P.S. I will get the electron movement diagram for the forbidden 1,3-product out to you guys sometime tomorrow.
My first day of tutoring Dr. Saadein's organic chem class: Th 7-8 p.m. Had about ~30-ish people. So far so good - I hope this many people can show up next week! I like the dynamics of you guys. You all seem pretty alert and cohesive. I'm quite encouraged with how things turned out so far. Come back next week for more cookies and FUN TIMES!! Organic chemistry is fun. :D
P.S. I will get the electron movement diagram for the forbidden 1,3-product out to you guys sometime tomorrow.
Why is organic chemistry useful to those not majoring in it?
Organic chemistry is set up to help you learn to think critically, as a chemist would. This discipline is hard to learn, but incredibly useful in the research/medical field later.
Organic I – big picture
Largely, organic I is giving you a foundation to understand how molecules typically react and behave. Additionally, you learn some foundational mechanisms that will be built upon in organic II.
Organic II – big picture
Organic II builds upon organic I’s foundation to teach you how to synthesize small molecules, predict products, reagents needed for products and understand how the molecules are reacting. This will ultimately teach you to begin thinking critically, as a scientist, based on how molecules behave.
What is the most important part of organic study?
Working practice problems regularly and often is essential to succeed in organic. Work the ones in the chapter, at the ends of chapters and the ones online, if you feel you need more practice.
A good study system
Try to build up two “toolboxes” as you read, take notes and do problems.
Toolbox 1 – a list, on a poster board or notebook, of how molecules behave. This includes mechanisms, the reasons for the mechanisms and rules like Markovnikov and Zeitsev. This list will actually become easier and easier to remember the more problems that you do.
Toolbox 2 – reagents; a great starting point for this list are the blue boxes at the end of the chapters that summarize the key reactions. You will need to know these! Chapter 11 and 14 end chapter blue boxes are good summaries, but reagents are not always listed. Pencil them in if you like. I also highly recommend putting these on a poster board somewhere.
Note: All mechanisms have similarities. Use these to create a “family tree” of mechanisms and help organize them in your mind. Straight up memorizing mechanisms is not near as helpful as understanding how they work. Based on what you know of how molecules behave – with resonance, carbocations, nucleophile strength, steric hindrance, etc. one can usually even “guess” mechanisms correctly if one cannot remember them specifically.
How to get the most from reading:
As you read, note the following….
1. Naming – helps to read line by line; also, summarize in notebook for reference
a. Work problems
b. Check solns
c. Fix mistakes in red on your notebook and put why it is wrong
d. Ask questions
2. Note all reagents and list them in your 2nd toolbox
a. Note how they work
b. whether they are strong/mild
c. what conditions they work on
d. what kind of substrates they require and why
e. other similar conditions
3. Note all mechanisms – most all of them, you will need to know; below are a few questions to get you started on what to pay attention to
a. Note why the mechanism works the way it does
b. Does it need a primary or tertiary nucleophile?
c. Are there any special conditions?
d. Under what conditions does it work best?
e. Does it need high or low temperatures?
f. Does it give mixtures of products?
g. Does it form a carbocation? Why/why not?
h. Does it function via SN1, SN2, E2, E1 or some of each? Under what conditions?
4. Making notes in the margins of your book or highlight the key pts above
5. DO the practice problems in the chapter, go over the solutions, and mark mistakes in red ink.
6. NOTE: The green boxes in the back of chapter 11 are very useful in formulating a mindset to thinking about problems. Also see my diagram, as a starting point for problem solving.
Online Review and Practice Problems:
Organic Chem study guide, do’s and don’ts: http://www.chem.arizona.edu/mcgrath/courses/studyguide%202.pdf
More orgo study advice: http://www.chem.arizona.edu/mcgrath/courses/studyguide%202.pdf
Topical review site: http://www.education.com/study-help/study-help-chemistry-organic-chemistry/
Sites offering practice problems:
1. http://www.studyorganicchemistry.com/ (lists the sites #2-4)
Here’s a promising online study site that I found: http://masterorganicchemistry.com/summary-sheets/
15-1 and 15-2 Stability of Dienes
· Understand difference between isolated double bonds, conjugated double bonds and cumulated double bonds and how each affects a compounds stability.
