Molecular Biophysics - Science topic

Did you find the solution? I am in the same trouble.

I just found people use ethanolamine in the SA coating.

No, the standard free energy of hydrolysis of the phosphodiester bond in DNA is -5.3 kcal/mol. It requires energy to forge a phosphodiester bond, while to break one requires only enough energy to overcome the activation energy barrier, which is lowered by enzymatic- , acid- or base catalysis. Under physiological conditions, hydrolysis is further facilitated by the high water concentration.

Indeed, in the case of a tip-scanning AFM the incident laser beam should follow the tip scanning motion, to record throughout the deflection signal for the same spot on the cantilever backside. This can be achieved by integrating the laser diode with a kind of tube (with its long axis parallel to the z-axis) that carries the cantilever holder at its lower end and is kind of hinged at its upper end. The scan piezos would act on the entire tube, incl the laser diode, in a plane between the tube's upper and lower ends. Whether or not your AFM system works exactly the same way I cannot tell for sure though.

Papain or other specific protease-functionalized gold nanoparticles may be useful.

Not without an impact on performance due to shared access to the PCIe bandwidth, but yet a reasonable practice.

It was really dependent of your system's resolution and your material used for calibration. For example, if the resolution is about 1cm-1 or larger, then your caibration has to be acceptable.

Practically, all main suppliers' products would give you a range of acceptable calibration wavelengthes instead of a single one for calibration. This will take, e.g. laser wavelength stability, material's Raman wavelength variability, etc. into consideration, in order to make your system in working order and reliable after calibration.

DNA fragment to be cloned must be isolated, you do with PCR or restriction enzymes isolated from bacteria, which are able to recognize and cut specific sequences creating "Sticky" or "Blunt" DNA ends.

The plasmid is digested with restriction enzymes, opening up the vector to allow insertion of the target DNA. If the isolated DNA of interest and the plasmid or vector are digested with the same restriction enzyme, their sticky ends will be complementary. The two DNAs are then incubated with DNA ligase, an enzyme that can attach together strands of DNA with double strand breaks.

Following ligation, the recombinant DNA is placed into a host cell like E. Coli, in a process called transfection or transformation with high concentration of calcium or heat shocks.

Finally, the transfected cells are then cultured but some may not contain a plasmid with the target DNA because the transfection process is not usually 100% successful and the appropriate cultures must be selected with markers usually for antibiotic resistance. When the treated cells are plated on a petri dish of nutrient agar containing the antibiotic, only the rare transformed cells containing the antibiotic-resistance gene on the plasmid vector will survive.

Further analysis of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of a process PCR or restriction fragment analysis, both of which need to be followed by gel electrophoresis and/or DNA sequencing. DNA sequence analysis, PCR, or restriction fragment analysis will all determine if the plasmid/vector contains the insert. Restriction fragment analysis is digestion of isolated plasmid/vector DNA with restriction enzymes. If the isolated DNA contains the target DNA, that fragment will be excised by the restriction enzyme digestion. Gel electrophoresis will separate DNA molecules based on size and charge.

I hope I was helpful Golla Ramanjaneyulu

Hi Anh Vo, sorry for the late answer. Virial is the sum of forces arising from bonded and non-bonded interactions whereas pressure is calculated from the kinetic energy of the system and the virial (refer to gromacs manual for details). Since stress in force per unit area, we consider only virial and not pressure.

Souparno Adhikary Optimal performance typically requires pinning mdrun lines to specific CPU cores and GPUs. You can think of it a sort-of "divide and conquer" approach. I'd recommend running your simulations in parallel on separate GPUs because crosstalk between them is likely slow.

I'm guessing here but I think you could run at least 4 simulations (2 per GPU) with that setup before you start losing performance. Here's an example part of an old script I use to run 4 simulations on a Broadwell node with 32 cores and 2 GPUs (0,1).

##########################################################

gmx mdrun -deffnm production -ntmpi 2 -ntomp 4 -gputasks 00 -pin on -pinoffset 0 -nb gpu -nstlist 120 -pme gpu -npme 1 &

gmx mdrun -deffnm production -ntmpi 2 -ntomp 4 -gputasks 00 -pin on -pinoffset 8 -nb gpu -nstlist 120 -pme gpu -npme 1 &

gmx mdrun -deffnm production -ntmpi 2 -ntomp 4 -gputasks 11 -pin on -pinoffset 16 -nb gpu -nstlist 120 -pme gpu -npme 1 &

gmx mdrun -deffnm production -ntmpi 2 -ntomp 4 -gputasks 11 -pin on -pinoffset 24 -nb gpu -nstlist 120 -pme gpu -npme 1 &

wait

#########################################################

I think the standard flags/options here are: "-pin on", -pme gpu, "-npme 1", and a -nstlist at/over 100. I'd start by benchmarking short runs with different combinations of the -ntmpi, -ntomp, -pinoffset, and -gputasks flags.

