Unnatural amino acid mutagenesis in molecular neurobiology
The nature of the interface between chemistry and biology
The general tenor of biological research is less reductionist than our usual work, and the position of most chemists is even more so. The general method that unites the disparate elements of this thesis is unnatural amino acid mutagenesis, performed on integral membrane proteins, in living cells.
Nonsense suppression and molecular neurobiology – a felicitous combination
Nonsense repression is used to introduce unnatural amino acids by loading the repressor tRNA with a synthetic amino acid. Center: repressor tRNA (anticodon shown in red) and mRNA containing a stop codon (also red) at the site of desired synthetic amino acid insertion.
Implementation of the suppression method in molecular neurobiology
- tRNA synthesis
- General considerations
- Essential controls
Factors associated with translation include codon context, read-through of the stop codon, and effects of truncated proteins. A second difficulty that can arise at the translational level is reading through the stop codon. 21.
Uses of unnatural amino acid suppression in molecular neurobiology
- Structure-function studies
- Dynamic in situ manipulation of ion channels
Pam England in this laboratory in 1997.29 The experiments presented here were performed with her very helpful assistance and many ideas were developed with her guidance. Ken Philipson, who generously shared authorship of a paper in the American Journal of Physiology for my help in the early stages of the project.32.
Tapping into the alpha subunit of the nAChR usually works very well, relative to the null control. Analysis of the conserved glycosylation site in the nicotinic acetylcholine receptor - possible roles in complex assembly.
Cation- π analysis using fluorinated Trp derivatives
Introduction
Bottom: Side and top views of the receptor with subunit binding site residues highlighted in red rendered as pin diagrams. In addition to the muscle-type receptor found at neuromuscular junctions, there are neuronal forms of the receptor.5,7,8.
Results
- Nicotine dose-response to Fx-Trp at α 149
- Nicotine dose-respone to fluorination at other binding site Trp residues
- N-methyl-nicotinium dose-response
- Noracetylcholine dose-response
- Variation of pH with tertiary ACh
- TMA dose-response
- Serotonin binding to 5-HT 3A R substituted with Fx-Trp series at W183
- Binding of tertiary and quaternary 5-HT analogs
- Involvement of hydrogen bonding at W183 investigated
- Efficacy
- Results from neuronal nAChR
In the case of nicotine, the pKa of the pyrrolidinium nitrogen is 7.8, quite close to physiological pH.22 Thus, it is quite reasonable to assume that the microenvironment at the nAChR binding site may be sufficiently basic to induce deprotonation. To test the hypothesis that fluorination affects not only the cation-π-bonding ability of the residue at α149, but also the protonation state of nicotine, a quaternary analog of nicotine, N-methyl-nicotinium, was prepared by Niki Zacharias.22 .
Discussion
- Anomalous behavior of nicotine
- Similarity between ACh and serotonin
- Expected behavior of variously alkylated ammonium compounds
- Experimental behavior of primary and substituted ammonium compounds
- Potential acidity of protonated nicotine
- Behavior of tertiary and quaternary nAChR agonists
- Conclusions
The EC50 rises, in fact, in a fairly regular manner with decreasing cation-π binding ability of the arom. Right panel: Plot of log [EC50/EC50(wt)] for ACh (O), nicotine ( ) and N-Menicotin () in nAChR versus the calculated cation-π binding energy of the series of fluorinated Trp derivatives.
Attempts to introduce unnatural amino acids into neuronal nAChR
- Motivation
- Expression of α 4 β 2 in oocytes
- Initial attempts at wild-type recovery by nonsense suppression
- Increasing translational efficiency
- Promoting folding, assembly, and transport to the plasma membrane
- Alternative strategies for increasing expression
- Conventional mutagenesis experiments
- Future directions
This technique is valuable here because membrane preparations from the plasma membrane of the cell can be compared with those from the cytoplasm. The affinity of the muscle-type receptor for ACh is significantly greater than its affinity for nicotine, as noted above. The αR55W mutation occurred in the muscle receptor, and the W88R mutation occurred in hα4. They measured whole-cell currents in response to different concentrations of ACh and nicotine, hoping to observe a change in the normal pattern of strength of ACh and nicotine.
The potency difference between ACh and nicotine at the muscle receptor can be seen from the behavior of the wild-type receptor in response to three concentrations of each agonist, 1 µM, 10 µM and 100 µM.
Experimental methods
- Electrophysiology
- Unnatural amino acid suppression in muscle nAChR and 5-HT 3A R
- Molecular biology of α 4 β 2
- Introduction of the HA epitope into h α 4
- Planning for four-base codon suppression
As a negative control, truncated 74 nt tRNA or truncated tRNA ligated to dCA was co-injected with mRNA in the same manner as fully charged tRNA. During attempts to introduce unnatural amino acids into α4β2 nAChR, numerous constructs were created and obtained. It may be that this increase in suppression was due to improvements made in the arc lamp arrangement by James Petersson during this period.