· Cumulated double bond systems are also called allenes
· See Fig 15-1. It is useful for comparing energies
· SEE PROBLEM: 15-1
o NOTE: In the problem, there is an error. In (b), the first structure has 3 double bonds. It should have only 2. The double bond on the bottom left should be crossed out.
15-3 Molecular Orbital Picture of a Conjugated System
· Resonance energy = the extra stability given by a conjugation in a molecule
· Molecular orbitals = current theory to explain where the extra stability due to conjugation comes from
· Example: in 1,3-butadiene, each carbon has sp2 hybridized, overlapping p orbitals
· The p orbitals have two lobes; each lobe is opposite in the phase of its wave function
o +/- signs, as well as different colors are used to indicate this in text
o Orbitals of opposite phases cancel each other out; think troughs and crests having destructive interference, like in physics, except opposite phased orbitals cancelling would be in 3D
· Since they are conjugated, these overlapping p orbitals combine to make one big molecular orbital (MO), because of resonance. The electrons are delocalized over the whole molecule.
· These molecular orbitals describe how all the atoms in the molecule bond together as a whole. They are kind of like one big orbital for the entire molecule.
· The number of molecular orbitals a molecule has = the number of p orbitals that combined to make it. Incidentally, this is always going to be an even number, because you will always have resonance between pairs of carbons.
· Half of a molecule’s molecular orbitals will be bonding orbitals (the majority of the p orbitals interact constructively – most stable) and half will be anti-bonding (the majority of the p orbitals interact destructively – less stable)
· Electrons enter MOs in order of stability – most stable first, least stable last.
· Bonding MOs have lower energy than in the original p orbitals, and are preferred. Antibonding MOs have higher energy than the original p orbitals and are not preferred.
· Only two electrons can fit inside any one MO
· In a molecule with two double bonds in resonance, there are four delocalized electrons, four p orbitals, thus four MOs. Two of those MOs are bonding, two are antibonding. Thus, two electrons enter the 1st molecular bonding orbital and two electrons enter the 2nd bonding molecular orbital. The anti-bonding orbitals are empty. This creates the most stable arrangement.
ONLINE MATERIAL RELATED TO SECTIONS 15-3, 15-8, 15-13 ON MOLECULAR ORBITALS
1. π Molecular Orbitals of Ethene: http://www.chem.ucalgary.ca/courses/351/Carey5th/Ch10/ch10-6-1.html
2. Molecular Orbital Theory: http://www.chemistryexplained.com/Ma-Na/Molecular-Orbital-Theory.html#b
3. Molecular Orbital Theory – thought it explained the interference concept pretty well: http://www.grandinetti.org/Teaching/Chem121/Lectures/MOTheory
4. Molecular Orbital Theory – another perspective: http://chemwiki.ucdavis.edu/Theoretical_Chemistry/Chemical_Bonding/Molecular_Orbital_Theory
15-4: Allylic Cations
· The allylic position is anything bonded to a carbon that is next to a C=C double bond
· Allylic cations are more stable than substituted cations, because they are next to a double bond and thus stabilized by resonance
· See problems 15-4 and 15-5
15-5: 1,2 and 1,4 addition to conjugated dienes [MECHANISM 15-1]
· Because of resonance, hydrogen halides (such as HBr) added across one of the double bonds of a diene can result in two different products, depending on which C the positive charge is on after the H is added.
