Science topic

Alanine - Science topic

A non-essential amino acid that occurs in high levels in its free state in plasma. It is produced from pyruvate by transamination. It is involved in sugar and acid metabolism, increases IMMUNITY, and provides energy for muscle tissue, BRAIN, and the CENTRAL NERVOUS SYSTEM.
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I have some interest in the interaction between protein A and B but I barely know about proteomics so I leave the question here.
To specify the exact interaction sites, I made four site-directed mutated plasmids having a GST tag using the quick-change method. The mutated sites are on the cold shock domain of protein B. Because I read this phrase "systematic alanine scanning mutagenesis has revealed that the substitution of an amino acid residue by alanine in these hot spot regions lowers the binding affinity by at least 2 kcal/mol (Bogan and Thorn 1998).", I changed every mutagenesis site into alanine.
And then I did a GST pull-down assay after co-expression of MYC-A and GST-B (treated with RNase, DNase, and MNase). This is the question. I could not understand the results I got from this experiment. The affinity between protein A and mutated B(all four mutations!!!) is so much higher(>100 folds) than that between protein A and wild-type protein B. They interact much stronger via these mutated sites. Do you have any idea what it is? And can it be a clue for finding exact interaction sites?
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Reading about biotechnology database and bioinformatics directory in this article may help you find your way through your research
This article published by Stanford university
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Can we also count, say the total number of alanine residues in a PDB file or any other other residue for that matter?
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Hi Prathvi,
In Chimera you can go to Tools -> General Controls -> Command Line
Here you can pass the command "select :HOH", which will select the water molecule and at the bottom of the window you can see the selected atom numbers too.
Hope this helps
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i am doing a point mutation of tyrosine to alanine leading to 3 alanine in a row in the peptide sequence.
the structure before mutation is alpha helix, will the mutation may lead to disruption of the alpha helix?
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As a matter of fact, according to: Pace, C. N., & Scholtz, J. M. (1998). A helix propensity scale based on experimental studies of peptides and proteins. Biophysical journal, 75(1), 422–427. https://doi.org/10.1016/s0006-3495(98)77529-0Alanine has the highest helix propensity. In other words your helix becomes even more stable.
An indication can be obtained by using a secondary structure prediction program like:
Just changes the FASTA form of your sequence and see whether a drastic change is to be expected or not.
Best regards.
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When I tried building the peptide sequence "KEPAPTT" on Pymol, I get the following error after trying to add alanine:
PyMOL>editor.attach_amino_acid('pk1', 'lys')
PyMOL>editor.attach_amino_acid('pk1', 'glu')
PyMOL>editor.attach_amino_acid('pk1', 'pro')
PyMOL>editor.attach_amino_acid('pk1', 'ala') Selector-Error: Invalid domain selection name "_tmp_editor_dom". Selector-Error: Invalid domain selection name "_tmp_editor_dom". Selector-Error: Invalid domain selection name "_tmp_editor_dom".  Error: Invalid input selection(s)
I cannot find any information on why this error occurred. If it helps, the way I tried to build this peptide is simply pressing Alt K, Alt E, Alt P, etc. under the 3-button editing mouse mode, which seemed to work perfectly fine in tutorial videos I found online. Does anyone have any information on why this error is occurring?
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You can use Maestro to build your peptide from sequence.
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I want to look at all my alanines in my protein chain and want a selection in VMD just for this. I don't know what their resID numbers are but want to be able to select them all by the fact they are all alanines.
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Type resname ALA
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First, I synthesized by SPPS with Fmoc strategy the linear peptide : D-Glu(OtBu)-L-Lys(Alloc)-D-Glu(OtBu)-L-Ala-D-Glu(OtBu)-L-Lys(Alloc)-D-Glu(OtBu)-L-Ala. After cleavage from the resin (2-chlorotritylchloride resin), the linear peptide was cyclized in PyAOP/DIEA/DMF. The cyclic peptide was obtained but cyclization was not completed so some linear peptide was left. Then I deprotected all OtBu of the side chains with TFA/H2O, and I obtained a mix of 3 products : deprotected linear peptide, deprotected cyclic peptide and a product with a mass of +71 Da compared to the deprotected cyclic peptide in LC-MS (it could be a +53 Da compared to the deprotected linear peptide). I suspected that it was a problem of Alanine so I changed the 2 Ala by 2 Leu in the sequence, but I still had this +71 Da. I also changed the conditions of cyclization but this +71 Da product was still here. Could it be a reaction on the double bond of Lys(Alloc) ? Does anyone has a clue ? Thank you !
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Which dipeptide has 71 mass? which aminoacid with amide group has MW71?
