Name: residue-specific exchange of proline by proline analogs in fluorescent proteins: how "molecular surgery" of the backbone affects folding and stability
Uploaded: 2022-02-03T13:30:00
Duration: 10 min 31 s
Description: Watch this Scientific Journal Video about Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability at JoVE

This work was supported by the German Research Foundation (Cluster of Excellence &#34;Unifying Systems in Catalysis) to T.F. and N.B. and by the Federal Ministry of Education and Science (BMBF Program &#34;HSP 2020&#34;, TU-WIMIplus Project SynTUBio) to F.-J.S. and T.M.T.T.

The authors disclose all and any conflicts of interest.

In nature, manipulations with protein structures and functions typically occur due to mutations, the phenomenon that leads to an exchange of an amino acid identity at certain positions in the protein sequence. This natural mechanism is widely applied as a biotechnological method for protein engineering in the form of mutagenesis, and it relies on the repertoire of the 20 canonical amino acids involved in the process. The exchange of proline residues is problematic, however. Due to its special backbone group architecture, it is hardly interchangeable with the remaining 19 residues for replacement72. For example, proline is typically known as a secondary structure breaker in polypeptide sequences because of its poor compatibility with the most common secondary structures, i.e., &#945;-helix and &#946;-strand. This proline feature is easily lost when the residue is mutated to another amino acid from the common repertoire. The replacement of proline with its chemical analogs offers an alternative approach, which enables to keep the basic backbone features of the parent proline residue while imposing bias on its specific conformational transitions or producing modulations of the molecular volume and polarity. For example, it is possible to supply bacterial cultures with analog structures such as hydroxy-, fluoro-, alkyl-, dehydroprolines, structures having variable ring sizes and more, thus facilitating the production of a protein containing specific proline residue alterations.
The selective pressure incorporation (SPI) method described in this study allows for a global, i.e., residue-specific replacement of all prolines in the target protein with related chemical analogs. The importance of the method is reflected by the fact that SPI allows creating sequence changes inaccessible to common mutagenesis techniques. For example, it allows the production of a target protein containing rather small structural changes that may typically not exceed one or two atom replacements/deletions/additions, as demonstrated in this study. Such protein modifications are dubbed &#34;atomic mutations&#34;73,74. In a fluorescent protein such as GFP, the result of this molecular intrusion can be seen in the velocity of folding, local polarities, protein packing, stability of the involved structural features. The changes in the absorbance and fluorescence properties are produced indirectly due to the impact on protein folding and residue microenvironments. The precision of the molecular changes performed by SPI is typically much higher, as compared to mutations of prolines to other canonical residues, the latter being typically detrimental for the protein folding, production, and isolation.
As a production method, the SPI approach uses the substrate tolerance of the aminoacyl-tRNA synthetase pocket towards chemical analogs of the native amino acid. The synthetase is responsible for the correct identification of the amino acid structure, while the incorporation into proteins occurs downstream in the translation process. Instrumentally, the protein production, isolation, and purification in SPI are performed in a way typical for any other recombinant protein expression techniques; however, with some additions to the protocol as follows: Proline, which is bound for replacement, is provided at the beginning of the fermentation process, such that the cells can grow and develop their intact cellular machinery. However, the cell culture is not allowed to reach the maximal optical density, to keep the cells in the logarithmic phase optimal for protein expression. There are two major variations of the SPI method at this point. In the first one, the concentration of proline is adjusted in the initial growth medium (a chemically defined medium) such that depletion of proline happens without any external intrusion. The cells exhaust proline in the medium before they can exit the logarithmic growth phase, and then, subsequently, the analog is added, and the protein of interest production is induced. In the second version of the method, the cells are grown in the medium containing proline until the middle of their logarithmic phase. At this point, the cells should be taken out and physically transferred into another medium, which no longer contains proline, only the analog, with subsequent induction of the protein of interest. In both versions, the analog and the protein induction reagent are provided to the pre-grown cells. The isolation and purification of the wild-type protein are performed in the same way as for the variants. In principle, every available Pro-auxotrophic strain can be used as an expression host. Nevertheless, expression tests to identify the most suitable host are advisable. Also, tests of different chemically defined media can be used to optimize protein yield.