15-7 Allylic Radicals [MECHANISM 15-2]
· Allylic radicals are stabilized by resonance delocalization and can give multiple products
· Review the mechanism of radical reactions – there are three steps – initiation, propagation and termination. Here there are 2 propagation steps because there are two possible resonance intermediates
o Propagation step 1
o Propagation step 2
o Regeneration of Br2
· Note that at high concentration adds across double bonds – if one has double bonds in the substrate, to avoid this, a low concentration of bromine is needed – and NBS is used
· N-bromosuccinimide (NBS) reacts with HBr to regenerate Br2 – keeps bromine concentration low by forming just enough bromine to keep the reaction going
· See problem 15-9, 15-10, 15-11
[Skipping 15-8, 15-9, 15-10, as they are not on the test]*Must confirm w/ Dr. Saadein for this year.
15-11 Diels-Alder Reaction [MECHANISM 15-3]
· For this section, understand the mechanism of and be able to draw Diels-Alder reaction products
· The diene is electron rich and acts as the nucleophile, attacking and donating electrons to the electron poor dienophile
· Diene reacts in the s-cis conformation, not s-trans
· What is s-cis versus s-trans? (end section 15-3)
· S-cis and s-trans describe the orientation that two conjugated double bonds can have toward each other. They can rotate around the intervening single bond to give either conformation. Both of these two allow overlapping p-orbitals, making them preferred. S-trans is slightly more stable, because there isn’t as much steric hindrance between the hydrogens on the same side of the molecule, as in s-cis.
· Electron adding groups can be placed on the diene and electron withdrawing groups can be placed on the dienophile to make them more reactive
· Some problems make require you to reconstruct the original diene/dienophile from a Diels-Alder product
· Problem Tip: A Diels-Alder product always contains one more ring than the reactants. The two ends of the diene form new bonds ot the ends of the dienophile. The center bond of the diene becomes a double bond. The dienophile’s double bond becomes a single bond (or its triple bond becomes a double bond).
· Problem Tip: To deconstruct a Diels-Alder product, look for the double bond at the center of what was the diene. Directly across the ring is the dienophile bond, usually with electron-withdrawing groups. (If a single bond, the dienophile had a double bond; if double, the dienophile had a triple bond.) Break the two bonds that join the diene and dienophile and restore the two double bonds of the diene and the double (or triple) bond of the dienophile.
· See problems 15-14, 15-15
· The Endo Rule: the endo position (dienophile part of the ring in the endo position, H-bonds in the exo position) is most stable, due to secondary overlap of the p-orbitals in the double bonds of each ring
· Remember that the 1,3-product is forbidden, due to impossible bonding/resonance patterns; 1,2 and 1,4-products are typically produced
· See problems 15-17, 15-18
· Above, important chart – summary below:
· Note that the diene in the top two squares does not change and the dienophile in the bottom two squares does not change
· The chart is saying that (on top) if you have a dienophile in cis or in trans, this will affect which way the bonds are pointing in the product
o Cis dienophile gives cis (meso) product
o Trans dienophile gives trans (racemic) product – because either group could be pointed up or down, as long as they are on opposite sides of each other
· (on bottom squares) If the diene has symmetrical substituents (left) it gives a cis(meso) product
· (on bottom squares) If the diene has asymmetrical substituents (right) it gives a trans (racemic) product
Key Points from Chapter 15-12: The Diels-Alder as an Example of Pericyclic Reaction
*Note: When doing problems dealing with molecular orbitals, remember the following…
Molecular Orbital Basics
· Molecular orbitals show all the different bonding and anti-bonding interactions that can exist between the molecule’s p orbitals in their double bonds.
· HOMO = highest occupied molecular orbital
· LUMO = lowest unoccupied molecular orbital
· HOMO* = new highest occupied molecular orbital in the molecule’s excited state
· A molecule will have twice as many p orbitals as it has double bonds; two p orbitals = one double bond
· The molecule has the same # of MOs as it has # of p orbitals
· Each p orbital contains one electron
· Thus, a MO will have as many electrons to fill it as it has p orbitals
· Electrons will always fill the lowest energy MO first, then the next lowest, etc.
Determining Forbidden and Allowed Cycloadditions
· MOs can explain why some reactions are allowed and others are not.
· In a cycloaddition, the diene – or electron donator – will always donate electrons from its HOMO (the first electrons “off the top” so to speak).