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Hello to everyone. We have a protein structure and we suppose there is a hinge zone. We did an eLNeme run and we obtain a moviment in this zone. For the paper we want to demonstrate that this residues compounds a hinge region. We are thinking to mutate an Alanine and Threonine to Phenilalanine to increase the hydrophobic cluster in this zone and stop to displacement. But we dont know if this experiment will run well ... So if anyone knows an experiment (if it is possible not using monoclonal antibodies)?
We are thinking that the hinge region are (more less) 7 residues.
Thank you so much
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Feel free to ask any questions as I am the first author.
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I am preparing L-Methyl 2-(3,5-dinitrobenzamido)-3-phenylpropanoate. I checked the CD spectra of L- phenyl alanine methyl ester and L-Methyl 2-(3,5-dinitrobenzamido)-3-phenylpropanoate. The amino acid ester shows a peak at 218 nm in ethanol, but no peak for the later product.
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Adel Amer Thank you
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As I checked some papers like the following one, the authors consider an amino acid as hot-spot if mutation to the alanine change the DeltaDeltaG > 2 kcal/mol.
My question is what about DeltaDeltaG < -2 kcal/mol? If a mutation change the DeltaDeltaG less than -2 we can consider that amino acid as hot spot?
Thank you,
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The hotspot of a protein can be defined as the single most amino acid responsible for binding of incoming molecule. This is newly developed concept coming out of CARd analysis from carbon value. According to CARd one, amino acids mostly aromatic one are responsible for the binding and also available for effective locking by adjoining portion by forming internal COD. Effective mean carbon distributed according to principle of cohesiveness of arising from carbon score of 31.44 percent.
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If yes, how to eliminate remaining KOH when the Schiff base has formed?
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Salicylic aldehyde with dissolved in ethanol alanine dissolved in aqueous solution and mixed and added ethanol sodium hydroxide to form Schiff base whether possible give suggestion
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Are there any validated data adressing the efficacy / efficiency of :
1. natural treatments (e.g. Zinc, pygeum africanum, urtica dioica, alanine, glutamic acid, glycine; selenium etc)
2. and / or antiflammatory drugs (eg.diclofenac) to treat benign prostatic adenoma?
3.Association of both approaches
Thank you for your responses
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Please go through the following RG links.
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Hello researchers,
Is PROCHECK the correct tool to check the stereochemical quality of small peptides? My peptide of interest: polypeptide with 16 alanine residues.
Many thanks,
Neena
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Dear Neena,
to me it is not clear what you want to do. Do you have NMR data (spectra, chemical shifts, 3D structure calculated...) which you want to use to check for enantionmeric purity of your 16-Ala peptide? My experience tells that it will be nearly impossible to rule out a complete epimersation of one (or several) peptide bonds conclusively. If you have too many NMR signals (side-peaks) their presence will tell you that side-products with wrong stereochemistry are likely, but it still will be very difficult to decide on details of the wrong stereochemistry.
Kind regards
Alfred
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Hi
I am calculating the rotational constants of L- alanine with different basis set using HF and B3LYP method, I found 3 rotational constants and I don't know what it corresponds.
Anyone can help
thanks
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Rotational constants are related to the moment of inertia of the molecule about the three rotational axes. The dipole moment vectors indicate the dominant direction in which the partial charges in the molecule are distributed. I assume you know this already, so could you please elaborate on what you are trying to find out? Thanks.
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Raised alanine aminotransferase and alkaline phosphatase level due to drug induced liver injury is resolved spontaneously in some cases through a process of adaptation. What is/are the underlying mechanisms for these adaptations? Is it due to scavenging effects of dietary antioxidants?
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Depending on the duration of injury and the histological location of damage, drug-induced liver injury (DILI) is categorized as acute or chronic, and either as hepatitis, cholestatic, or a mixed pattern of injury. The hepatitis pattern is characterized by hepatocyte necrosis and is associated with a poor prognosis. There are three types of acute cholestatic drug-induced injury: bland cholestasis is the result of abnormal biliary secretion, and is not accompanied by significant hepatocellular damage; cholestatic hepatitis (mixed type) refers to cholestasis with concomitant hepatic parenchymal damage; and the third form of acute cholestasis is defined by the presence of bile duct injury or cholangiolitis. Medications may cause chronic cholestasis through two additional mechanisms: through the obliteration of bile ducts, also known as the vanishing bile duct syndrome, or by extrahepatic biliary obstruction, known as secondary sclerosing cholangitis.
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For example purity of amino acid leucine/ phenyl alanine/ serine etc.