There are certain requirements regarding the chemical analogs that need to be considered for SPI, such as solubility and concentration. The metabolic availability and uptake of amino acids are dependent on the number of dissolved molecules in the medium. To increase the solubility of a particular compound, slightly acidic or alkaline conditions can be chosen. Since the artificial molecules can cause growth inhibitory effects due to their cell toxicity, the concentration should be lowered to a minimum in order to avoid cell stress75.
A minor weakness of SPI is the decrease of incorporation efficiency with larger numbers of positions that need to be exchanged. In principle, a reduction of the amino acid frequency within the target biomolecule by site-directed mutagenesis can solve this problem. However, the structural and functional properties of a desired protein might be affected by changing the primary structure.
As mentioned before, SPI allows residue-specific&#160;replacement of the canonical amino acid. This implies that non-canonical amino acids are inserted into every position of the canonical amino acid within the target protein, including conserved residues that are indispensable for protein function or folding. Alternative methods for site-specific incorporation are the only possibility to overcome this issue3. In the past few decades, the orthogonal pair method has been developed that can produce proteins containing modified residues at predefined sites. The most common modification of this method is known as stop codon suppression. This method is based on an engineered orthogonal translation system dedicated for site-specific incorporation of synthetic amino acids76. More than 200 amino acids with different side-chain modifications have been incorporated into proteins to date using this approach77. However, these translation systems are still not suitable for insertions of proline analogs into target proteins. Furthermore, the method&#39;s performance is considered low in the case of minor amino acid modifications because some background promiscuity of the aminoacyl-tRNA synthetase typically remains in the engineered translation systems.
Using SPI, we produced a number of &#946;-barrel fluorescent protein variants and studied outcomes of the exchange of proline with its unnatural analogs. In the case of proline replacement with R-Flp and Dfp, a dysfunctional protein was produced by the expression host. The effect is likely produced by protein misfolding. The latter may originate from the C4-exo conformation promoted by R-Flp, which is unfavored by the parent protein structures27. With Dfp, the misfolding is likely to be produced by the diminished velocity of the trans-to-cis peptide bond isomerization at the proline residue27. The latter is known to be among the limiting steps in the kinetic profile of the protein folding that affects &#946;-barrel formation and subsequent chromophore maturation. Indeed, for both amino acids, R-Flp and Dfp, the protein production resulted in an aggregated and insoluble protein. Consequently, chromophore formation could not occur, and the fluorescence was lost entirely. With S-Flp and Dhp, however, proper protein maturation was observed, resulting in fluorescent protein samples for each analog/protein combination. Despite some modulations in the absorbance and fluorescence features of the protein, these largely remained similar to those of the wild-type proteins. The effect of the amino acid substitution was revealed in the refolding kinetics studies. The latter showed a faster refolding in the case of replacement with S-Flp. Model studies have shown that this residue may generate some improvement in the trans-to-cis amide rotation velocity and lead to the formation of the C4-endo conformation. Both these factors are likely to contribute to the beneficial kinetic effects of this residue in EGFP. In contrast, Dhp produced kinetic folding profiles maximally similar to the parent protein. The diversity of the outcomes produced by mere atomic mutations in the examined fluorescent proteins illustrates the potential of the SPI production method in altering target protein properties. The protein alterations induced by proline replacement with the analogs have further implications in the engineering of enzymes78,79,80&#160;and ion channels81,82, as well as in general engineering of protein stability.
The basic limitation of the SPI method is its &#34;all-or-none&#34; mode in exchanging proline resides with related analogs. It would be of great advantage to be able to select precisely, which proline residues should be replaced with the analogs, and which ones should remain unmodified. However, at present, there is no technique that could perform such a sophisticated production using a microbial production host. Chemical synthesis of proteins83,84, as well as cell-free production85,86, are the two alternative methods that can produce position-specific proline modifications. Nonetheless, their operational complexity and low production yields make them inferior compared to the production in living cells. As of now, SPI remains the most operationally simple and robust approach for the production of complex proteins bearing atomic mutations. By introducing unnatural amino acid substitutes, the method allows modifying protein features in a targeted manner, as exemplified here by alterations in folding and light absorption/emission of fluorescent proteins generated by proline replacements.