Triplex DNA hybridization is dependent a lot on the excess of the third strand if it’s an intermolecular triplex and on pH if it’s pyrimidine-based triplex. I have observed triplex formation of 24 Y-R-Y triplex (Y- polypyrimidine Strand) even in pure tris buffer (without any extra salt) however upon annealing. Without annealing, I have demonstrated that 50 mM NaCL was enough to form triplex With about 80% yield. The triplex was dissociating upon separation of the third strand as observed by a specific peak shape corresponding to multiple signatures (mobilities) in capillary gelbelectrophoregrams. Upon building up the ionic strength, proportion of triplex increased.

I suggest you to read my papers on Electronic pH switching of triplex reactions (https://pubs.rsc.org/doi/c5ra02628h) and

Arg Nε-Hε and at low pH Arg Nη-Hη and Lys Nζ-Hζ are visible in HSQC but these are aliased/folded peaks. Go for an open Sweep Width (in 15N dimension) HSQC where these peaks will appear at their exact 15N ppm values and you can identify them easily. If you go to the R/K side chains' 15N plane in CBCANH, you will observe CD and CG of R or CE and CD of K. The Trp side-chain usually shifted downfield and appear near the bottom left corner.

Start with the assignment, you will understand which one is the backbone NH because in CBCANH and CACB(CO)NH they will show only the CA and CB (these you have to identify based on their C ppm values). But the side chains will be different.

Best wishes.

"What could be the reason for not imaging DNA using SEM?"

In general, TEM and AFM have higher resolution and they are more suitable tools for such tasks. In addition, an AFM can operate in liquid and makes it possible to study mechanical properties of molecules at physiological conditions.

Depending on what your need is - if you are working with a structure that has been deposited in the PDB, you may want to use the sequence associated with that file in the PDB, which (usually) is the sequence of the construct used - including residues that are not resolved in the structure because of disorder. This sequence is also to be found in the SEQRES records in the header of the PDB file. If you need exactly the sequence represented by the coordinates, the servers listed by

parmbsc1 is an improved DNA parameters but not for protein. You can either use amber14sb or amber99sb. But I cannot see a good reason why these versions of amber will perform bad/differently to charmm36. Terminal base pairs are always flexible. I would suggest extending the length of the DNA by 3-5 base pairs to mitigate there effect on core DNA behavior. Can you post a vmd snapshot of the final structure?

before starting with trjconv, first make sure that your reference structure (must be tpr as Justin has suggested, tpr is required for -pbc mol) is also not broken (pbc fixed). I recommend using tpr used for energy minimization. Dump the gro/pdb to ensure that the protein-dna complex is whole. A possible work flow...

gmx trjcat prun.xtc -n index.ndx -o protein-dna.xtc (removing water first assuming water is not of interest)

gmx trjconv -s protein-dna.xtc -pbc mol -o nojump-protein-dna.xtc -s em-protein-dna.tpr

or

gmx trjconv -s protein-dna.xtc -pbc nojump -o nojump-protein-dna.xtc -s em-protein-dna.tpr

---that can fix most issues, if not then go with this

step1: gmx trjconv -s protein-dna.xtc -pbc whole -o whole-protein-dna.xtc -s em-protein-dna.tpr

step2: gmx trjconv -s whole-protein-dna.xtc -pbc cluster -o cluster-protein-dna.xtc -s em-protein-dna.tpr

output of step2 should fix even the most crazy pbc issues....

##### RMSD calculation for dna-peptide

better calculate the rmsd for protein and dna separately. Dump an index with protein backbone and dna-backbone and then use gmx rms

One more biomolecular-modeling.com/FF/index.html

Dear Richard,

Thank you very much for your insightful suggestions. I will consider to incorporate a positive control (sfGFP) and its amber mutant (150) into my study. I have another concern, does the media component will have a profound impact on the expression level? I have tried to use LB media as well, however it did not give a good result.

Regards,

Anshuman

The force spectroscopy with AFM is a really sensitive technique, have you have consider the measurement of the cantilever constant for each experiment?, and for each cantilever?. Have you have consider the temperature difference for each experiment?. Any way, If you already had been fixed this variables you always can try again to make a reproducible experiment. 

Dear S. Xavier

As we know that any molecule, be it protein or ligand, is made of atoms and atoms itself is made of nucleus (protons & neutrons) and electrons revolving around it S, P, D, F subshells.