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I am confused about what it is exactly you are trying to achieve. The Sørenson formol titration, as I understand it, is used to determine the protein content in a sample. How can you use it to determine the purity of an amino acid? For example, if your contaminant was another amino acid it would titrate in the same way as the pure amino acid (https://en.wikipedia.org/wiki/S%C3%B8rensen_formol_titration). Do you know anything about the origin of the sample and its likely contaminants?
It is pointless to use a method which is not fit for purpose.
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Hello researchers,
For MD simulations of a peptide in vacuum, I want to find OPLS force field compatible parameters for modified alanine residue in the peptide (with the carbonyl oxygen protonated). Such a protonation site is possible in gas phase.
I was wondering do I use the mktop program suggested by gromacs?
Thank you,
Neena
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manually editing the topology.
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I would like to separate L-phenylalanine from D-phenylalanine as well as L-alanine from D-alanine. The problem is that we don't have a reversed phase chiral HPLC in the lab and the regular chiral HPLC doesn't separate them.
I tried to derivatize the amino acids to inject in the chiral GC but it didn't seem to work... no separation, I tried many concentrations/conditions...
Any suggestions/experience?
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Hi Anna,
You can separate them using polysaccharide-derived chiral stationary phases (Chiralpak IA, IB, IC) as fluorenylmethoxycarbonyl derivatives or nitrobenzoxadiazole derivatives. Please see these papers:
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My wild type protein is of 70 kDa (tagged to EGFP) and the mutant protein with one amino acid change (Lysine to Alanine). The mutant protein band is positioned considerably at higher position than the wild type. The 1st lane is of mutant protein whereas the rest of the lanes contain different concentrations of wild type protein.
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If you have any tag GFP, STREP, His it will save time for your project. As you can see the picture, you still see something sticked to the well. Your protein will be denatured or not by using SDS - it is not possible to directly conclude it without checking it. SDS will help samples to enter the lanes, and if used in the right way it would be mild enough for fluorescence detection. When we make SDS-PAGE gels, we add SDS means we can decide the percentage of SDS to make our gel- you can check 0.1%, 0.5%, 1% and 2% .
It was found that GFP is stable even with 1% SDS, check some papers -
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Hi all.
A similar question to this has been asked several times, however there is subtle difference.
I would like to change e.g., an alanine to a glycine (or any amino acid) in a .gro file AND to the corresponding .itp file (which means all bond atom numbers of coordinations must be updates).
Going back and forth using pdb2gmx does a great job for 99% of the protein but it doesn't put in a new set of atoms for the substitution and it sometimes proves difficult given the number of little bespoke changes I need to make to this heavily post-translated modified protein.
I've yet to find anything that will deal with both .gro and .itp files. Any suggestions would be appreciated.
Thanks
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Anthony Nash you should try with pmx module from Daniel Seeliger (https://github.com/dseeliger/pmx). pmx is a toolkit for free-energy calculation setup/analysis and biomolecular structure handling. With this module is perfectly possible to generate a dual topology (in this case alanine/glycine) for your system and run the proper simulation either with glicine or alanine on your protein. For instance, suppose alanine correspond to the state 1 and glicine to state 2. In the .mdp file you can control which of these aminoacids will appear in the protein by changing lambda parameter (i.e. lambda=0 --- alanine, lambda=1 --- glicine)...
only one question: you want to change alanine into glycine accross the MD simulation or simply you want run two different simulation (i.e. one with alanine and other with glicine)
hope this helps!
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I have a question regarding which residues should I mutagenise into. By using bioinformatics method, I have identified some putative amino acid residues (Isoleucine, Methionine and Leucine) that may mediate this interaction.
However, the problem is that this interaction is potentially a hydrophobic interaction. Normally one would mutagenise something into Alanine to avoid side effect on the protein conformation. But Alanine itself is slightly hydrophobic, so I'm not sure if doing this can answer my question?
I can mutagenise it to something hydrophilic but I'm not entirely sure if this is a good idea because this can potentially change the protein conformation.
So what is your suggestion?
Any help would be appreciated.
Thank you
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Dear Ann
there are examples of mutageneiys applied to hydrophobic surfaces of dimeric protein to induce monomerization.
For example in the past i worked with Human Cu,Zn Sod1 and for this proteins there is a characterized F50E/G51E/E133Q mutant ) that is monomeric but active and structurally similar to the dimer.
If there is the structure solved for your dimeric protein you can try to predict the effect that your hypothetical mutation will have on protein stability with bioinformatic tool. A simple (but in my experience powerfull tool) is POPmusic ( ) that is now available in DENZIME platform (https://soft.dezyme.com/). I used it in the past to design some stabilizing mutation with good result.
However found the right mutant it is something not obvious and probably you will need to screen a panel of mutants and you will need to set up a protocol to check the protein stability/or activity. In this case i reccoment you PIPE CLONING approach for rapid generation of single point mutant with out the use of any specific mutagenesis kit.
