduction So far we have been mostly concerned with the proteins themselves. The techniques (NMR or X-ray diffraction) used to determine a structure have some differences, both in terms of how they see the protein and of how the results are presented. This lab will aquaint you with proteins from this point of view. Objectives 1. Use RasMol to locate short distances in α helices and β sheets in ribonuclease A. 2. Use RasMol to identify short distances that determine the folding topology of ribonuclease A. 3. Use RasMol to view coordinate files containing ensembles of NMR structures. 4. Use RasMol to view unique features of structures determined using X-ray crystallography. Brandén & Tooze 2 nd Edition: Chapter 18 Data files This lab uses files contained in the downloadable self-expanding archive: http://www.biosci.ki.se/courses/sbb/cl6.exe NMR Due to the regular nature of α helices and β sheets, distances between certain proton pairs will be < 5Å and can thus be used to identify these secondary structural segments in the absence of a full three-dimensional structure. The Nuclear Overhauser effect (NOE) results from dipolar relaxation between spins. This effect is very sensitive to the distance between the spins and under favourable conditions, provides an atomic ruler within the molecule. Scalar coupling, a phenomenon in which nuclear dipoles sense the spin orientation of neighboring dipoles through electrons shared in covalent bonds, also provides geometric information. A number of scalar coupling constants have been empirically correlated with intervening dihedral angles. Internuclear distances measured by the NOE and dihedral angles measured by scalar couplings together form the basis of a web of conformational restraints that allow the determination of macromolecular structure. Part 1 Short distances in a helices Elements of regular secondary structure can usually be identified in the early stages of analysis of protein NMR spectra. Scalar coupling constants, 3 J HN/Hα, as well as intraresidual and sequential 1 H NOEs in these segments display certain characteristic values. 1. Open ribonuclease A in RasMol and select a segment of the polypeptide backbone in an α helical conformation. The script 5rsa_nmr1.txt is provided for this (Note that the script will load the coordinate file and select an appropriate representation. - 1 -

Now find the distances from a HN (e.g. residue 9) to its neighboring protons in the helix. RasMol> set picking distance RasMol> set picking monitor HINT: On the PC version, if you hold down the shift key after this selection, clicking on subsequent protons measures the distances from the first atom selected. Repeat the exercise this time beginning with H α of.residue 9. Be sure you measure the distances between the Hα (i) and HN(i+1) pair and the Hα(i), HN(i-1) as well as Hα(i), HN(i); Hα(i), HN(i+2); Hα(i), HN(i+3); Hα(i), HN(i+4); and Hα(i), HN(i+5). When you are finished, you can remove the monitors with the command RasMol> monitors off 2. Measure the distances between HN(i), HN(i+1) pairs. Compare it to HN(i), HN(i+2); HN(i), HN(i+3); HN(i), HN(i+4); HN(i), HN(i+5). 3. If you identify pairs with distances <5 Å, measure the distances in each pair of that type in the helical segment. 4. Summarize your results by answering the question What types of short distances (and to which atoms) are expected for an α helix? strand? Within a β sheet? JH α H β2 Part 2 Short distances in b strands/sheets 1. Repeat the measurements in Part 1 with a segment of a β sheet (e.g., residues 42-46, 81-85, 98-102). Measure the distances from the Hα and HN protons of a residue (e.g., 83) in the center of sheet to its neighbor protons. The script 5rsa_nmr2.txt is provided. 2. Summarize your results by answering the question What short distances are expected between protons within a β 3. How can parallel and anti-parallel sheets be identified? JH N H α JH α H β3 Part 3 Coupling Constants and backbone torsion angles f,y, and y The three-bond coupling constant between the intraresidual alpha and amide protons, 3 J HN/Hα, is an important conformational restraint for secondary structure determinations as it can be directly related to the backbone torsion angle φ through the empirical Karplus relationship (see graph at left). As seen in the previous computer labs, helical and extended conformations have very different values for φ (-60 and -120, respectively) which result in differences in 3 J HN/Hα. 1. Return to the previous two scripts and look at the relationship between Hα(i), HN(i) and the coupling constants 3 J HN/Hα. - 2 -

