The overall goal of this procedure is to determine the structure of a Metallo chaperone protein mimetic peptide complexed with copper. This is accomplished by first preparing the sample in an oxygen free environment. The second step is to acquire the nuclear magnetic resonance or NMR spectroscopy data showing interactions among hydrogen atoms.
Next, the spatial interactions are mapped onto a linear peptide template. The final step is to find a representative low energy structure that fits the data. Ultimately, NMR derived structures are used to determine the mode of binding and to perform structural analysis on the copper bound complex.
The main advantage of this technique over existing techniques such as x-ray crystallography, is that you can use it to look at weekly binding complexes and also at molecules and complexes that don't crystallize. Though this method can provide insight into copper binding proteins. It can also be applied to other systems such as other metal chaps to study how proteins enable safe delivery of essential, yet potentially toxic metal ions in the cell line environment.
To begin prepare samples in an oxygen free environment to prevent the copper from oxidizing to prepare the APO sample dissolve approximately one to two milligrams of the peptide in 450 to 500 microliters of Deuterated NMR grade solvent for the copper reacted sample dissolve the same amount as the APO peptide with an EQU molar amount of metal salt in 450 to 500. Microliters of the NMR solvent filter each solution using a center glass filtration paper or any other technique that suits the compounds under investigation and does not absorb them. This is essential to remove any metallic particles, which will affect the homogeneity.
Transfer the solutions to NMR tubes and close the samples in the tubes. Before leaving the glove box, take the samples out of the glove box and seal them. Record the one dimensional proton spectra of the APO and copper reacted samples in the NMR machine and compare.
The APO peptide is flexible and shows an average of confirmations, but upon reacting with copper, the bound peptide amides have a more rigid structure. Therefore, the copper containing peptide spectrum should show a significant change in the chemical shift in the amide region, and the peaks may become resolved. Set up optimized, cozy tox, nosy or rosy NMR experiments under identical conditions as described in the text protocol and run in sequence.
Run one dimensional experiments in between each experiment to make sure that the sample composition remains constant throughout data acquisition. After processing the data as discussed in the text protocol, prepare a set of cozy and to spectra overlaid on the noea and rosy spectrum. To assign all NOE peaks in the spectrum.
Start with assigning peaks that overlap toxis signals in the fingerprint region. As this will facilitate subsequent peak assignment entries with the Sparky program record the assigned peaks translate H alpha to amide protein coupling values into dedal angles. Also translate peaks into distance constraints by integrating the peaks from within the program and translating them using an interaction of known distance.
If the peaks overlap or automatic integration methods cannot be used, the peaks can be labeled as strong, medium, weak, or very weak by visual estimation, and these designations can be translated into distances up to 2.5, 3.5, 4.5, and 5.5 angstroms respectively. In import the distance constraints and dedal angles with the correct format for explore. Explore will search confirmational space to find structures that adhere to canonical chemical geometry.
In addition to the experimentally found distance constraints to generate an ensemble in which none of these parameters are violated. This will constitute the starting ensemble. Perform the first structure determination run without using any constraints to the metal to find which residues participate in metal binding without any bias.
Introduce the constraints gradually to facilitate identifying mistakes in assignment as well as NOE energy and simulated kneeling parameters as described in the text protocol before minimizing the structures using conjugate gradient energy minimization for 4, 000 iterations, create a final ensemble of usually 50 members performing introduction of constraints in an iterative fashion on the entire ensemble report the number of each type of NOE interaction found. Finally, create an ensemble of structures that adhere to canonical chemical geometry and the empiric NMR derived constraints Report. The total number of confirmations, the number of these that have violations of the NM Rived constraints and the RMSD of the entire ensemble, including both backbone and all heavy atom RMSD values.
Analyze the low energy ensemble and determine which residue side chains are in correct proximity to each other to be able to bind the metal ion. Once these have been determined, repeat the analysis, including the copper binding data. In addition to the NMR derived distance constraints, now add metal binding constraints to the residues determined.