· In a cycloaddition, the dienophile – or electron acceptor – will always accept electrons into its LUMO (first empty shell, essentially).
· When determining whether or not an addition reaction is allowed, keep in mind that the bottom half of the p orbitals in the diene always react with the top half of the p-orbitals in the dienophile (see diagram below)*SEE CLARIFICATION POST REGARDING THIS DIAGRAM.
· A reaction is thermally forbidden when the symmetry of the p-orbitals results in non-bonding reactions instead of bonding ones. The electrons cannot flow easily from one to the other (see figure below).
· However, the same reaction can be “photochemically allowed” – when UV light hits ethylene, it excites one of its electrons in the HOMO into the next molecular orbital (or LUMO).
· Thus, the LUMO is now occupied and in this brief transition state, it becomes the new HOMO, called HOMO*.
· HOMO* of an excited ethylene can donate its electron to a ground state ethylene LUMO, in an allowed reaction.
· In most cases, photochemically allowed reactions are thermally forbidden and thermally allowed reactions are photchemically forbidden.
o See problems 15-19, 15-20 and 15-35
Conjugation and UV absorption
· The energy difference between HOMO and LUMO decreases as the length of conjugation increases.
· A compound that contains a longer hcain of conjugated double bonds absorbs light at a longer wavelength (see the example of β-carotene in text).
· Only conjugated double bonds interact to give lower absorption frequencies. Isolated ones do not.
· An additional conjugated double bond increases the wavelength absorbed by ~30-40 nm, while an additional alkyl group increases it ~5 nm
o See problem 15-22
Going to Oxford last Friday was a treat. I'm going to start tutoring organic chemistry for Dr. Saadein again starting next week, and worked that out with him. I got to talk to my professors about in-cell NMR. Being chemistry focused, they'd not heard of it before. I explained what I knew of it and finished reading one and a half of the two papers I brought that I hadn't read. I left one that I had read w/ Ms. Harmon & kept the other two.
The most wonderful part though: I learned that an anonymous donor had paid the remaining 12 million that was needed for building the new science building... I've never been SOOO happy!! Oxford will get its new science building AND its new library!! Dr. Parker had told me just a few months ago that he thought the whole thing would be tabled and wouldn't happen until after he retired. Construction will be starting soon. I wanted to get up and dance. Mom had been praying for exactly that since she heard Dr. Parker's fears, and I'd been praying for that for about a year.
After lunch, I also got to mine Ms. Harmon's computer for in-cell NMR articles ... *makes heavenly awe sound*. I SO miss not being connected to a school and getting to look up any journal article that I want. I found LOTS - about 20. I was surprised there weren't more.
After that, I got to watch Ms. Harmon's scientific microwave actually run. Never seen that. It cuts chemical reactions that take days to minutes. I think everyone should use one. But, it is expensive (~$40k) and the technique got a bit of a bad wrap before the scientific ones came out, I think.
See this link for video of the microwave: https://www.facebook.com/photo.php?v=10151433916615225&set=vb.736455224&type=2&theater
See this link for video number 2 of the microwave - much easier to see it moving in this one: https://www.facebook.com/photo.php?v=10151433919565225&set=vb.736455224&type=2&theater
I got to help my mother-in-law judge her elementary school's science fair projects: I did 5th grade and a large portion of 4th. I thought I could do more than that, but I got too tired. I was amused by some of the purpose and hypothesis statements. All the projects were really cute. None of them really had controls though. The ones that weren't actually experiments we skipped.
Review: Looking into live cells with in-cell NMR spectroscopy
Philipp Selenko *, Gerhard Wagner
Journal of Structural Biology 158 (2007) 244–253
NOTES (excerpts from the journal spliced together, along with my own comments) - I'm here hitting the highlights of the article, particular the parts I found most interesting, with comments.
Labeling of proteins with 15N and 13C, over-expression in bacteria, and NMR of the crude extract. Harder to do in EU cells because of more expensive growth media, difficulty of over-producing the bacteria, poor incorporation of the labeled atoms….