Whenever we perform protein - ligand docking, what we basically check is the conformation of ligand with which it is binding to the receptor protein and try to quantify that binding energy between them using various force field equations.

Now, whenever the ligand interact with protein, at atomic level, it is the electrons which are being involved in formation of covalent or non-covalent bonding.

These pi-alkyl, pi-pi T shaped and pi-Sulphur interactions comes in the broad category of non-covalent interactions.

In pi-alkyl interactions there is interaction of pi- electron cloud over an aromatic group and electron group of any alkyl group.

In pi-pi T shaped interaction there is interaction of pi- electron cloud between two aromatic groups but in a T shaped manner, i.e, sidewise electron cloud of 1 ring and head on electron cloud of other ring.

In pi-sulphur interaction pi electron cloud of aromatic ring interact with lone pair of electron cloud of Sulphur atom.

Hope that will help you understand the above interactions.

regards

Tanuj Sharma

Prepare an index group with only the P atoms of your head groups. Feed that and the trajectory into gmx density. Compute the density along the bilayer normal (-d z) of the P atoms. Calculate the distance between the peaks in the density profile. This way you get the average P-P distance, which is used to describe bilayer thickness. (for example here: http://aip.scitation.org/doi/abs/10.1063/1.472323) You might want to increase the number of slabs and use the center and symm functions of gmx density.

In chemistry, 'like prefers like' and thus non-polar solvents (such as hexane/heptane) will tend to be miscible with non-polar components (and will contain very little free water). Similarly for polar solvents.  Now APTES is an aminosilane frequently for silanization of surfaces.  It's highly polar and thus will favor other polar solvents such as DMF, THF (as mentioned above), DMSO etc. This also means that it will favor moisture absorption as water is a polar solvent. Thus your request for the 'best anhydrous organic solvent' contains a dichotomy - you'll find a solvent that has little water content (e.g. hexane) but with have limited solubility of your material.  My suggestion is to take a suitable solvent (that actually dissolves your material in the correct quantity - [C] and spin rate govern the final thickness - and dry over anhydrous sodium sulfate or similar to reduce any water content.

Prefer using PBS buffer of pH 7.35  with approximately ionic strength of 0.5m NaCl, as the  interaction is strong at this pH. As the pH becomes lower, the interaction decreases.

Hope this works for you.

Best wishes

Hi Isaac Karimi,

Thank you for your reply to my query. I got the structure now.

Amber has good nucleic acid-specific force field corrections, especially those based on developments by the Orozco group (parmbsc0 in 2007, parmbsc0/OL15 in 2015, parmbsc1 in 2016), and most protein-DNA papers I see refer to this particular sets of parameters - it has become a standard to some degree, in the same way that CHARMM is preferred for lipid and perhaps protein simulations. So while it might not be perfectly obvious that one particular FF is in every regard better than the other, there seems to be a clear field-specific preference in terms of usage. Look up the linked paper for a more in-depth discussion.

In terms of .mdp settings, I don't think you need to make any significant changes to the simulation protocol when adding DNA to your system.

Hi Sunil,

You should try antechamber module in AmberTools, and ACPYPE.

Is there any particular reason you want to use fluxer.py?

I could recommend you some other tools:

-flux that will graphically show you where water is flowing

-the newest tool of my research group, AQUA-DUCT, that will trace water molecules and also gave you some statistic data

-CAVER 3.0 taht will gave you some geometerical data of the tunnel

Best regards,

Karolina

No. You need an electronegative atom attached to it to induce a dipole. For example, methane is nonpolar but methanol is polar.

There are implicit and explicit solvent models. In the implicit one, you do not actually define (polar) solvent molecules. Instead, a homogeneously polarizable medium is assumed and the main parameter used is the dielectric constant of the solvent. So, in such a model there is no distance cut off.

If you define polar solvent molecules explicitly near the motif, generally a distance cut off of 9 angstrom is used to consider dipole-diople interactions in the molecular mechanics calculations.

Hello Hina Ahmad, may I know what is the result?

first extract your protein from the trr. file using this command gmx_mpi trjconv -s prod.tpr -f prod.trr -o nojump.xtc -pbc nojump  . after that you can use the VMD input.gro nojump.xtc . after opening the protein go to Graphics and choose Representations .   Drawing method choose cartoon. Coloring method choose Secondary structure and run all steps. You can visualize changes on  your protein. DSSP tool also gives information about secondary structure evolution for each residue as well. 

While the distance seems to be a bit large, it is impossible to answer this question without knowing what kind of molecules are we talking about (proteins? other biomolecules? small organic systems?) and how is the distance calculated (the smallest distance between two atoms of molecules?).