right-handed α helix φ = - 57 right handed 3.10 helix φ = - 60 antiparallel β sheet φ = -139 parallel β sheet φ = -119 left-handed α helix φ = 57 3 J HN/Hα = 3.9 Hz 3 J HN/Hα = 4.2 Hz 3 J HN/Hα = 8.9 Hz 3 J HN/Hα = 9.7 Hz 3 J HN/Hα = 6.9 Hz 2. Can a proton coupling constant be identified that could be used to define ψ? What about the use of a heteronuclear ( 13 C, 15 N) coupling constant? Part 4 Long-range NOEs and Protein Structure Due to the compact nature of proteins, the folding topology brings atoms close in space which may be from residues far apart in the amino acid sequence. These short distances are invaluable for determining the 3D structure of the protein. 1. Use the script 5rsa_nmr3.txt to illustrate how the topology of the polypeptide fold brings distantly-related portions of the chain close in space. In particular focus on the segment 4-11 and 116-121.What short distances do you see? Could this be used to determine the conformation of ribonuclease? 2. Now lets look at how many protons in ribonuclease are within the NOE distance of at least one other proton. Load the coordinate file of ribonuclease again (5rsa.pdb). RasMol>select RasMol>monitors off RasMol>wireframe RasMol>select hydrogen and within(5.0, (hydrogen and!solvent)) and!solvent RasMol>cpk 100 Try to make the opposite selection that is all the protons in ribonuclease that are not within 5.0 Å from another ribonuclease proton. Do you find any? What does this tell you about short distances and protein structure determination? Viewing NMR Specific Features with RasMol You have by now grown accustomed to viewing single molecules using RasMol. As you will come to appreciate, threedimensional structures determined using NMR techniques are very often represented as groups (ensembles) of structures which are optimally superimposed on one another. RasMol can also be used to view ensembles of NMR conformers. However, be warned, the program is not very well suited to this type of analysis. The PDB standard for NMR coordinate files is that each conformer is designated with a MODEL number and conformers within the coordinate file are separated with TER and ENDMDL. In RasMol version 2.7.1, opening a coordinate file will automatically open all models in the coordinate file. - 3 -

The syntax of RasMol atom expressions allows the selection of individual molecule conformations if present in an NMR file. The simplest form of the atom expression is the syntax "*/1" to select model 1 from the coordinate file. The most general form of atom expression is now CYS14/1.SG which denotes the gamma sulfur of residue cysteine-32 in of model 1. Part 5 BPTI The NMR solution structure of BPTI is a good example of a high precision NMR structure. The structure was calculated using a total of 766 constraints of which 642 were NOE-derived distance constraints, 0 hydrogen bond constraints, 9 disulfide bond constraints, and 115 torsion angle constraints. The RMSD of backbone atoms 2-56 is 0.43 Å. Use the script 1pit_m.txt to explore the 20 conformers that make up the NMR structure of BPTI. Note that in this file, I have generated the mean (average) coordinates and added it to the file 1pit_m.pdb as model 21. RasMol> script 1pit_m.txt 1. Are all the side chains determined with equal precision? 2. What types of side chains are well-defined? Not well-defined? Does this correlate with hydrophobicity? Position on the inside/outside of the protein? 3. Are all disulfide bonds determined with similar precision? 4. Is the mean (average) structure a good representative of the ensemble? For what purposes would it be? not be? Use script 1pit_nmr1.txt to look at an example. Part 6 Protein G The NMR solution structure of 56 residue Protein G is arguably the highest precision NMR structure available today. It is a good example of what can be achieved using modern NMR techniques. The experimental input data consisted of 1058 constraints of which 854 were NOE-derived distance constraints, 60 hydrogen bond constraints, and 144 torsion angle constraints. The RMSD of backbone atoms is 0.25 Å. Use the script 1gb1.txt to explore the 60 conformers that represent the NMR structure of Protein G. I have placed the energy minimized mean structure at the end of the file 1gb1_m.pdb as the 61 th model. RasMol> script 1gb1.txt 5. Are all the side chains determined with equal precision? 6. Why are 60 structures needed? 7. Is the energy minimized mean structure a good representative of the ensemble? Part 7 Cyclophilin Out third example of an NMR structure is that of cyclophilin. You have also looked at this protein before in the complex with cyclosporin. Now we will focus on the protein which was calculated using a total of 1980 NOE-derived distance constraints. - 4 -