Micro-injection vs Protein Over-production…. with in-cell NMR Advantages and Disadvantages:
Advantage of protein over-production
Disadvantage of protein over-production
Advantage of micro-injection
Disadvantages of micro-injection
This approach, however, is restricted to a few eukaryotic cell types, which can be manipulated in such a way, and requires the labeled protein samples to be sufficiently soluble at high concentrations, since the maximum injection volume per single cell is typically on the order of nano-liters.
Alternatively, one could envisage intracellular sample
delivery by means of cell-permeable synthetic vectors. We
are particularly intrigued by the potential application of
‘Trojan’ peptide tags, which confer efficient cell membrane
transduction activities to a wide range of fused protein substrates
(Derossi et al., 1998; Dietz and Bahr, 2004). These
internalization peptides are composed of short, positively
charged amino acid residues, which can be genetically engineered
to be part of virtually any recombinant polypeptide
(Li et al., 2002). Upon labeled expression and purification
of tagged fusion proteins, these substrates are simply added
to the growth medium of a variety of cultured laboratory
cell lines and readily internalized. In theory, this method
should be generally applicable to a wide range of eukaryotic
cells and quantitatively accomplishable for a large
number of cells.
The method of choice for eukaryotic in cell NMR measurements will hence be dictated by both the suitability of the protein of interest for either approach and by the overall biological question to be addressed.
Difficulties of in-cell NMR in EU cells
The major obstacles for the selective labeling of recombinant proteins with NMR-active isotopes in eukaryotic cells
have been …
Growth media for NMR labeling in E. coli are simple in their composition, easily prepared and, depending on the type of labeling, relatively cheap. Bacteria will also incorporate isotopes with high efficiency (_98%). Labeling media for eukaryotic cells are sophisticated, they must often be obtained commercially for satisfactory results, and they are expensive.
Tumbling rate and line widths gives information about cellular binding partners
In general, small proteins display large tumbling rates, which
lead to slow overall relaxation and narrow NMR line
widths (Fig. 3a). Molecules of larger size tumble more
slowly, relax faster and exhibit broader resonance signals (Fig. 3b).
Any molecule in a cellular environment exhibits
a reduced tumbling rate due to intracellular viscosity
and hence displays broader NMR line widths (Fig. 3c).
In the absence of protein binding to endogenous cellular
factors, a direct comparison of individual protein line
widths in buffer and in in-cell NMR experiments will
readily yield a qualitative estimate about intracellular viscosity.
Upon sample binding to cellular components, the
resulting protein complexes can either display tumbling
rates that correspond to the sum of their individual
masses (Fig. 3d), or individual contributions to a mixed
set of rates, when the interaction is restricted to a subset
of residues (Fig. 3e).
The latter results in residue specific line broadening, which yields
information on the dynamics and localization of the cellular interaction.
Binding to quasi-static cellular structures like organelles or membranes
results in severe line broadening (Fig. 3f), which can serve as a
qualitative indicator for the kind of interaction.
Many biological binding events are dynamic and
modulated by cellular signaling, which often leads to
transient and interpretable changes of NMR line widths.
Getting around the complicity of the cellular environment and binding partners
It is apparent that a biologically active protein can experience
any of the aforementioned conditions, and superpositions
thereof, in a cellular environment. Complicated or poor quality in-cell
NMR spectra are the likely result.
In such cases, the researcher needs to reduce the complexity
of the system under investigation, which can be
achieved either by ‘chopping up’ full-length proteins into
individual domains in order to selectively probe differential
biological activities or by introducing site-directed
mutations that abolish certain functional characteristics
and similarly enable one to discriminate between specific
Example: Indeed, given the multitude of cellular binding partners for
ubiquitin in Xenopus oocytes, in-cell NMR experiments for
the wild-type protein yielded poor quality NMR spectra.