The HA epitope was successfully introduced into pSP-α4, at a position in the M3-M4 loop [PPQQPLEAE*KASPHP] that is homologous to the traditional 347 position (see Chap. 3) in muscle alpha, and also at the C-terminus immediately before. the TAA codon [WLAGMI*].
This approach relies on the incorporation of the unnatural amino acid Npg via nonsense suppression. Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor. In the Americas, some of the most dramatic masks were carved by the people of the Northwest Coast.
Incorporation of caged cysteine and capped tyrosine into a transmembrane segment of the nicotinic acetylcholine receptor.
Analysis of protein conformation
Introduction
In the work presented in the previous chapter, unnatural amino acids were used to make more subtle side chain perturbations than are possible using conventional mutagenesis. However, the range of side chain substitution allowed by the ribosome allows unnatural amino acids that cause much more radical transformations. In this chapter, nonsense suppression was used to chemically modify proteins both dynamically and after isolation from the membrane.
In the first case, nitrophenylglycine (Npg) was introduced to photolytically cleave the polypeptide backbone at the site of amino acid introduction.1 The second set of experiments investigated the possibility of site-specific photoinduced cross-linking mediated by the unnatural amino acid benzoylphenylalanine (Bpa).2 In the third study, hydroxy acids were inserted in the backbone by nonsense suppression.3-5 The resulting ester bond is unstable to strong base treatment, allowing for the incorporation of hydroxy acids to be used for protein linkage mapping.4 Finally, cysteine and tyrosine residues with bulky photo-removable protecting groups were used to induce conformational changes in a sensitive transmembrane region of the nAChR.6.
Site-specific backbone cleavage in nAChR Cys loop using Npg
- Site-specific nitrobenzyl-induced photochemical proteolysis (SNIPP
- Previous results with Npg in the nAChR
- Experimental design
- Results
- Conclusions from non-alpha Npg cleavage
This experiment seems to confirm the prevailing opinion about. significance of the Cys loop. In addition, it was verified that the HA tag could be introduced at a homologous position in each of the four nAChR subunits. Collection of membranes from such oocytes followed by Western blotting always led to the detection of subunits in the membrane fraction.
Thus, interpretation of the results obtained by photolysis of receptors containing Npg in non-alpha Cys loops remains difficult.
Site-specific photocrosslinking with Bpa
To gain temporal control over the state of the tyrosine side chain at position 242, the unnatural amino acid Tyr(ONb) at position 242 was incorporated into Kir2.1. Irradiated oocytes co-expressing wild-type Kir2.1 and v-Src showed no decrease over the course of the experiment. Effect of coexpression of v-Src and exposure to PAO, but not oocyte irradiation.
The protein-conducting channel in the membrane of the endoplasmic reticulum is open laterally to the lipid bilayer.
Using hydroxy acids to establish Cys-Cys connectivity of the P2X2 receptor
- Experimental design
- Results
- Future directions
Use of photolabile side chains to induce dynamic conformational change
- Experimental design
- Results
- Future directions
In the case of Bpa, the photoactive side chain was included for cross-linking purposes. As noted above, the conformational change resulting from ligand-gated ion channel agonist binding is mediated by gating in the transmembrane regions, resulting in channel opening. Suppression in the alpha subunit proved to be quite ineffective, although better results were obtained in both the beta and gamma subunits.
As will be seen in the next chapter, caged amino acid side chains can also be used to control certain post-translational modifications.
Interactions of leucine residues at the 9' position of the M2 domain of AChR probed using unnatural amino acid mutagenesis. Such a motif has been demonstrated to regulate the expression of the sodium channel ENaC in oocytes.77. Here, Kir2.1 was chosen as an experimental system because there is a clear reduction in current that is dependent on a single tyrosine in the intracellular C-terminal tail of the receptor, Y242.
Ribosome-mediated incorporation of a non-standard amino acid into a peptide by extension of the genetic code.
Unnatural amino acids with caged side chains
Introduction
- Caged compounds
- Caged amino acids, particularly tyrosine
- Caged proteins
The protected connection is thus often referred to as being 'caged' or 'masked'. Irradiation of the system ('de-caging') removes the protecting groups and restores the intrinsic efficiency of the molecule. A system that relies on a small molecule is primed by diffusion of cage compounds near the active site. Since the encapsulated compounds lack the efficiency of the natural substrate, the investigated process is held up until it is experimentally initiated.
