What does the squiggly line in the amphetamine structure mean?

Amphetamine has a chiral center at the carbon attached to the wavy bond, the amine, the carbon branching off to the phenyl group, and a hydrogen atom (not shown). Because of this, there are two enantiomers of amphetamine shown below with their IUPAC names.

Chemists sometimes use a wavy bond when they want to represent both enantiomers in a single figure. This might be useful shorthand if you need to represent a racemic mixture (a mixture of both enantiomers), if the stereochemical identity of a compound is unknown, or in an article where you’re referring to both of the two forms in a general sense. Another acceptable way of doing the same thing without drawing as much attention to the stereochemistry is to use a regular, straight line for the bond.

You could also write this as (±)-1-phenylpropan-2-amine to represent that fact that the name refers to two distinct chemical compounds.

See also: http://en.wikipedia.org/wiki/Substituted_amphetamine
A thorough list of official IUPAC rules for drawing chemical compounds (including wavy bonds and more) can be found in this PDF: http://www.iupac.org/publications/pac/2006/pdf/7810×1897.pdf

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What are some essential websites for biologists?

It’s funny how little overlap there is between different fields within biology; I’ve heard of almost none of the others posted. I’ll add some sites that I use a lot for molecular biology, biochemistry, and/or structural biology.

DNA and cloning tools

  • BLAST NCBI – search the NCBI database for nucleotide or protein sequences and align them (http://blast.ncbi.nlm.nih.gov/)
  • Reverse complement – quick and simple way to get the reverse complement of a DNA strand (http://www.bioinformatics.org/sms/rev_comp.html)
  • ExPASy translate – quick and simple DNA to protein translation (http://web.expasy.org/translate/)
  • NEB cutter – design restriction digests, useful for cloning (http://tools.neb.com/NEBcutter2/index.php)
  • OligoCalc – a simple Tm calculator for primers (http://www.basic.northwestern.edu/biotools/oligocalc.html)
  • Invitrogen primer designer – can make primer designing a bit easier (http://tools.invitrogen.com/content.cfm?pageID=9716)
  • PrimerX – QuikChange primer design tool for site directed mutagenesis (http://www.bioinformatics.org/primerx/)
  • Miscellaneous other DNA tools (http://www.bioinformatics.org/sms2/)

Protein tools

  • Protein absorbance calculator for measuring concentration by UV vis spectroscopy (http://www.scripps.edu/~cdputnam/protcalc.html)
  • ExPASy MW and pI calculator (http://web.expasy.org/compute_pi/)
  • Buffer calculators for those too lazy to do basic chemistry (http://www.protocolpedia.com/index.php?option=com_content&view=article&id=76&Itemid=82) & (http://www.currentprotocols.com/WileyCDA/CurPro3Tool/toolId-3.html)

Structure tools and databases

  • PDB – database of nearly all published protein structures (http://www.pdb.org/pdb/home/home.do)
  • BMRB – biological NMR structure database (http://www.bmrb.wisc.edu/)
  • EM databank – for electron microscopy work (http://www.emdatabank.org/)
  • Crystallographic spacegroups – fun reference for space groups (http://img.chem.ucl.ac.uk/sgp/large/sgp.htm)
  • SAXS profile calculator – convert a crystal structure into SAXS data for comparison (http://modbase.compbio.ucsf.edu/foxs/index.html)
  • Circular Dichroism (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml)
  • Mass Spectrometry tools (http://prospector.ucsf.edu/prospector/mshome.htm) & (http://prowl.rockefeller.edu/) & (http://www.cbrg.ethz.ch/services/MassSearch_new)
  • PSIpred – put in a sequence and get back a secondary structure prediction (http://bioinf.cs.ucl.ac.uk/psipred/)
  • Protein modeling (http://www.proteinmodelportal.org/)
  • PymolWiki – good guide for using Pymol, not useful if you prefer Chimera (http://www.pymolwiki.org/index.php/Main_Page)