Only upon mutating conserved binding sites of known
interaction surfaces, did ubiquitin provide interpretable
in-cell NMR data. This work thus serves a fine example
for what kind of biological studies will be amenable to
in-cell NMR approaches.
Note that: The necessary concentration of injected protein for the minimally sufficient experimental NMR signal has to be evaluated empirically for each sample.
Other factors affecting the minimal amount of labeled compound needed for a minimal NMR signal…
Injection concentrations are typically in the range of 0.5–3.0 mM.
It is evident that a compromise between the
experimentally achievable signal-to-noise, the duration of
individual NMR experiments, the cellular concentration
of labeled proteins and its physiological relevance, will
have to be found if in-cell NMR measurements in X. laevis
oocytes are to yield biologically meaningful results.
1H, 15N correlation pulse-sequence,
isotope-edited correlation techniques
Solvent suppression was achieved by a Watergate NMR pulse-sequence
2d scan was described as follows:
Fig. 5 (b) Identical samples as in (a) but recorded as 1D 1H(15N) hetero-nuclear single quantum coherence (HSQC) correlation spectra, using a Watergate version of a standard, sensitivity-enhanced HSQC pulse-sequence with the same number of scans as in (a).
1d NMR provides too much noise, but 2d isotope edited technique provides a clearer picture
Fig. 5a shows the characteristic appearance of 1-dimensional
proton-only NMR spectra (no isotope-edited correlation
‘filter’) of a 15N-labeled protein sample in its pure
form (top panel), resuspended in crude Xenopus egg extract
(middle panel) or upon oocyte injection (bottom panel). It
is apparent that by conventionally recording all NMR signals
from 1H nuclei only, these spectra do not allow to discriminate
between resonances from endogenous, cellular
components and the ones from the labeled compound.
Moreover, it is evident that the quality of in-cell NMR
experiments, recorded in this mode, is too poor to provide
any information on the injected protein sample. When,
however, these same samples are measured with the application
of a 1H, 15N correlation pulse-sequence, similar 1-
dimensional traces selectively display NMR resonances
from the labeled protein only.
The good quality of these background-suppressed spectra
enables the selective detection of protein NMR signals under
extract and in-cell conditions (Fig. 5b). Changes in NMR line widths of
individual resonance signals in these different aqueous solutions
are readily visible (Fig. 3).
What will cause in-cell NMR to fail
Once cellular proteins engage
the labeled specimen in too many generic interactions, or
scavenge the NMR-active protein into complexes of molecular
weights too large for detection by conventional solution-
state NMR methods, the envisaged in-cell NMR
approach is likely to fail. In most instances, such unfavorable
binding behaviors are easily pre-assessed by NMR
experiments in cellular extracts (Serber et al., 2006).
This future application sounds FASCINATING to me
In-cell NMR analyses of intrinsically disordered proteins
Intrinsically disordered proteins (IDPs) represent a
growing class of gene products (Dyson and Wright,
2005), which are characterized by lack of secondary and/
or tertiary structure in their pure forms and at physiological
pH (Uversky, 2002). IDPs are estimated to account for
_20% of all human proteins (Dunker et al., 2000) and exert
important functions in key cellular processes (Dunker
et al., 2002). A significant number of IDPs are implicated
in human protein deposition diseases, in which a normally
soluble polypeptide forms insoluble aggregates in a subset
of cells and precipitates in the form of amyolid fibrils
(Fink, 2005). Little is known about the general 3-dimensional
conformation of IDPs in cellular environments and
it is tempting to speculate whether unfolded protein conformations
are preserved under native, intracellular conditions.
Could it be possible that some IDPs are not per se
structurally deficient protein entities but that their
unfolded state results as a consequence of the isolated
in vitro experimental setup employed for their characterization?
In-cell NMR experiments on the natively unfolded
FlgM protein indeed suggest a more folded conformation
in the cellular environment of live bacteria (Dedmon
et al., 2002). Are there cell type specific differences in the
conformation of unfolded proteins and to what extent do
different cellular environments modulate the pathological
conversion of IDPs during amyloid formation? In-cell
NMR spectroscopy appears to be a most appropriate tool
to address these questions in live cells.