Add appropriate parameters to describe the metal ion and its topology. Input the appropriate physical information such as mass bond lengths with other atoms, angles, and non bonding repulsion parameters into the parameter file. Add the binding information to the topology file.
This information includes which bonds are formed and broken, and which formal charges are changed as a result of binding. Finally, acquire an ensemble of structures as before the solved ensemble represents the confirmational space adopted by the peptide. During the NMR measurement, import all confirmations of the structure to the Mal Mall program to create a starting ensemble.
Examine the ensemble to determine the local stability of the molecule. Determine the backbone and side chain RMSD values by selecting subsequent four residue regions along the sequence and having the program calculate the RMSD to the lowest energy structure or the mean, determine which regions of the molecule show local stability by plotting the local RMSD as a function of sequence, overlay the ensemble along this region of the molecule and use this ensemble for further analysis. Choose low energy confirmations that adhere to the NMR derived constraints.
These will form the low energy ensemble record and report the number of confirmations in the ensemble, the criteria for choosing them and the RMSD values. If the metal binding mode has not yet been determined, analyze the low energy ensemble and determine which residue side chains are incorrect. Proximity to each other to be able to bind the metal ion.
Use KYMERA to determine intramolecular distances between atoms suspected for metal binding. Calculate the average distances in the ensemble once these have been determined. Repeat the analysis including the copper binding data.
Examine the ensemble and determine local secondary structure within the molecule using the default search parameters of the MAL mall program. Next, import the ensemble into kymera. Secondary structures are held by hydrogen bonding and indicate stable regions of the molecule.
Determine hydrogen binding using the kymera tool. Continue structural analysis as detailed in the text protocol. Then sum up all the structural findings to reveal how they reinforce each other To study copper binding protein models, the structure of the conserved binding sequence of a protein within the derived linear peptide was determined by solution state NMR, the amide region of the peptide from 6.7 to 8.5.
PP M showed an expansion upon reaction with copper to 6.6 to 9.0 PP M.Line broadening due to slight copper oxidation is evident in the baseline. Shown here is an overlay of the fingerprint regions of Roy Toxi and cozy spectra of the copper bound peptide. The sample was stable with time and the spectra were well resolved and gave 81 NOE interactions that were acquired by a rosy experiment.
Since the molecule gave near zero NOE interactions in the NO C experiment, the ensemble of the peptide derived for the reacted sample, but using no constraints to the metal gave 47 out of 50 non structures with an RMSD value of 1.44 and 2.07 angstroms on the backbone and heavy atoms respectively. Of these 13 low energy conformers were chosen for further analysis with RMSD values of 0.25 and 0.61 angstroms on the backbone and heavy atoms respectively. The local RMSD plot showed a region of stability between residues three and seven In addition to the rigid C terminal region, including a prolene residue, this region is found in a bend confirmation between residues four and seven in all confirmations.
The bend confirmation is stabilized by hydrogen bonding between backbone donors and acceptors glycine five and three anine two, as well as cysteine six and cysteine three. This bend is also evident in cystine three and sine seven by the reduced coupling values in this region. The confirmations were superimposed over this region and analyzed for possible binding residues when considering cystine three, cystine six and methionine one as potential binding residues.
The shortest sulfur to sulfur atom distance was that between the folate groups of cysteine three and cysteine six, copper binding was introduced and the calculation was repeated to give the ensemble used for analysis. The low energy ensemble of the copper bound peptide shows that the end terminal amine is proximate to the bound copper. Shown here is the electrostatic potential distribution isosurface with positive potential shown in blue and negative potential shown in red.
The arginine residue extends from the backbone of the peptide forming a positive lobe of electrostatic potential, whereas the backbone carbon yields are arranged in a line forming a less prominent negative electrostatic potential Once mastered, a structural determination can be done in about a week of NMR time and another few days of workup in order to obtain an ensemble of confirmations that can be used for structural analysis. Following this procedure, other peptide mutans and different conditions can be analyzed in order to answer additional questions that address the conditions required for different degrees of binding and release of the copper ion.