A possible tool to check how much the mutation will affect the protein stability is in my opinion the DSF but of course only structural characterization or a positive result in a activity assay (if the monomer could be active ad the dimer) will prove you that mutation alter only the oligometizzation state and not the monomer folding,
Good luck
Manuele
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I am doing crystallization of a protein. I have tried many screening kits but couldn't get crystals. The theoretical pI of protein is 5.9 and the pH of buffer I am using is 8.0 (20mM tris-hcl, 100mM NaCl). I also tried mutations of some lysine and glutamate to alanine, methylation of lysine, deletion of some residues that are predicted to be disordered. However, none of these produced crystal. The percentage of glutamate and lysine in the protein is 9.9% and 7% respectively. The molecular weight of protein is around 55 kD. I have also tried crystallization screening with the substrates as well as substrate analogue of the protein. Still no crystals. I am getting precipitate, phase separation or clear drops. Is there anything I can do to get the protein crystallized? or is it the buffer I am using is not good? or is there any method that I can use to check whether the protein can be crystallized or not? Thank you...
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Hi there!
I was going to write agreeing with the other people around here, but seeing as you already have some initial hits, perhaps my experience with optimization will be useful:
1. Finely tune the components of your condition. For this, perform a systematic screen. Say for example your condition is 0.1 M Buffer pH 8, 200 mM CaCl2 and 10% PEG400. In that case, I'd test from pH 7 to pH 9, from 100 mM to 500 mM CaCl2, and probably from 5% to 25% PEG400.
2. Screen for PEG length. The number behind the PEG corresponds to the average molecular weight in the PEG mix. I'd suggest you test slightly larger, and slightly shorter PEGs. So, in your case, PEG400 gave initially good results; test what happens with PEG200 to PEG600, for example.
3. Seeding! I've had a lot of success using initial crystals as seeding stock. In that case, I do both microseeding and streak seeding (owning cats is a requirement for any self respecting crystallographer, in my humble opinion).
4. Seeds are good in any condition. What I mean is that I've had it before where my initial crystals grew in condition A, I processed them into seeds, and after using them as an additive for robot assisted crystallization, my good, final crystals appeared in condition B.
5. Now for a bit of self promotion. In 2014, we published a paper in Angewandte Chemie describing the use of crown ethers as crystallization additives to help stabilize otherwise disordered lysines and hydrophobic patches. The reason we started with this project was because we noticed that, at times, short PEGs (like PEG400 or PEG200), co-crystallized with proteins in this ring-like C-shapes. Of course that's very similar to a crown ether, and indeed, we saw that, in many cases crown ethers can act as an improved version of short PEGs. I'd recommend you check out the paper (Lee, CC & Maestre-Reyna, M 2014, Angewante Chemie), and see if it may be useful for you.
Good luck!
Manuel
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Hi
we mutated a tyrosine residues to alanine near the active site of an enzyme, which resulted a 3.6 fold increase in KM and 3.75 decrease in catalytic efficiency (kcat/km) as compared to wild type enzyme but the kcat and vmax is almost same to that of the wild type.
is it possible that mutation can only affect the binding affinity of the substrate and show no significant effect on turnover number or Vmax ?
if it is possible then how someone can explain the reduction in binding affinity by mutating the tyrosine to alanine near the active site ?
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Hi there,
Yes it is perfectly possible if the residue is stricly involved in substrate binding and has no catalytic role. In your case phenol side chain of tyrosine can contribute to both hydrophobic (stacking) and polar (hydroxyl) interactions with substrate whereas alanine can not as its methyl side chain is only hydrophobic but much smaller than phenyl ring of tyrosine.
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Hello i am looking for causes of hyperlactatemia (energetic disorders) with secondary hyperglycinemia (2 times its normal value) , PDH and PC deficit dont fit the profile nor NHK or other causes of hyperflycinemia (normal ketones, normal organic acids); other findings include low glucose (50 mg/dl) and silghtly elevated alanine, isoleucine and leucine, normal renal function. History of seizures, uses Valproic Acid.
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hipofisis tumor?
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I performed deletion mutagenesis along the c terminal region of a protein targeting two specific leucine residues, and showed no change compared to wild type, as assayed by dual luciferase-renilla. However, when the leucine residues are substituted with alanine, there is a significant change. Alanine is smaller non-polar amino acid compared to leucine, but I'm having difficulty understanding why this would yield a significant result compared to knocking out the residues all together...
Additionally, these residues are on a C-terminal alpha-helix, followed by a region of intrinsic disorder, before protein end.