To solve the problem of specificity, proteins containing caged amino acids can be synthesized through either solid-phase methods or by semisynthesis followed by ligation of the unnatural amino acid-containing portion to the rest of the protein.
Using nonsense suppression to introduce caged amino acids into proteins
- Expression systems
- Caging groups
- Side chain uncaging
- Choice of receptor
- Assay
- Precedent for introducing caged amino acids by nonsense suppression
- Application of caged tyrosine to ion channels
In vivo unnatural amino acid mutagenesis provides a way to solve the problem of specificity and biological context of a caged protein. The presence of pendant groups has been shown to attenuate the response of the receptor to acetylcholine. The experiments described in Chapter 3 with caged cysteine and tyrosine incorporation into the transmembrane domain of the M2 nAChR are an example of caged amino acids incorporated into membrane-spanning protein domains.
The effectiveness of caged tyrosine in both of these contexts led to a consideration of using caged tyrosine to control the accessibility of this particular residue in a functioning ion channel.
Incorporation of caged tyrosine into the potassium channel Kir2.1
- Introduction
- Results
- Discussion
- Summary
- Experimental methods
Importantly, PAO-treated and irradiated oocytes co-expressing Kir2.1 and v-Src showed a mean decrease in min current after irradiation. Importantly, Table 4.4 shows that the current decrease observed in oocytes expressing v-Src and Kir2.1-Y242-Tyr(ONb) was accompanied by a significant decrease in capacitance. The oocyte on the left was injected with v-Src and Tyr(ONb)-repressed Kir2.1-Y242TAG.
Current and capacitance data from representative oocytes coexpressing v-Src and Kir2.1-Y242TAG, suppressed with Tyr(ONb) and treated with PAO.
Probing the structure and function of the tachykinin neurokinin-2 receptor by site-specific biosynthetic incorporation of fluorescent amino acids. Effects of the protein-tyrosine-phosphatase inhibitor phenylarsine oxide on excision-activated calcium channels in Lymnaea neurons. An intermediate state of the GABA transporter GAT1 revealed by simultaneous voltage clamp and fluorescence.
Irradiation itself reveals the wild-type phosphoamino acid, placing the phosphorylation state of the protein under direct experimental control.
Caged phosphoamino acids
Introduction
- Design of caged phosphoamino acids
- Non-hydrolyzable analogs
- Mechanism-based phosphatase inhibitors
The three unnatural amino acids discussed here, caged phosphorylatable residues, caged phosphoamino acids, and caged nonhydrolyzable phosphoamino acid mimetics, provide a complete set for analysis of phosphorylation of a particular side chain in a protein. The simplest design for caged phosphoamino acids involves protecting the phosphate oxygen atoms with photoremovable protecting groups. Semi-synthetic peptides and proteins containing caged phosphoamino acids require transport into cells to be used to investigate signal transduction.
Introduction of caged phosphoamino acids by unnatural amino acid mutagenesis has the advantages of site specificity and compatibility with in situ use.
Synthesis of caged phosphoamino acids
Therefore, an even simpler route was then used to generate caged phosphoserine 9 (Figure 5.8) and phosphothreonine 12 (Figure 5.9).
Synthesis of caged non-hydrolyzable phosphoamino acid analogs
- Difluorophosphonate intermediates
- Future prospects
A number of methods exist for the formation of aliphatic carbon-phosphorus bonds.67,71-81 Most of them are useful for the generation of phosphonates, although a number can be used to prepare difluorophosphonates.67,71-81 The triflate displacement methodology employed by Berkowitz and others has been selected for the synthesis of serine and threonine analogs.41,61-. In most cases the phosphates are protected as the diethyl ester, although Berkowitz has reported benzyl- and allo-protected difluorophosphonates.41,83. An important intermediate for the copper(I)-mediated cross-linking central to the Shibuya and Burton pathways is the dialkyl bromodifluorophosphonate.
The ethyl phosphodiester can be deprotected under the conditions of Rabinowitz, McKenna, and Jung (TMSI/TFA) to give the dianion.55,56 Conditions analogous to those used in peptide coupling have been reported to generate.
Identification of appropriate biological systems for analysis by caged pAA
- Tyrosine phosphorylation
- Serine phosphorylation
Progress toward controlling phosphorylation with unnatural amino acids
- Tyrosine phosphorylation
Synthetic methods
- General experimental procedures
- Bis(nitrobenzyl) diisopropyl phosphoramidite
- Boc-Tyrosine-OtBu
- Boc-pTyr(ONb) 2 -OtBu
- pTyr(ONb) 2
- Nitrobenzyl phosphite
- Nitrobenyl H-phosphonate
- dCA-4PO-pTyr(ONb) 2