The RMSD of all backbone atoms is 1.07 Å for cyclophilin which makes it the least precise of the three structures presented here. Use the script 1cys.txt to explore the 22 conformers that represent the NMR structure of cyclophilin. RasMol> script 1cys.txt 1. Is the precision of the polypeptide chain constant throughout the sequence? Does the precision correlate with structure? What is the structure of the regions which are less precisely determined? 2. Throughout the exercise, we have discussed precision. What about accuracy? How might accuracy defined? Is it better to be precise or accurate? 3. Having looked at three structures of very different size and precision, what determines the precision of the structures? Part 8 Conformational Constraints To get some feeling for the relationship between some of the experimentally-derived NMR constraints and the polypeptide conformation. First you will see the slowly exchanging amide protons of BPTI. Then C α atoms are color-coded according to the magnitude of 3 J Hα/ HN ( 3 J Hα/ HN <6 orange; 6< 3 J Hα/HN <7 green; 3 J Hα/ HN >7 violet). RasMol> script amide.txt Questions: 1. Do the slowly exchanging amide protons correlate with structure? Position inside/outside of the protein? Involvement in hydrogen bonds? 2. Do the 3 J Hα/HN coupling constants correlate with structure? Position inside/outside of the protein? Involvement in hydrogen bonds? 3. Would these coupling constants alone be a good predictor of secondary structure type? Viewing X-ray Specific Features with RasMol Introduction By now, we have been looking at quite a few structures. In this section of the lab, we will focus on specific features present in structures determined by X-ray diffraction of single crystals. These features will help you assess the quality of the structures and very often provide new insights into the molecules. The coordinate files of structures determined by X- ray crystallography contain all the information necessary to recreate the crystal of the molecule. Thanks to symmetry, one does not have to specify the coordinates of every molecule in the crystal. It suffices to specify the coordinates of the asymmetric unit and give the symmetry of the crystal form. While multiple structures may seem an unnecessary complication when you are getting started, there is useful information to be gained by their comparison. - 5 -

Part 9 Symmetry (optional) To get a feeling for the interrelationship between neighboring proteins in the crystal, the crystal environment for one molecule of BPTI was generated using the symmetry parameters contained in the header of the coordinate file. The unit cell of this crystal form has P 2 1 2 1 2 1 symmetry. Try to see the relationship between the neighboring molecules along the x, y, and z axis. Do you see any crystal contacts that might influence the structure? Chorismate mutase is an essential enzyme in the pathway for the synthesis of aromatic amino acids. The protein from Bacillus subtilis is a trimer of identical subunits. In the structure 2CHS, there are 4 trimers in the asymmetric unit. As each monomer of each trimer has an identical sequence and is independently determined, one can compare all twelve monomers to get an idea of the variability of the conformation. (Visit the course site and follow the link for this lab to a superposition of the 12 structures.) Part 10 Occupancy The occupancy of atom is a measure of the fraction of molecules in the crystal in which atom actually occupies the position specified in the model. If all molecules in the crystal are precisely identical, then occupancies for all atoms are 1.00. Occupancy is included among refinement parameters because occasionally two or more distinct conformations are observed for a small region like a surface side chain. The model might refine better if atoms in this region are assigned occupancies equal to the fraction of side chains in each conformation. For example, if the two conformations occur with equal frequency, then atoms involved receive occupancies of 0.5 in each of their two possible positions. By including occupancies among the refinement parameters, we obtain estimates of the frequency of alternative conformations, giving some additional information about the dynamics of the protein molecule. We also make the model more accurate, which contributes to progress in the refinement. Correction for missing electron density can take the form of a reduced occupancy for an atom or group of atoms in the structural model. One example is found in one crystal form (5PTI) of BPTI where side chains of two amino acids (7 and 52) are found to have multiple conformations. What is the occupancy of each form? RasMol> wireframe off RasMol> restrict protein RasMol> set bondmode or RasMol> backbone 60 RasMol> select (7 or 53) and sidechain RasMol> wireframe 40 RasMol> select (7 or 53) and *.*;A //selects alternate conformation A RasMol> color green RasMol> select (7 or 53) and *.*;B //selects alternate conformation B RasMol> color yellow 1. Are all the side chains determined with equal precision? 2. What types of side chains are well-defined? Not well-defined? Does this correlate with hydrophobicity? Position on the inside/outside of the protein? - 6 -