Misc. protocol and tool sites

  • ExPASy – many useful tools to name them all, new ones added occasionally (http://expasy.org/)
  • Protocol-online – molecular biology protocols and reasonably populated question and answer forum (http://www.protocol-online.org)
  • Open WetWare – a wiki for molecular biology protocols (http://openwetware.org/wiki/Main_Page)
  • Current protocols – more protocols and some nice tools too (http://www.currentprotocols.com/)
  • VADLO – more protocols (http://www.vadlo.com/)
  • MolBio Tools – misc molecular biology tools (http://molbiol-tools.ca)

Other

  • MSDS – check if you are going to die after something spills (http://www.msdsonline.com/msds-search/)
  • NIH reports – find out if you need to penny pinch next semester (i.e. whether your grants were renewed) (http://projectreporter.nih.gov/reporter.cfm)

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Do people produce protein for crystallography using cyanobacteria as the expression system?

The E. coli expression system crowds out most others for crystallography. You need a lot of protein to set trays and get crystals and this is the easiest system for getting high yields. There are times when a protein needs to be expressed outside of bacteria, for example if it requires alternative mRNA splicing. The most common alternative expression systems are yeast, insect cells with baculovirus, and mammalian cell lines. I was able to track down statistics on expression systems from the Protein Data Bank (http://www.pdb.org).

Below is just the top of a very long list of expression systems used out of the 68,266 crystal structures deposited in the PDB that list an expression organism. Most are probably crystal structures. You can see the full list here: http://www.pdb.org/pdb/statistics/histogram.do?mdcat=entity_src_gen&mditem=pdbx_host_org_scientific_name&numOfbars=50&name=Expression%20Organism%20(Gene%20Source)

I added in some numbers for reference. The drop-off in expression systems is pretty steep and I suspect most of the lower tail consists of false positives anyway. A brief summary of the most common expression systems for protein structure determination:

  1. Bacteria (various E. coli strains, B. subtilis) ~89%
  2. Insect cells (S. frugiperda, T. ni) ~4%
  3. Yeast (P. pastoris, S. cerevisiae) ~2%
  4. Mammalian cells (H. sapiens, CHO cells, M. musculus) ~2%
  5. Cell free systems ~0.3%

I searched for various cyanobacteria genera in the “Expression Organism” category of the PDB advanced search (located here:
http://www.pdb.org/pdb/search/advSearch.do). There were two hits but one was a virus released into a cyanobacterium and then solved with TEM (2XD8) and the other was a false positive. I didn’t do an exhaustive search but it seems unlikely that there are any. It’s certainly not a common expression system if there are.

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Why do some PCR kits include GC Enhancer solution? What does this chemical do?

PCR reactions work by raising and lowering the temperature of the components over multiple cycles to accomplish a few different tasks.

  1. Denaturation - The temperature is raised high above the melting point of the DNA and the two complementary strands of DNA will separate.
  2. Annealing - The temperature is lowered to about 3-5 degrees below the Tm of the primers causing them to pair with their complementary DNA templates.
  3. Elongation – The temperature is raised high enough such that DNA polymerase can begin adding new nucleotides to pair with the template starting with the primer but not so high that the DNA is denatured.
  4. Repeat these steps many times and you’ll roughly double the amount of DNA each time you repeat.

A major problem for amplifying GC rich sequences is that the melting temperature is much higher for guanine and cytosine than it is for adenine and thymine. This is due to the increased stability of the three hydrogen bonds between G and C compared to the two between A and T. DNA primers that have high GC content are more likely to prime to the wrong DNA sequence giving you the incorrect product because their Tm’s are a bit higher than 3-5 degrees above the annealing temperature. GC rich regions of DNA may also form secondary structures that disrupt elongation.