Because in vacuum, protein structures are often unstable. Watch the trajectory and you'll probably see structural changes, or at minimum large oscillations in the positions of those atoms (which is what RMSF is directly calculating).

Hi,

although this is a very interesting topic, I think you have to specify your questions a bit more.

Which methods do you have at hand? Experimental or computational ?

What do you consider as "biophysical" ?

In principle, for biophysical studies, you should consult light scattering techniques (DLS, SLS, SAXS), neutron scattering, magnetic resonance (NMR, EPR), Rheology, Calorimetry (DSC, ITC), electron microscopy, force microscopy ... and many more ... so its too much to do it all at the same time ;). Simply pick up a good one, that you have at hand ...

In case you want to explore some computational methods I suggest to use YASARA Structure. With that program you get nice parameters quite easy. Unfortunately, it's not Freeware .. you have to check ... 😉

kindly,

Jörg

CHARMM General Force Field (CGenFF)

Soteras Gutierrez et al. (2016) Bioorg Med Chem 24: 4812-4825.

---

Not a Gromacs/CHARMM user.

Thus far, I have managed to find: The mfold Web Server

i though that you can get rid of  the interaction between protein with high pKa and membrane of E-coli by changing the pH of buffer solution used in extraction with best chances

As suggested by Benalla you should look for system requirement of the simulator you will use and specially :

is the chosen simulator compatible with Cuda?

is there a version for multi node (parallel version)?

Hi,

tipically commercial solutions for resuspending DNA are almost quite similar between each other. They are essentially slightly alkaline solutions (pH 8) with or without a cation chelator (tipically EDTA). The effects on downstream steps are difficoult to predict without knowing witch experiments you are running. I have to advise you that a buffer containing EDTA could reduce the yield of subsequent enzymatic reactions that often need metal cations. After DNA isolation using commercial kits, I usually resuspend DNA pellet in sterile water to avoid this problem and always worked well.

I have deposited amphiphilic peptides, spread as monolayers on water in a Langmuir–Blodgett trough, onto 1mm thick, 2cm diameter quartz slides successfully. You will need to stack a few together to get reasonable signal to noise. I measured these in grad school using a Jasco J500. Stacks were flipped by hand and accumulated scans added.

In order to make a tip-dip bilayr you need to be careful about buffer solutions inside and outside you pippette. Below please find a clip from Biosensors and Bioelectronics 22 (2007) 1605–1611, that describes tip-dip mebrane we use for functional reconstitution of many channels and receptors.

Lipid bilayers containing pure 1,2-diphytanoyl-sn-glycero-3- phosphocholine were formed on the tip of patch pipettes using the tip–dip technique. The bilayers were formed in asymmetric saline condition by the successive transfer of two lipid monolayers on the tip of the patch pipette. The pseudoextracellular solution (bathing the cis-side of the membrane) contained 125mM NaCl, 5mM KCl, 1.25mM NaH2PO4, and 5mM Tris–HCl at pH 7.4, while the pseudo-intracellular solution (inside the patch pipette) contained 110mM KCl, 4mM NaCl, 2mMNaH2CO3, 0.1mMCaCl2, 1mMMgCl2, and 2mM MOPS at pH 7.4. A 2:1 (mol/mol) mixture of 0.6 μg AmB and 0.12μg cholesterol dissolved in a chloroform–methanol (2:1, v/v) solvent was sonicated together with 10μg phospholipid (in hexane) and 10μL of pseudo-extracellular solution in order to form liposomes. The emulsion was then carefully transferred to the surface (air–water interface) of the pseudo-extracellular solution bathing the cis-side of the membrane and allowed to equilibrate for 1 min. Incorporation of AmB–cholesterol into the membrane was achieved by dipping the tip of the patch pipette into the emulsion.

Are you asking about "Hydrogen exchange mass spectrometry for the analysis of protein dynamics" e.g. http://www.ncbi.nlm.nih.gov/pubmed/16208684

Basically, the speed of hydrogen-deuterium exchange of protein amide protons is dependent both on the static and the dynamic solvent accessibility of the different amide protons. Mass spectrometry can be used to measure the time course of hydrogen exchange, while NMR is used to determine exactly which protons exchange in which timeframe.

Thank you both,  Maurice and Vladimir for your replies. The data are still quite discrepant, but I think I will be able to make something out of them.

Hi Vishal:

It's pretty easy. You know the molar ratio, and you also know the molecular weight of PbCl₂ and CH₃NH₃I, right? Now for example, if you use DMF as the solvent, take into account the density of it (probably 944 mg/ml). Then you can calculate how much of each material you need to blend by simple calculation.