Classical site-directed mutagenesis allows permutation of any existing gene-encoded protein sequence by codon manipulation at the DNA level. To study protein folding and stability, it is often desirable to replace similar amino acids with similar counterparts. However, traditional protein mutagenesis is definitely limited to structurally similar replacements among canonical amino acids such as Ser/Ala/Cys, Thr/Val, Glu/Gln, Asp/Asn, Tyr/Phe, which are present in the standard genetic code repertoire. On the other hand, there are no such possibilities for other canonical amino acids such as Trp, Met, His, or Pro, which often play essential structural and functional roles in proteins1. An ideal approach to study these interactions in the context of the highly specific internal architecture of proteins and their folding process is to generate non-disruptive isosteric modifications. Indeed, when isosteric amino acid analogs of these canonical amino acids, also known as non-canonical amino acids (ncAAs), are inserted into proteins, they allow for subtle changes even at the level of single atoms or atom groups such as H/F, CH2/S/Se/Te known as &#34;atomic mutations&#34;2. Such &#34;molecular surgery&#34; produces altered proteins whose properties result solely from the exchange of single atoms or groups of atoms, which in favorable cases can be analyzed, and the detected changes can be rationalized. In this way, the scope of protein synthesis to study protein folding and structure is extended far beyond classical DNA mutagenesis. Note that proteins generated by site-directed mutagenesis are usually referred to as &#34;mutants,&#34; whereas proteins with substituted canonical amino acids are referred to as &#34;variants&#34;&#160;3, &#34;alloproteins&#34;4, or &#34;protein congeners&#34;5.
The green fluorescent protein (GFP), first identified in the marine organism Aequorea victoria, exhibits bright green fluorescence when exposed to ultraviolet-to-blue light6,7. Today, GFP is commonly used as a highly sensitive labeling tool for routine visualization of gene expression and protein localization in cells via fluorescent microscopy. GFP has also proven useful in various biophysical8,9,10 and biomedical11,12 studies, as well as in protein engineering13,14,15. Rigorous analysis of the GFP structure enabled the creation of numerous variants characterized by varied stability and fluorescence maxima16,17. Most of the GFP variants used in cell and molecular biology are monomeric proteins both in solution and in the crystal18. Their principal structural organization is typical for all members of the GFP family, independent of their phylogenetic origin, and consists of 11 &#946;-strands forming a so-called &#946;-barrel, while a kinked &#945;-helix is running through the center of the barrel and bears the chromophore (Figure 1A). The autocatalytic maturation of the chromophore (Figure 1B) requires the precise positioning of the side chains surrounding it in the central place of the protein; many of these side chains are highly conserved in other GFP variants19. In most fluorescent proteins from jellyfish such as Aequorea victoria, the green-emitting chromophore consists of two aromatic rings, including a phenol ring of Tyr66 and the five-membered heterocyclic structure of imidazolinone (Figure 1B). The chromophore, when properly embedded in the protein matrix, is responsible for the characteristic fluorescence of the whole protein. It is located in the center of the structure, while the barrel structure insulates it from the aqueous medium20. Exposure of the chromophore to the bulk water would result in fluorescence quenching, i.e., loss of fluorescence21.