Part 11 Temperature Factors The temperature factor or B-factor can be thought of as a measure of how much an atom oscillates or vibrates around the position specified in the model. Atoms at the end of side chains are expected to exhibit more freedom of movement than main chain atoms. Diffraction is affected by this variation in atomic position, so it is realistic to assign a temperature factor to each atom and to include the factor among parameters to optimise during least-squares refinement. From the temperature factors computed during refinement, we learn which atoms in the molecule have the most freedom of movement, and we gain some insight into the dynamics of our largely static model. If the temperature factor B is purely a measure of thermal motion at atom j, then in the simplest case of purely harmonic thermal motion of equal magnitude in all directions (called isotropic vibration), B is related to the magnitude of vibration as rmsf = 3B 2 8π Thus if the measured B is 26 Å 2, the rms fluctuation is 1.0 Å. B values of 6 and 1.6 Å 2 correspond to rms fluctuations of 0.5 and 0.25 Å. But the B values obtained for most proteins are too large to be seen as reflecting purely thermal motion and must certainly reflect disorder as well. Analysis of B-factors in important in analysing the structure and assessing its quality. In general, B-factors for water molecules should be < 35Å 2. For covalently bonded atoms, B- factors should change gradually along the chain with B < 7 Å 2. Atoms or atomic groups with high B-factors are indicative of high mobility in the crystal and can be modelled with multiple conformations. 1. Obtain the coordinate file 3B5C and make a plot of B-factors of the C α atoms as a function of the amino acid sequence. 2. RasMol can interpret the B-factors as a range of colors from blue (low) to red (high). Color the protein according to temperature and correlate the picture with your graph. Part 12 Internal Water Molecules By now, it should not be surprising to find cofactors buried in the interior of some proteins. Water can also be integrated into the architecture of macromolecules. One well-studied example is the 4 internal water molecules in BPTI. RasMol> wireframe off RasMol> restrict protein RasMol> color white RasMol> cpk RasMol> select solvent and (60-62,73) RasMol> color temperature RasMol> cpk Can you see the water molecules? RasMol> slab Turn the slab off and focus on the contacts between the protein and water. Try to deduce the positions of the hydrogens by inspecting the protein-water contacts. Make a sketch of the three waters and add the hydrogens. Feel confident about your assignment? The water - 7 -

hydrogen positions are known from a neutron diffraction study (5PTI). How does this agree with your assignment? - 8 -

Answer sheet Use this form to fill in answers to the questions appearing in the text of the lab as you go through it. NMR What types of short distances (and to which atoms) are expected for an α helix? What short distances are expected between protons within a β strand? Within a β sheet? How can parallel and anti-parallel sheets be identified? Can a proton coupling constant be identified that could be used to define ψ? What about the use of a heteronuclear ( 13 C, 15 N) coupling constant? NOEs. What short distances do you see? Could this be used to determine the conformation of ribonuclease? Protons in ribonuclease that are not within 5.0 Å from another ribonuclease proton. Do you find any? What does this tell you about short distances and protein structure determination? BPTI. Are all the side chains determined with equal precision? What types of side chains are welldefined? Not well-defined? Does this correlate with hydrophobicity? Position on the inside/outside of the protein? Are all disulfide bonds determined with similar precision? - 9 -

Is the mean (average) structure a good representative of the ensemble? For what purposes would it be? not be? Protein G. Are all the side chains determined with equal precision? Why are 60 structures needed? Is the energy minimized mean structure a good representative of the ensemble? Cyclophilin. Is the precision of the polypeptide chain constant throughout the sequence? Does the precision correlate with structure? What is the structure of the regions which are less precisely determined? Throughout the exercise, we have discussed precision. What about accuracy? How might accuracy defined? Is it better to be precise or accurate? Having looked at three structures of very different size and precision, what determines the precision of the structures? Do the slowly exchanging amide protons correlate with structure? Position inside/outside of the protein? Involvement in hydrogen bonds? Do the 3 J Hα/HN coupling constants correlate with structure? Position inside/outside of the protein? Involvement in hydrogen bonds? Would these coupling constants alone be a good predictor of secondary structure type? - 10 -

X-RAY Crystal contacts in BPTI? Occupancies of BPTI aa 7 and 53? Are all the side chains determined with equal precision? What types of side chains are welldefined? Not well-defined? Does this correlate with hydrophobicity? Position on the inside/outside of the protein? How does your hydrogen bond assignment agree with that is found in 5PTI? - 11 -