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Hi there,
What are the residues immediatly downstream the 2 Leu? Are they similar (ie. bulky hydrophobic residue)? In this case deletion might not be deleterious because the immediate neighbouring residue is able to play the same role as the Leu within the structure. Can you modelize the helix and the impact of deletion on its overall structure? In case of alanine, methyl side chain would be too short to efficiently replace Leu.... BTW what do you mean by "significant change" about the ala mutants? Is it about expression/stability of the protein or about it's function?
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Hi,
I am willing to mutate 5 specific consecutive amino acids to Alanine in a certain insert using site-directed mutagenesis in one PCR reaction only. I'm scanning a certain area of my protein to see wether it is involved in an interaction and I want to go 5 mutations, if not another 5 etc... Did anyone try it before and worked or are there better alternatives? I've read several publications but they're mostly for high throughput scanning and random mutations.
Thank you
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I don't think there's a way to mutate 5 consecutive amino acids in one PCR reaction. The main reason being primers. You intend to mutate 5 consecutive amino acids, which means 15 consecutive nucleotides. The minimum requirement for good primer annealing is 20-22 nucleotides with more than 90% complementarity. In your case, if you want to introduce substitution mutations in 15 nucleotides, you decrease the chances of primer annealing at the site of your interest because the primers would not be entirely complementary. Also, you might increase the chances of non specific primer annealing at multiple sites in your plasmid resulting in non specific amplification.
There are kits that are available from NEB and Stratagene that allow multiple mutations at multiple sites but far from one another, but not next to each other. You can check their websites for further information on these kits.
The more reliable and sure shot method of SDM is sequential mutations at a single site. First round, mutate one amino acid, then mutate the second one on the already mutated clone and so on till you mutate all the five.
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I want to couple the amino acid with Alkyl chains by Decarboxylation of amino acids.
my questions are
1. can we remove the carboxylic acid group having Pka ranging from 3-4.5
2. how to prevent the Alaninate and ester formation which are the major side product.
3. can we carry out Decarboxylation in pure water as solvent
4. can we get the Decarboxylated product at room temperature using photocatalyst (and which kind of photocatalyst would be best)
5. how can we inhibits the byproduct formations i-e ester formation?
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This is a fairly new paper in very high end journal which could allow you to couple a carboxylic acid and an alkyl halide together by decarboxylating the carboxylic acid into an alkyl radical. The reaction requires Blue LED light (we bought ours from Amazon), an Iridium photocatalyst, and a Nickel cross coupling catalyst.
They actually use a lot of amino acids in their substrate scope so I think this would be very helpful to you.
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I want to do an MD simulation of a dipeptide which contains two alanine and in one of the alanine in place of the methyl group a CH2CN group is present. I have attached the .gro file of my structure here. I am using OPLS-AA force field. But when I am running grompp I am getting the following error-
NOTE 1 [file minim.mdp]:
With Verlet lists the optimal nstlist is >= 10, with GPUs >= 20. Note
that with the Verlet scheme, nstlist has no effect on the accuracy of
your simulation.
Setting the LD random seed to 1981633587
Generated 332520 of the 332520 non-bonded parameter combinations
Generating 1-4 interactions: fudge = 0.5
Generated 332520 of the 332520 1-4 parameter combinations
ERROR 1 [file topol.top, line 152]:
No default Angle types
ERROR 2 [file topol.top, line 188]:
No default Ryckaert-Bell. types
ERROR 3 [file topol.top, line 192]:
No default Ryckaert-Bell. types
ERROR 4 [file topol.top, line 196]:
No default Ryckaert-Bell. types
I believe this is because in my ffbonded.itp file there is no definition of angle type and dihedral type for the following combination of atoms -
CA CB CZ
N CA CB CZ
HA CA CB CZ
C CA CB CZ
Therefore, my question is, from where can I get the parameters needed to edit the above-mentioned angle type and dihedral types in my ffbonded.itp file? I have attached my topol.top file and ffbonded.itp file here. Any suggestion regarding this will be highly appreciated.
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AMBER requires the same approach as any force field - one needs to generate appropriate parameters. AMBER99sb doesn't automatically support this residue, either. One would have to proceed through a parametrization protocol, presumably aided by antechamber or other utilities, and then still verify that the generated topology (which by default may be a combination of standard protein and GAFF atom types, which may not be preferred) is valid. It may be "easier" to generate a topology this way, but it is always difficult to verify and refine it.
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i mutanted the  cysteine to alanine, but the protein is inclusion body. why the protein is inclusion body which is soluble before the mutant? And is  there some other amino acid is similar to the  cysteine except  to be Hydrogen receptor?
