---------Ab Initio determinations and comparison with the experimental observations

• Main focus on the effects of the denaturant SmallUbiquitin-like Modifier (SUMO) protein.

• This protein has shown to have some unique denaturationcharacteristics. Instead of going into a fully random coilstate, some of the residues in this 88-residue long proteinshow propensities to form secondary structure even afterdenaturing them.

• The main objective of the study is to prove that theseproteins become trapped into the local minima state thusproving that there may exist some secondary conformationin a denatured SUMO protein.

Chemical Shift

• Tetramethylsilane (TMS) = Most shieldedTMS

Normal Phenomenon

Expected outcome

Energylandscapeinprotein

High energy zone-place where

unfolded proteins normally exist.

Local Minima(place where SUMO protein is assumed

to exist after denaturation).

Global minima (place where

most of unfolded proteins exist).

WhyWorkonSUMO?????§ Small Ubiquitin-like Modifier or SUMO proteins are a family of

small proteins that are covalently attached to and detached from otherproteins in cells to modify their function.

§ The function performed by SUMO proteins is known as Sumoylation

§ Sumoylation is a post-translational modification involved in variouscellular processes such as transcriptional regulation, apoptosis, proteinstability etc.

§ SUMO proteins are similar to Ubiquitin, and Sumoylation is directed by anenzymatic cascade analogous to that involved in Ubiquitylation. Incontrast to Ubiquitin, SUMO is not used to tag proteins for degradation.

§ Ubiquitin’s NMR property has been studied by both NMR and Ab-initio methods .

AboutdSmt3 fromDrosophila…….

� This protein is 88 residues long and has four Beta sheets and two alpha helices.

� It has methionine at its N-terminal end and Glycine at its C-terminal end.

DifferencesbetweenSUMOandUbiquitinLong and flexible N-terminal extension of

SUMO

C-teminal di-glycine motif

FunctionsofSUMO………….

vNucleo-cytoplasmic transport.

vTranscriptional regulation.

vRegulation of intracellular localization.

v Interplay between Ubiquitin and SUMO signaling.

vRole of SUMO in Mitochondrial Fission.

vRole in Cell-Cycle.

Experiments performed………......

Approach…………………………….Ø Preparation of protein structures and their Topologies

Ø Giving dynamics to the proteins and simulating the denaturing conditions.

Ø Calculating the NMR properties through Ab-initio methods

Ø Comparing with the experimental data

Ø Checking out structural propensities with help of 13Cchemical shifts

Preparation of protein structures……….

� Software used – Cyana

� Obtained the structure from Protein Data Bank

� For the sake of convenience and ease of dynamics, SUMO protein was divided in to five fragments.

� These fragments were divided based on their propensity to form secondary structures.

Fragment Residue numbers

Fragment 1 1-12

Fragment 2 11-32

Fragment 3 31-53

Fragment 4 52-72

Fragment 5 71-88

FilesusedinCYANA……….� First need to create .CCO file of the FIVE fragments

.CCO file------------

32 HIS H HA 6.7277 3.20E+0032 THR H HA 6.9968 1.20E+0033 PRO H HA 6.4720 1.20E+0034 LEU H HA 6.8444 1.20E+0035 ARG H HA 6.9625 1.20E+00.......53 THR H HA 7.5359 2.20E+00

Init.cya File

.cya is a batch file which contains a set commands.

Rmsd range := 31.....53Cyana.libRead seq third.seqSwap = 0

Batchfile

Ø ‘Seed’ asks the program to generate 1000 topologies

Ø The last two commands creates the 20 best topologies

Five topologies generated From Cyana

� Each topology resembles one conformer of the fragment

performing molecular dynamics on each of the topologies

Using

GROMACS………………

� Although normally represented as static structures, proteins are infact dynamic.

� Most experimental properties, for example, measure a timeaverage or an ensemble average over the range of possibleconfigurations the molecule can adopt.

� One way to investigate the range of accessible configurations is tosimulate the motions or dynamics of a molecule numerically. Thiscan be done by computing a trajectory, a series of molecularconfigurations as a function of time, by the simultaneousintegration of Newton's equations of motion.