The Tm is the point at which half the DNA molecules will be separated at equilibrium and is a good measure of what sort of temperature range you need to drop below for your primers to anneal. The Tm depends on the entire sequence of the DNA primers as well as the salt concentration. The free energy between a DNA template and matching primer with a lot of Gs and Cs is much larger than one with more As and Ts and so it also has a higher Tm. The purpose of the enhancer solution is to bring the Tm of GC rich regions closer into line with AT regions so that the primers anneal quickly and uniformly.

I couldn’t find the exact contents of commercial PCR enhancer solutions, but presumably they work in a similar ways to published academic solutions like the addition of betaine.[1] When betaine is used as a cosolvent, it reduces the gap between the stability of AT and GC base pairs that is a consequence of having two verses three hydrogen bonds. This brings the Tm of GC rich DNA sequences closer into line with the Tm of AT rich sequences. Below are the melting profiles of a GC-rich DNA sequence without betaine (1) and with 5.0M betaine (2). Notice that the Tm is shifted lower and is sharper reflecting a narrower melting range of GC and AT base pairs.

Betaine’s effect on DNA melting is likely due to two competing thermodynamic forces.

  • Betaine has a sequence independent destabilizing effect on all DNA base pairing interactions due to its charged nature. This reduces the stability of hydrogen bonds and lowers the Tm of AT and GC rich regions by roughly the same amount. With this effect alone, GC rich regions would still have higher Tm’s relative to AT rich regions.
  • Betaine also binds preferentially to the major groove of AT sites due to a slight hydrophobic effect between betaine and thymine’s major groove methyl group. This has a sequence dependent stabilizing effect that increases the stability of the double strand DNA in AT regions relative to GC regions.

The overall result of both effects taken together is that the stability (and with it the Tm) of double stranded AT rich regions drops, but the stability of GC rich regions is drops even further. The curve below shows the Tm of DNA strands vs. mole fraction of GC content. The top curve (filled triangles) is without betaine and the bottom curve (open circles) is with the addition of 5.6M betaine. Note that the Tm in the presence of betaine is roughly independent of GC content, which was the original goal.

Other cosolvents that function as GC-rich enhancer solutions may work in slightly different ways, but presumably they all bring the stability of GC base pairs closer in line to AT base pairs making the overall Tm less dependent on GC content. It’s worth noting that there are other tricks to help deal with GC rich DNA that fall outside the scope of the question. In the end, it’s hard to predict your PCR yields and playing around with the conditions through trial and error is the best way forward.

[1] W. A. Rees, T. D. Yager, J. Korte, and P. H. von Hippel. Betaine Can Eliminate the Base Pair Composition Dependence of DNA Melting. Biochemistry 1993, 32, 137-144. doi:10.1021/bi00052a019 (http://pubs.acs.org/doi/pdf/10.1021/bi00052a019)
[2] J. Marmur, P. Doty. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. Journal of Molecular Biology, 1962. 5, 109-118. 10.1016/S0022-2836(62)80066-7.
(http://www.sciencedirect.com/science/article/pii/S0022283662800667)

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How does monoamine oxidase (MAO) cleave monoamines?

Monoamine oxidase, like most protein enzymes, catalyzes reactions largely by positioning the necessary molecules in such a way that they react much faster than they would just by bumping one another randomly. This positioning is accomplished by folding into a structure that binds the components tightly and in the correct orientation. After that it’s just the chemistry that causes an amine to donate hydrogens to a nearby flavin molecule, converting into an imine and then an aldehyde soon after.

The Biology

Below WT human monoamine oxidase A (PDB: 2Z5X) is shown as a cartoon representation in green. An FAD molecule (red) is covalently attached to Cys-406 (blue). IThe reversible inhibitor harmine (purple) is bound in the active site where a monoamine like dopamine or norepinephrine might bind.[1]

Below residues Tyr-69, Asn-181, Phe-208, Val-210, Gln-215, Cys-323, Ile-325, Ile-335, Leu-337, Phe-352, Tyr-407, and Tyr-444 which form a hydrophobic pocket that fits the monoamine substrate (or in this case the inhibitor) are highlighted in cyan. There are also interactions with the aromatic rings of the inhibitor that help lock it in place (not shown).