Good luck!

Dilution heats are most often due to either buffer mismatch or ligand dissociation. In the first case the buffer composition (including pH) of the cell and syringe samples are different - the bigger the difference the larger the heat. The type of buffer does not really matter - the recommendation about PBS in the first answer actually refers to the buffer ionization heats (nothing to do with dilution). Best way to match the buffers is co-dialysis assuming both protein and ligand are large enough for that. In the second case - dissociation, the ligand forms oligomers (dimer for example) at the high concentrations in the syringe and these dissociate upon injection (dilution). This is an equilibrium process by itself and cannot be simply subtracted from the protein-ligand titration curve. How to distinguish between the 2 – do a control experiment, inject ligand into blank (buffer). The simple buffer mismatch tends to be a constant (or slightly sloped) offset that can be easily subtracted from the data. The dissociation will look more or less curved (depending on the diss. Kd). The best practice is to try to minimize the dilution heats by proper sample preparation. If this does not work you can try to subtract them from the data no matter how large they are – assuming mismatch I would do a linear fit of the control titration and subtract this fit from the protein-ligand titration. I am not sure how this type of analysis works in practice on the TA software, on the MicroCal (Malvern Instruments) data analysis this is very straight forward and in the case of the new PEAQ software it is automatic. Since you are dealing with low affinity sample I’d recommend the following white paper: http://www.malvern.com/en/support/resource-center/Whitepapers/WP140701-studying-low-affinity-fragments-of-ligands-by-itc.aspx . More general ITC method articles are:  http://faculty.missouri.edu/~tannerjj/bchem/fbdd-pdfs/perozzo-%20thermodynamics%20of%20proteins.pdf and the attached.

I also had the same doubt with fibronectin coating. You can put a small  glass coverslip or a round piece of parafilm on top of your droplet to make it flat an uniform.

just for self-citing:

Yes (as above) but there is more to it than that. It depends on what you are trying to achieve. If you are trying to mimic real life, then you need to know that proteins (if you are talking about bundles of transmembrane helices) do not sit well in the membrane on their own, the lipid molecules do not pack nicely round these bundles, That is why, in real life, you encounter cholesterol molecules which pack around the helix bundle and help to embed it into the bilayer. Crystal structures of membrane proteins often include cholesterol molecules that were on the surface of the protein and this helps you to place them correctly in your starting structure. You probably need a few more cholesterol molecules than the crystal ones, however. Nobody really knows how many. Another issue is that most lipid bilayer systems, certainly in eucaryotes are asymmetric - the head groups on the surface of the cell are polar but overall not charged while the head groups on the cystosolic side are negatively charged (which explains the "positive inside" rule). If you do not include all this in the system you are trying to simulate then you are really wasting a lot of CPU, because that is the way life is in reality and if you want to simulate it, you need to get these things right. Good luck !

If you take a look at P. G. DE Gennes' book (Chapter 10, page 511), you can notice that based on the analogy he made between smectic liquid crystals and superconductors, the magnetic vector potential for superconductor is analogous to small variation of smectic director in x and y direction. Therefore the vector potential is delta(n). Attached you can find extra info.

As the others pointed out, your situation is not really uncommon.

The interaction with the high affinity ligand is stabilizing the protein conformation that provides the best interaction energy. By doing so, it effectively quenches protein dynamics so even the folded truncated mutant looks "more folded". There is extensive literature on this kind of phenomena.

Regarding the protein getting folded upon truncation of the 30 aa tail, it is also possible that the very presence of the tail makes it difficult to detect at all the signals of the folded part in the full construct. Your experiments with the WT protein are at a much lower concentration, and here even some non-specific oligomerization promoted by the unfolded tail may easily make your signals too broad for detection. Keep in mind that the signals from the folded part are much broader than the sharp signals of the unfolded tail. Thus, in the WT protein spectra you should really be looking for apparently low intensity, but well dispersed signals. Instead, the unfolded region will be high intensity, poorly dispersed. You'll have to set your RG correctly and look for signals where you now expect them based on the spectra of the truncated form.

A way to measure peptide / protein orientation in membranes is by attenuated total reflection IR spectroscopy (ATR-FTIR).

You use a IR transparent crystal as support for a lipid mono- or bilayer and mount it into a flow trough cell. You pump in your protein solution. You measure the light reaching the detector after multiple (total) reflections on its way through the crystal. The evanescent wave developing at each reflection is decaying within the first micrometer enough to see the membrane and proteins inserted or attached. You check the spectra for the amide bands of your helices and/or beta sheets.