MOLECULAR DYNAMICS ON PROTEINS

The main Purpose of Molecular Dynamics of SUMO wassimulate the exact denaturing conditions on it virtually by an in-silico approach

• Proteins in solution are considered to dynamic

• It is difficult to study their motions, behavior, structural flexibility insolution

• The structure of small proteins can be solved and studied by theconventional technique of X-RAY CRYSTALLOGRAPHY.

• X-RAY techniques require strict periodic boundary conditions which isvery difficult to obtain in a non crystalline structures.

• Molecular dynamics simulations can predict the state of a protein insolution and save these states in the form of a trajectory.

• MD can predict the movement of large proteins in the solution which isnot possible in X-ray.

• MD can simulate the exact condition of the existence of a protein.

• Structures obtained after MD simulation can be regarded as best energyminimized and geometrically optimized structures thus allowing themto be used in various experiments-------NMR, Docking, protein-ligand interactions.

Usefulness of MD in the experiments……

• Subjected the best topologies to molecular dynamic simulations in 8Murea, which will simulate the conditions of denaturing the proteins. (useof GROMACS).

• The mean structures were calculated from the best trajectories.(use of MolMol)

Calculating the NMR properties through Ab-initio methods……….

Tools used:

v Gaussview 3.1

v Gaussian 03

v Argus lab

v Swiss PDB Viewer

Approach• Took the fragments and added hydrogen's to them.

• From each of the residues specified the cut-off distance of 3A

• Gave stringent calculations for residues falling in these range.

• Fragment layer based approach

• Using different basis sets

• Calculated the shielding tensors for these layers.

• comparing with experimental results

Building input files for NMR calculationsHydrogen Bond distance: 1.6 - 2.0A0

Covalent Bond distance: 0.96 – 1.0A0

Van der Waals radii: Always less than 2A0

Electrostatic and ionic interactions: Always less than 1.5 A0

Done with the help of Swiss PDB viewer.

Step 1:� One of the fragments of the particular topology taken.

Step 2: The amino acid whose NMR has to be calculated was selected.

Lys residue whose NMR had to be calculated was selected (marked in red

Step 3 : selecting the neighboring residues.

Step 4: setting the cut of radius as 3Å

Step 5: keeping the residues which fall within the cut-off radius and discarding the rest.

Three residues (R6,K7,L8) falling within 3Å and the rest is discarded

Step 6: Subjecting the retained residues to Gaussian calculation.

� Opening the cluster in Gaussview

Add hydrogen to the cluster if not added.

Setting up Gaussian calculations

NMR chemical shift calculations were performed on the trajectories using Gaussian03.

Accuracy of the calculations

Name of the checkpoint file

Memory used

Output from Gaussian

Results…………

Results of Molecular dynamics

Difference in the energy before and after Molecular Dynamics

NMR results

Criteria for checking the accuracy of the Basis Set

Correlation plot

Assigning Structural Propensities

Formula

Final outcome

In silico results Experimental results by TIFR group

The Residues 32 to 34 shows β Sheet propensity as it contains three residues in sequencethat show a downward trend in the graph. On the contrary the residues 41 to 47 show acontinuous upward trend in the graph, which is indicative of α-helical propensity.

Conclusion� Thus it can be concluded that the we can try using In-silico approaches to

compute NMR for a Biological molecule.

� The fact that the structures can still be recovered from the denatured SUMOis quite promising.

� This can provide a new breakthrough in protein folding pathway.

Future Prospects� Can be used on any other protein with no constraints on its size.

� It will then be possible to map the whole protein in the denatured state for thehidden structural propensities.

� The ab initio calculations can be correlated with the experimental NMR data.

� This work can be used as a model to study the denaturation kinetics and thefolding pathways of the SUMO and other bigger proteins (more than 70residues).

� This project can lead to the development of a valuable computationalapproach to gather more insights on the protein folding pathways.

Acknowledgements

• Dr. S. Ganapathy , Scientist Emeritus, Central NMR, Pune

• Prof. R.V. Hosur, Department of Chemical Sciences, Tatainstitute of fundamental Research, Mumbai.

• Dean and Professor Dr. D.A. Bhiwgade D.Y. Patil Institute ofBiotechnology and Bioinformatics, Navi Mumbai.

• Dr. Arpita Gupte, Dr. Madhavi Revankar, and Mr. Shine D,Padmashree Dr. D.Y. Patil institute of Biotechnology andBioinformatics .