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Hi there,
If the cysteine is implicated in a cystine bridge necessary to stabilize the native structure of the protein then the mutation prevents the bridge to occur and therefore may be the reason for the protein not to fold properly and finally aggregate in inclusion bodies. A more neutral mutation from a chemical content point of view would be to replace Cys by Ser but even though cystine bridge wouldn't occur in this mutant. If it is only about hydrogen bonding serine should be able to replace efficiently the cysteine residue.
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How to dissolve 4-borono-phenylalanine close to physiological pH? It usually dissolves in solutions above pH 10! Any suggestions or thoughts are welcome!
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Thank you all and we did it by increasing the pH to 10 in fructose and readjusted the pH to near 7 by HCl.!It worked fine..
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Can I mutate serine to leucine to mimic de-phosphorylation?
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I concur with Huanting Liu. Ser to Ala mutation would be better approach and the reviewers will be fine with that when you submit that work for publication. You may also also create Ser to Asp (S to D) mutant which is a phospho-mimicking mutant and might give you a good phenotype (if you get any!).
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Hello everybody,
I've done some literature search on alanine metabolism. I always found it as some kind of "garbage can" for ammonia in cells other than hepatocytes (like in the physiological glucose-alanine cycle between muscle and liver).
Now I wonder if there is another function for alanine biosynthesis beside depleting ammonia from the cell or for simple protein synthesis. Furthermore, I would like to know if there is evidence for other alanine biosynthesis pathways beside glutamate-pyruvate transamination by GPT/ALT in animal cells (I found at least three in E.coli).  
Does anybody know some literature or is perhabs an expert on his/her own in this topic?
Thanks a lot,
Maren
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Interesting paper in Nature this year (2016, 536 (7617): 479) on a role of alanine produced by stellate cells in supporting malignant growth (fuels TCA, Non-essencial AA bio, and lipid biosynthesis in pancreatic cancer cells).
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Normally I have seen mutation to reduce binding of any residue to Alanine or Glycine (since the short size abolished binding). But I found a paper for hCypA where they do mutation of the Histidines to Glutamines. Is this a regular approach?, I would understand that maybe for conserving the structure is need to have a NH, but wouldn't be similar for Argenine? or other residues with NH in side chains?. Also in the structure I don't see salt bridges and it is for two Histidines.
The paper doesn't say why they didn't use Alanine (the mutants for other type of residues is to Alanine). I just wonder if the Histidine to Glutamine is something well know and why is better than mutation to Alanine.
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Hi there,
His to Gln mutation allows to keep a similar backbone on the side chain up to epsilon nitrogen of the imidazole ring (3 C and one N). The same applies for His to Asn mutation for mimicking delta nitrogen of histidine (2C and 1N). These mutants mimick the non protonable form of the amines from imidazole ring. Typically if the binding is not dependent on the charge of the imidazole ring then binding should also occur for the corresponding mutant (but may be reduced).
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Mutated a Arginine residue with Alanine by SDM in the active site and the protein became insoluble. WT protein was found soluble but the mutated one was found in pellet fraction.
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Dear Eniyan,
It sems to me that you provide the answer to your own question : obviously, in your case, the single mutation you introduced in your protein is sufficient to make it insoluble. The behaviour of proteins is basically unpredictable; that's what makes them interesting
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Hi guys, I am going to work on bacteria that is coated with poly(alanine) hydrochloride polymer.
I am searching for protocols on how to create and maintain the bacteria. I only managed to find literature on how to coat the bacteria with the polymer. However, there are no steps describing how should I grow it or keep it.
Should I just treat it as any normal bacteria and grow in nutrient media or keep in glycerol stock? Or should I take any extra precautions??
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Ubaidah - this is an interesting question.  So that I understand, are you proposing somehow associated the polyalanine to the surface (that is, covalently, or by adsorption, or some other method)?  My answer assumes that to be the case, rather than finding a means by which bacteria are modified to synthesize polyalanine and display it on their surface - which would be a really good trick.
I don't believe you will be able to display the polyalanine on a culture you are trying to propogate.  My thinking is this:  at 'time 0' you complete the surface coating procedure.  After that point, no additional polyalanine is deposited on the bacterial surface.  After the coating you allow the organisms to start dividing.   After one doubling time, you have twice as many cells (and thus twice as much bacterial surface area) but the same amount of polyalanine as at time 0.  You've now reduced your original coating efficiency by 50%.  After the second doubling, your efficiency has fallen to 25% of the time 0 value.  For some organisms, this could take place in only one or two hours.  Soon there will be very little coating, all on the basis of dilution of the polyalanine through expansion of the total culture surface area.
The above answer assumes that the polyalanine is also not available as a metabolic substrate.  If the species of bacteria you're working with could also utilize the polymer as a nitrogen or carbon source, the coating performance might be even less.
Wish I had a more positive answer.  