With some assumptions on the system it is possible to calculate an order parameter for the helices and/or beta sheets of your protein in contact with the membrane.

The proteins were designed with Rosetta so the proteins should be at a minimum in sequence space with respect to the Rosetta energy function. We showed this with one of the designed proteins made in https://www.researchgate.net/publication/258031760_An_Evolution-Based_Approach_to_De_Novo_Protein_Design_and_Case_Study_on_Mycobacterium_tuberculosis 

where the crystal structure of the designed protein has a lower energy in the force-field used to design the protein  then the native structure (D.Shultis, Y.Zhang, submitted). However, only 3 of the 5 proteins in that paper are correctly folded. The incorrectly folded and correctly folded proteins have similar energies. This means that our force-field is over-reporting the stability of designed sequences when they are designed with that same force-field. Although this is not Rosetta, the overreporting of stabilities when the same force-field is used for design and assessment is probably a general effect and also true for Rosetta and other design programs. 

What this means is that you need an alternative energy function to assess the stability that is at least orthogonal in some respects to Rosetta. The MD force-fields are a good indication here. If the MD indicates the natural proteins are stable and the design ones are not, you have fairly good evidence that Rosetta is over-reporting the stability of the designed proteins. If the MD shows the native proteins are not stable, MD is probably not useful, provided the native protein actually has a single, well folded structure (see below).

One important point. Your proteins are 20 amino acids in length. This means that they are actually peptides, which generally do not have a single well-folded structure. There are some exceptions though. 

We have unpublished theoretical calculations of bond dissociation enthalpy  of thiophenol at 298 K and it was 75-77 kcal/mole. We obtained this value several years ago using DFT calculations.

Near-UV CD and intrinsic fluorescence can be sensitive to tertiary structure changes and are easy to do. If you can make ¹⁵N,²H-labeled protein, 2-D NMR could be very informative. FTIR and MS with H-D exchange can also be used to detect conformational changes.

Justin, what do you mean with hard to find? Aren't we talking about the DSSP versions available from this side? http://swift.cmbi.ru.nl/gv/dssp/ Both old and new DSSP are offered for download there.

Hi Tarak,

The principle component is just a vector desribing the relative contribution of each c-alpha atom's x,y and z component to the correlated mode of motion corresponding to that component. (Assuming you calculated your covariance matrix using c-alpha atoms only). Thus the projection of a given simulation frame onto a given PC is just the dot product of the c-alpha coordinates of that frame with the PC vector. Often one will want to see the projection on the 1st and 2nd PC which results in a projection on a 2D plane: the coordinates in this plane are the dot product with the 1st PC on one axis and the dot product with the 2nd PC on the other axis. Does that make sense?

Pawel

Dear Karteek,

I think that you could try to employ the path collective variables (J. Chem. Phys. 126, 054103 (2007); http://dx.doi.org/10.1063/1.2432340) in combination with a free energy calculation technique like metadynamics or umbrella sampling. All of this is implemented in the plugin Plumed (www.plumed-code.org), compatible with several MD programs like gromacs, namd, lammps etc. Note that path CVs allows you a great flexibility by taking as input the whole set of atomic positions in each state (L/R). In addition, you may need to define some intermediate state, e.g., by linearly interpolating between them.

Hi John,

I would agree with Nicolas. The process of getting a good sample might take some optimisation, but it usually works. As long as you do not use the really large nano discs, you can feel relatively safe that you will only have one protein (monomer or whatever the native oligomeric state of your target protein is) per nano disc. There is simply not enough space in the smaller nano discs to fit much larger stuff in there. 

The gromacs tool do_dssp (freeware) uses the dssp program for a graphical output. In it's -cs output (scount.xvg) it gives you the secondary structure in the following form:

@ s0 legend "Structure"

@ s1 legend "Coil"

@ s2 legend "B-Sheet"

@ s3 legend "B-Bridge"

@ s4 legend "Bend"

@ s5 legend "Turn"

@ s6 legend "A-Helix"

@ s7 legend "5-Helix"

@ s8 legend "3-Helix"

# SS % 0.67 0.17 0.13 0.01 0.11 0.16 0.38 0.00 0.05

A diploma student of me accidently discovered such nanotubes on supported lipid bilayers. They are also formed in force-spectroscopy experiments on pore-spanning membranes (10.1002/smll.201101557). The tether force was independent of the tether length (force plateau) showed a "most probable" lifetime. In 2009 Daniel J Müller and coworkers published a nice review (10.1038/nchembio.181): The tether force is a measure of isotropic tension, bending rigidity and extension velocity (in combination with viscosity) of the cell membrane. Consequently you are right that the force depends on the diameter and when the properties of your membranes are known, you may calculate it (10.1038/nrm1784). 