Below is a transparent surface representation of MAO-A. You can still make out dopamine in purple (which I mutated into the structure assuming it fits in roughly the same way that the harmine inhibitor did) and FAD in red inside the protein.

If we slice the above image down the middle (below) you can see the internal pocket that FAD (red) fits into as a grayish shadow. You can also see dopamine in purple entering from the left side (marked by an arrow) through a small opening leading into the larger cavity containing the active site. The pore as shown is a bit too small for the substrate, however dynamic fluctuations in the loops near the entrance likely allow it to enter and exit.

The structure of MAO-A positions the substrate in the active site so that it contacts flavin directly. This drives a reaction that oxidizes the substrate’s amine group into an aldehyde group. The mechanism for this reaction is discussed in the section below. MAO-B likely works the same way but the pocket has a slightly different shape and therefore functions better with a different set of substrates.

The Chemistry

A post-translational modification of a cysteine residue with FAD near the active site of monoamine oxidase is necessary because FAD takes on an important chemical role as a proton acceptor in the catalysis. FAD is essentially a riboflavin molecule attached to a molecule of ADP as shown below.

Once the protein is chemically modified a monamine, like dopamine or norepinephrine, can use the flavin group as a proton acceptor and form an imine. One of a handful of proposed mechanisms (in other words, this is consistent with the structure but there are no guarantees that it is right) is shown below. In this “polar nucleophilic” mechanism the amine will attack the flavin as shown below. During the reaction the monoamine in red is converted into an imine.[2]

The imine will undergo immediate hydrolysis into an aldehyde in the presence of water as shown below. The incoming imine starts in red and remains in red as it becomes an aldehyde, releasing an ammonium ion in the process.

The positioning of the monoamine directly adjacent to flavin in the protein cavity, as shown in the crystal structures at the beginning, vastly speeds up these reactions. The enzymes work best for primary amines, like amphetamine (below, left), but not for the methyl-subsitituted secondary amines like methamphetamine (below, right) which has a second carbon coming off its amine group.

The issue is not so much the chemistry, which could proceed through the same mechanism shown above, but the biology. I’m not sure exactly what about the methyl group reduces the binding efficiency in this enzyme, but I think it’s safe to say that the presence of that extra carbon atom disrupts the interaction between the amine group and the flavin molecule, either because of its position or its effect on the position of methamphetamine in the binding pocket. Without the precise orientation of the correct chemical groups the enzymatic efficiency of MAO is significantly reduced.

This is similar to the way reversible inhibitors might work. The reversible inhibitor harmine, shown early on in the crystal structure, binds to and obstructs the active site of MAO-A without being oxidized. While it’s there, it prevents access of other monoamines and thus inhibits monoamine oxidation. Since it is bound non-covalently there is nothing preventing it from falling out of the pocket and allowing the enzyme to function normally. This is in contrast to irreversible MAO inhibitors that do bind covalently to the flavin molecule. The mechanisms of these irreversible inhibitors vary, but generally involve some kind of chemical reaction with the flavin molecule that doesn’t end in release of the inhibitor. Rasagiline, for example, will react covalently with the central nitrogen on the flavin group and remain there blocking the active site for the remainder of the protein’s lifetime.

[1] Son, S.-Y. et al. Structure of human monoamine oxidase A at 2.2-A resolution: the control of opening the entry for substrates/inhibitors. Proceedings of the National Academy of Sciences of the United States of America 105, 5739-44 (2008). http://www.pnas.org/content/105/15/5739.short
[2] Edmondson, D. E., Mattevi, A., Binda, C., Li, M. and Hubálek, F.. Structure and Mechanism of Monoamine Oxidase. Burger’s Medicinal Chemistry, Drug Discovery and Development (2005). http://onlinelibrary.wiley.com/doi/10.1002/0471266949.bmc111/full

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