6.2. In situ observation of post-translational protein
A limitation of in-cell NMR spectroscopy in bacteria is
the inability to study post-translational protein modifications.
Whereas the function of most eukaryotic proteins
is regulated by a variety of sometimes transient, covalent
modifications, mammalian proteins expressed in bacterial
organisms are typically not modified. Eukaryotic protein
modifications, with the exception of post-translational glycosylation,
involve the covalent attachment of small chemical
entities, acetyl-, methyl-, or phosphate-groups, which
do not greatly alter the overall molecular weight of the
modified substrates. This is particularly favorable for biomolecular
NMR analyses as the spectral quality of a
unmodified protein is likely to be preserved upon covalent
modification. In addition, post-translational protein modifications
greatly change the chemical environment of targeted
residues, which translates into large chemical shift
changes. In theory, transferring an unmodified protein substrate
into a eukaryotic cellular environment should result
in its post-translational modification by endogenous
enzymes and according to a specific, biologically relevant
pattern. In-cell NMR measurements should then enable
the in situ observation of the establishment of these modifications
in a time-dependent and residue-specific manner.
We have recently confirmed that these in vivo approaches
can indeed resolve cellular phosphorylation reactions in
X. laevis oocytes (Selenko et al., submitted). They point
to a plethora of possible in-cell NMR applications in
eukaryotic post-translational protein modification
In discussing micro-injection…
About 200 manipulated oocytes are required for an in-cell NMR sample, which corresponds to >250 lL in sample volume and, inside a Shigemi_NMR tube, will suffice to span transmitter- and receivercoil extensions of most commercial NMR spectrometer probes (Fig. 4e).
Considering the small injection volume per cell (50 nL), an oocyte sample requires only about 10 lL of labeled protein.
Question: in a sample NMR tube, one can be reasonably sure that each molecule is only influenced by itself and/or average interactions in an inert environment. But here, each protein or even, each protein group in each of the 200 cells could be being acted on differently and giving different resonances. How does one sort out all this noise?
Question: Water is not inert to H1 resonances. Cells are full of water. How does one get around the interactions on water on these cellular proteins or is that something that one wants to see? I'm assuming from this article that in a 2d NMR map, only the water directly interacting with the protein would be visible indirectly on the spectra, via it's influence on the N or C under observation.
Question: I need more information about 2d NMRs. I’ve only worked on the 1d kind.
Question: In traditional NMR, you have the reference compound TMS. What is the equivalent for in-cell NMR or is there one? If there is not one, how does one determine a reference point? Note that figure 5 description seems to imply a standard, or, I could just be reading that completely wrong.
Question: While I can see the advantages of micro-injection into X. laevis oocytes, I do wonder, since the injected protein is not native to that cell, I presume the interactions within that cell would be different from its host cell, thus not giving the true picture of how it may structurally relate to certain molecules.
ANS: Most biological reactions are executed with comparable activities in Xenopus cell extracts and can easily be modulated by the addition of small molecule inhibitors or activators. These extracts can also be selectively depleted of certain cellular components, and replenished with labeled, NMR-active substitutes, which enables the
isolated investigation of biological processes without compromising the qualitative nature of the experimental readout by residual endogenous activities.
What's going on in my corner of the science world? Find out here!
Rolling Statuses: Technical journal blog. Here you may discover what the daily life of a grad student looks like: day-to-day snippets of life, clutter, rolling statuses and unimportant fluff.
Progress Updates: Will include entries with more meaningful science.
Weekly lab report: My write-ups on what I did each week (I posted these publicly during my rotation but not as much now. That may change.)
Here is a link to collected writing, poster and presentation tips.
As of February 8, 2014 I have officially joined the Salaita lab!! Very exciting. Stay tuned for updates. "Micro Min" category equates to grad school journaling; most of these have moved to my status updates blog under Home tab. See "progress updates" on this blog for more important news.