The non-exponential decay of tether lifetimes indicates, that they to not simply detach from the cantilever/tweezers bead, but somehow collapse. This assumption is supported by recent theoretical calculations (10.1016/j.bpj.2012.03.048).

Hi Raed, you do not need use MOPAC before ORCA. Important is the right quantum chemical method. This paper can probably help you to select of a method attending your molecules. (Assessment of quantum-chemical methods for electronic properties and geometry of signaling biomolecules. DOI: 10.1002/jcc.21393). This paper find out some methods with minimal variation with respect to experimental data.

Electrophoretic separation in agarose is not just based on molecular weight and charge. The agarose forms a sieve and large structured molecules can become entangled in the pores of the agarose gel particularly if they are irregularly shaped. This is why DNA electrophoresis is generally carried out using denatured DNA. This allows the DNA to form more linear structures, that are less likely to be physically held up by the gel structure. 

It s not clear from your coments exactly what type of electrophoresis you are carrying out (other than it is DNA and on agarose of course) and the relative sizes of the scaffold to the origami  (though I believe the scaffold is generally many times smaller than the origami which would support this hypothesis) so I am not sure if this advice is relevant to your situation. But you could denature the DNA or move to acrylamide with very large pore size structure (low cross linking) in the hope that both molecules would be freer to move. I hope this helps.

You can find some information in the paper "Evolution in a family of chelatases facilitated by the introduction of active site asymmetry and protein oligomerization." (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017170/ )

Apologising for stating the obvious, but I do get frustrated when people start mixing quantum into very inappropriate settings. This is especially common in non-traditional medicine areas: I am sure people just want to use the word "quantum" to create an impression of complexity -- or sometimes genuinely mix up the (entropic) stochasticity with quantum uncertainty.

There is a basic criterion for the physical situation where quantum effects are relevant: this is when wave functions of interacting objects overlap. It first emerges in the statistical mechanics of ideal gas: the ratio N*L^3/V has to be >1 for the quantum effects to matter. Here N/V is the density and L is the de Broiglie thermal wavelength (square root of the ratio hbar^2/m kT). The de Broiglie wavelength of even the lightest Na+ ion at room temperature is about 0.1 Angstrom (according to my quick estimate just now), so it is much smaller than any of the distances involved in a membrane (or even membrane channel). So the answer is simple and inflexible: there are no quantum effects relevant in this, and most other biological processes.

Take a look at this option: http://einstein.cs.iastate.edu/PSHOMPPI/index.html

See this link for more information about the SUMO protease necessary to remove your N terminal SUMO fusion partner (leaving a Cys as your first residue if you clone it that way)

Yes. There are two methods, often used concurrently:

1) capacitance compensation circuitry

2) P/4 subtraction

Both serve to eliminate the capacitance charging transient, so that you can study the ionic currents alone.

The first approach is built into most modern commercial patch clamps. The capacitance charging transient is the current that flows in response to a voltage step applied from the patch pipette to the interior of the cell. The current enters the cell via the narrow aperture (so-called "series resistance") where the circular rim of the pipette connects to the cell interior, in whole-cell patch clamp configuration. Normally this current is applied by the measurement circuitry, so it is recorded along with any ionic (transmembrane) current. When the capacitance compensation circuitry is used and a voltage step command is applied, it is the compensation circuit that applies the current needed to charge up the membrane capacitor, relieving the measurement circuitry from having to supply that current. This results in the measurement circuitry reporting only the ionic current, without the capacitance charging current.

The second approach is based on the fact that the membrane capacitor roughly approximates a linear capacitor, which means that the capacitance transient that flows to charge the capacitor should have the same shape, but differ in size in proportion to the voltage step size. Simply put, to use this approach, one applies one or more small voltage steps -- too small to activate voltage-gated ion channels, and adds them up to get the predicted full sized transient -- and subtracts that full-sized transient from the current transient that results when a full-sized voltage step is applied. In the case of applying a full-sized voltage step, the current transient is the sum of both the ionic current and the linear capacitance transient, the latter of which can be subtracted out.

I will add some references later...

I am puzzled by this discussion, but maybe I have missed something. Two quite different issues seem to be being discussed. One is whether or not thoughts or ideas could be inherited genetically through DNA, in the way that we accept that behavioural traits like birds singing can. I would have thought this was fairly obviously the case. The other is whether thoughts could be transmitted in a Lamarckian fashion, where the thinking of thoughts by parents could encourage immediate offspring to have more of those thoughts via an effect on DNA. This is generally agreed to be totally implausible and unsupported by any evidence.