Best, John
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I have to determine the concentration of alanine in solution. The problem is, that I also have an amine (Dodecylamine hydrochloride) in it. I've tried an enzymatic assay specific for alanine, but  unfortunately DDAC is also determined.
Does anyone have any idea how to determine only alanine?
Many thanks for any suggestions in advance.
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Hi there,
Is there any D-alanine in your sample? Does it matter to quantify isolate L and D stereoisomers or total alanine content is enough? For total alanine determination aminoacid analysis is enough (HPLC separation of aas, postcolumn derivatization, identification and quantification). If interested in L and D determination, there are specific oxydases for L-ala and D-ala on the market...
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For lysine mutation
will the mutation affect the quartary structure of proteins?
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I don't think there are any generally applicable rules for predicting the effect of site-directed mutagenesis. Alanine-scanning mutagenesis is often done, but other changes may be made for specific reasons. Some changes are more conservative than others. Changing between a small side chain and a large one, or altering the charge of a side chain, is most likely to have a substantial effect on the secondary, tertiary, and/or quaternary structure, especially if that side chain is involved in interactions or folding. In some cases, even seemingly minor changes (e.g. Ser to Ala) can be devastating to the folding of the protein. If the change is made at the interface between subunits, a change in quaternary structure may well result.
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I want to substitute 5 amino acids to alanine. As Alanine has 4 codons, does it matter which ones I use? Its a mammalian protein to be expressed and studied in mammalian cells so I hope there won't be any problem of codon bias.  I have also searched the codon bias in human and seen that GCC is used 27% whereas GCG is used in 7%. So is it better to use GCC in my sequence. The alanine residues already present in the protein is encoded by all the 4 codons but GCA is used 12 times. So the protein has a different bias for codon usage than the whole organism. In such cases, how to decide?
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The sequence by Zhang seems a good choice; maybe I would substitute the last codon to GCC just to avoid a potential GCTGCA direct repeat-induced slippage
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Hi,I did my qPCR experiment in kidney tissue for alat2 gene ( mitochondrial alanine aminotransferase) and I got 3-4 fold increase as compared to control, but when I am doing my Western Blotting ,I am not getting any protein bands at al.Please suggest any reason or solution for this.,thank you
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Often, the main problem is to get a specific and sensitive antibody. Do you have a chance to check your antibody, e.g. by forced expression of your cDNA or has this already been done? A positive control may also help to optimize the conditions (bloking agent, dilution ...). 
One problem could be the low level of ALAT2. A PCR signal does not mean that there is much protein. Have you tried to enrich for mitochondria? Are you already using a sensitive detection system?
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Hi, 
I am using martini force field  and  Coarse-Grained simulations to calculate the salvation energy of alanine in water in the form of decoupling. I have previously followed Justin Lemkul's tutorial on free energy calculations and trying to implement the same method with coarse grained simulations but I am getting a positive value in the case of coarse-grained simulations. Which I think I is unlikely. I have made changes in some of the parameters in order to make it compatible with coarse grained simulations. I am attaching my mdp file for production run. If someone can help finding the problem. I will be grateful.        
Thanks, 
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Just some thoughts:
1) Alanine isn't actually very soluble, solvation energy will be relatively small.  If you have uncapped termini then it should definitely be negative, but I'm not sure martini even has special bead types for termini.
2) One residue?  With Martini?  This is basically a single bead, CG forcefields like martini are for studying collective behaviour of many residues.  My analogy is that you can treat a million water molecules as LJ spheres, but you can't usefully treat three water molecules as LJ spheres.
3) Perturbing dialanine to nothing is a pretty standard example for atomistic calculations.  Try one one of the many worked examples for atomistic dialanine that are out there and then see if you still want to move to CG monoalanine once you have that sorted.
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I am researching on the use of alanine/EPR dosimetry to measure absorbed dose for radiotherapy. I have been able to obtain my EPR spectrum with a Bruker Elexsys E500 Spectrometer. I am intending to use peak to peak analysis but would like to improve my readings by reducing background distortion. Any suggestions would be helpful. Thanks!
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If you have a SHQE resonator that is more sensitive but be sure that the sample is properly fixed in the cavity. Good luck
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I want to calculate binding free energy of antigen-antibody complex after 30ns MD simulation.
I used MMPBSA but it takes approx 12 days for 1 ns (500 frames) of antigen-antibody complex in which i also performed computational alanine scaning (2 amino residues were mutated) . so now i want to used any other software that can calculate   binding free energy of 30ns of antigen-antibody complex in less time.