Inheritance of ideas or thoughts cannot be unexpected if we accept that ideas reflect the dynamics of brain function, just as bird song does. A human being cannot have a thought of a red tomato unless it has inherited neural mechanisms that support such a thought. You may need a tomato, but then birdsong needs springtime; none of these things need to be considered in the absence of any external stimulus. And thoughts like 'I'm tired' do not need anything very external. We inherit the having of that thought.

There are complexities to this but a number of people, including Plato, Leibniz and Jerry Fodor, have suggested that all our concepts are there at birth. This is entirely feasible if the brain works like the immune system, where we have antibodies to every possible germ at birth (or at least we make them within 3 months). These systems are preset to be able to recognise anything that comes along. There is also later fine tuning in both so Fodor may have been exaggerating when he said that all babies are born with the concept of carburettor. But we are almost certainly all born with the idea of a circle, I would suggest. The mechanism is that of setting up connectivity in the brain that mediates recognition of certain types of dynamic invariance through differentiation processes - all pretty standard Hubel and Wiesel type stuff.

probably, the vector has been lost. You can use a univeral primer to sequence the clone and verify it.

Charmm force fields can be used in CHARMM program, but also in other programs such as NAMD, Gromacs, etc.

About the force field you can read more in the CHARMM software documentation site:

More about the force fields and downloading them (including references) you can find in this link

To accurately assess the contribution of water release due to burial of hydrophobic surface you will have to define the binding induced change in heat capacity, i.e. the temperature dependence of binding enthalpy. (See Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. USA 74, 2236-2240, and Spolar, R.S., Ha, J.H. and Record, M.T. (1989) Proc. Natl. Acad. Sci. USA 86, 8382-5)

Sturtevant outlines the biochemical rationale for observed changes in the heat capacity and Spolar and Record empirically define the effects of hydration change on observed heat capacity. Since then other groups have presented minor modifications to the Spolar and Record calculations.

Without understanding the temperature dependence of the thermodynamic parameters of the binding interaction all you can do is wildly speculate on the chemical rationale for the ITC-derived values.

Also, you must take into account all linked thermodynamic equilibria such as conformational and configurational changes and binding induced changes in protein ionization, which will contribute to your measured thermodynamic parameters.

Hi Sabya,

Thanks for your reply and nice explaination. But I think I'm looking for a more physical view of the lipid bilayer. I understand phosphatidylinositols are essential lipids mostly because they can interact with different peripheral proteins, and spontaneous curvature arised from protein-lipid interactions can be very complicated as you said.

But if we only look at the lipid bilayer, I think the shape of inidvidual lipids can also be interesting, at least biophysically. For example, the shape or spontaneous curvature of lipids will determine the prefered curvature of the monolayer. When two monolayers form a bilayer, the pressure profile should be somewhat related to the original shape of the monolayers. And the pressure profile across a lipid bilayer could also influence the insertion properties of proteins. The spontaneous curvature of different lipids in the wikipeida page such as for DOPE and DOPC, are mostly measured by x-ray diffraction from lipid hexagonal phases. So it should be a pure physical parameter of this lipid species, but again I agree with you about how protein-lipid interactions can make this much more complicated and also more meaningful.

Best,

Zheng

It also contains an aspartate and histidine in the triade which are bothdependent on pH and salt. The stability of the oxy-anion is potentially the most important step in hydrolysis transitiion state.

I would imagine that in systems where refractive index is utilized to measure the density or viscosity of a material there would be a direct correlation between T1 and T2 measurements obtained on the same materials. We have also found that PLS regression of NMR spectral variability, example would be in heavy vacuum gas oils in petroleum industry, correlates well to refractive index which is typically used to determine aromaticity (% carbon aromatic content ). The NMR experiment readily observes the relative amount of either aromatic protons or aromatic carbons in the sample that can then be correlated to refractive index.

What about GLEEVEC/imatinib to inhibit Abl kinase for the treatment of CML leukemia?

Developing the Synergy between Biophysics and Medicinal Chemistry to Deliver Better Drugs

First event: Strasbourg, France - October 15-18, 2013 www.novalix-conferences.org

Next: June 2015 to be announced

“Biophysics in Drug Discovery” is a core strength of NovAliX but is often relegated to the status of a side topic at other events. The NovAliX conference is an externally professionally organized event open to all, gathering together cutting-edge industrial and academic practitioners and suppliers spanning both biophysics and medicinal chemistry.

The inaugural event in 2013 covered:

Biophysical characterization of targets

Biophysical methods for identifying hits & optimizing hits into leads