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I have been working in sequence data from Sbe 1 in traditional and modern commercial maize varieties.I have found 12 SNP and when translating resulted in 6 amino acid polymorphism. Among those AA polymorphism substitution from alanine to aspartic acid has been found to be significant where alanine is found in the modern varieties and aspartic acid is found in the traditional as well as wild type. Therefore i am concerned in finding out whether this substitution of AA is accidental or is it really selected for? Can anybody help me with this?
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This only can be detected through using multiple sequence alignment with similar and homologous sequences in protein databases. See attached publication 
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I have seen various articles regarding the major amino acid source is glycine and alanine in case of silk fibroin from B.mori. I would like to know about the possibility of the residual functional groups in the fictionalization of our fibroin with other polymers? Are there any ways?
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Thank you for ur kind concern professor!!!
I mentioned Grafting in the sense??? like functionalisation??? coupling with other proteins or polymers by some chemistry??? 
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Our protease has better efficiency when we did substitution of a single alanine in the cleavage site. I think It is a unusual result and I could not find the related paper...If you know about it, please let me know. Thank you!
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in the case of rhomboids you increase the activity when you add alanine in the cleavage site
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An induced systemic resistance study needs an assay of these defence enzymes
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both the assay methods are different so you cannot expect the method of estimation will be the same you can follow the procedures given in methods of enzymology.
you have to clean the plant samples before going for estimations
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I have modeled a cysteine protease enzyme using different software. Though the model generated from Modeller performed better than the other software, as evaluated by using procheck, Q Means server and MD simulation, I was having doubts about the conformation of a particular residue (tyrosine129) in the active site. The particular tyrosine residue is important as it occupy the active site position and since there is no similar residue in the template (alanine or cysteine), it is hard to predict which one of the conformation generated by the software is correct. Please suggest on what basis should I decide which conformation is stable.
In the picture the tyrosine residue has a different conformation from different models generated from different tools.
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Thank you all for the important inputs. I have got the problem solved.
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After a nightmare of MR trials, I've finally got a resonable match using poly-ala model by first pruning (gedit) then chainsaw. Any recommendations of a good site for the next steps would be helpful to me. I know I have to mutate all residues back as well as build the remaining missing residues from the pruning. Your thoughts on what I should be looking for and how I know if the results are good and worth refining would be helpful also.
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The next step is to replace the residues to the correct side chains. It is very easy to get register errors (moving sections of your structure one residue forward or backward). First of all you can replace big, bulky side chains in well resolved regions - the interior of the protein, between helicies or sheets, rather than at the flexible loops. Tryptophan, tyrosine and phenylalanine are best for this, but if you have arginine, isoleucine or glutamate in interior regions these can also be helpful, as the densities are easily recognisable.
One you have a few residues you are absolutly confident of near each other you can mutate between these and get a good section of your protein. If you can now be sure of around 50% of your structure is in the right places you can then use this as a template to automatically build again and this should give you some better links between residues. Delete any loops that are poor and rebuild these, as alanines, re-refine and see if you can now see side chains. Remeber some side chains will noto resolve on the surface your protein so remain as alanine during refinement (you can set atom ocupancy to 0 so it is the correct residue, but not included in the model). If the R values are getting better then you are improving your structures, and MolProbity will help to tell you hw reliable your structure is.
 
I hope that helps,
Ellis
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I heard about the alanine scanning mutagenesis but not the other amino acid mutagenesis. Is there any specialty in alanine?
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Alanine (Ala) has propensity to form alpha helices but can also occur in beta sheets and is generally equivalent to simply truncating a side chain back to the beta carbon, which is the first side chain atom. The beta carbon position depends upon the backdone dihedral angles of the polypeptide so is really part of the main chain structure of the protein. Thus, alanine is generally an accepted single residue first choice for mutational scanning because it retains the beta carbon but no other side chain chemistry. Glycine which removes the beta carbon is unusually flexible and can take on polypeptide backbone conformations generally not allowed by other amino acids, so mutation to glycine will cause flexibility and possible conformational changes convoluted with the effects of removing the side chain atoms making interpretation more complex than for Ala. Replacing side chains with larger, more constrained (such as branched beta carbon side chains of valine and Isoleucine), more polar, differently charged, or more hydrophobic atoms may all cause changes in structures and conformation along with the side chain chemistry thereby complicating analyses of results more than Ala.
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What are the effects of various amino acids, both chiral adn achiral, both amino and imino acids, on egg sac production in ramshorn snails?
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I am strongly convinced that chirality (so glycine versus alanine) or the imino acid (proline versus alanine) plays a role in the olfactory/reproduction processes. We do NOT have a way to measure the differentiation of stem cells in the snails, so we can only examine reproduciton as a possible function of chirality and the chiral amoni acid's interaction with the olfactory sense that would in turmn influence the reproductive cycle. More when results out!