The proper folding of the barrel-like structure is essential for protecting the chromophore against fluorescence quenching22. Proline (Pro) residues play a special role in the structural organization of GFP23. Being unable to support a &#946;-strand, they constitute connecting loops responsible for maintaining the protein structure as a whole. Not surprisingly, 10-15 proline residues are found in both Aequorea- and Anthoathecata-derived GFPs; some of them are highly conserved in other types of fluorescent &#946;-barrel proteins. Prolines are expected to critically influence folding properties due to their peculiar geometric features. For example, in Aequorea-derived GFPs, of the ten proline residues (Figure 2A), nine form trans- and only one forms a cis-peptide bond (Pro89). Pro58 is essential, i.e., not interchangeable with the rest of the 19 canonical amino acids. This residue may be responsible for the correct positioning of the Trp57 residue, which has been reported to be crucial for chromophore maturation and the overall GFP folding24. The fragment PVPWP with three proline residues (Pro54, Pro56, Pro58) and Trp57 is the essential part of the &#34;lower lid&#34; in the GFP structure from Figure 1A. The PVPWP structural motif is found in several proteins such as cytochromes and eukaryotic voltage-activated potassium channels25. Proline-to-alanine substitutions at positions 75 and 89 are also detrimental to protein expression and folding and abolish chromophore maturation. Pro75 and Pro89 are part of the &#34;upper lid&#34; burying the chromophore (Figure 1A) and are conserved across 11-stranded &#946;-barrel fluorescent proteins23. These two &#34;lids&#34; keep the chromophore excluded from the aqueous solvent, even when the stable tertiary structure has been partially broken26. Such a specific molecular architecture protects the fluorophore from collisional (dynamic) fluorescence quenching, e.g., by water, oxygen, or other diffusible ligands.
In order to perform molecular engineering of the GFP structure, one should introduce amino acid substitutions in the primary structure of the protein. Numerous mutations have been performed on GFP, providing variants with elevated stability, fast and reliable folding, and variable fluorescence properties17. Nonetheless, in most cases, mutation of proline residues is considered a risky approach due to the fact that none of the remaining 19 canonical amino acids can properly restore the conformational profile of the proline residue27. Thus, an alternative approach has been developed, in which proline residues are replaced with other proline-based structures, dubbed as proline analogs28. Owing to its unique cyclic chemical structure, proline exhibits two characteristic conformational transitions (Figure 1C): 1) the proline ring puckering, a fast process entailing organization of the backbone, which primarily affects the &#966; torsion angle, and 2) the peptide bond cis/trans isomerization, a slow process impacting backbone folding via the &#969; torsion angles. Due to its slow nature, the latter transition is commonly responsible for the rate-limiting steps in the folding process of the whole protein. It has been shown previously that peptide bond cis/trans isomerization around some proline residues features slow steps in the folding of GFP variants. For example, the formation of the cis-peptide bond at Pro89 features the slow step in the process of folding because it relies on the bond transition from trans to cis29. A faster refolding can be achieved after replacing Pro89 with an all-trans peptide loop, i.e., by abolishing a cis-to-trans isomerization event30. In addition to the cis/trans isomerization, the pucker transitions may also generate profound changes in protein folding due to the backbone organization and packing within the protein interior27,31.
Chemical modifications result in alteration of the intrinsic conformational transitions of the proline residues, thereby affecting the ability of the protein to fold. Certain proline analogs are particularly attractive candidates for proline substitution in proteins as they allow the manipulation and study of the folding properties. For example, (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) are four analogs (Figure 1D) that differ minimally from proline in terms of both molecular volume and polarity32. At the same time, each analog exhibits a distinct ring puckering: S-Flp stabilizes the C4-endo pucker, R-Flp stabilizes the C4-exo pucker, Dfp exhibits no apparent pucker preference, while Dhp abolishes the puckering (Figure 1D)33. By using these analogs in the protein structure, one can manipulate with the conformational transition of the proline residues, and with this, affect the properties of the resulting GFP variants.
In this work, we set out to incorporate the designated set of proline analogs (Figure 1D) into the structure of GFP variants using the selective pressure incorporation method (SPI, Figure 3)34. Replacement of amino acid residues with their closest isostructural analogs is an applied biotechnological concept in protein design35,36. Thus, the effects of proline analogs in a model protein illustrate their potential to serve as tools in protein engineering37. The production of proteins containing desired analogs was performed in modified E. coli strains that are not able to produce proline (proline-auxotrophy). Thus, they could be forced to accept replacement of substrates in the process of protein biosynthesis38. This global substitution of proline is enabled by the natural substrate flexibility of endogenous aminoacyl-tRNA synthetases39, the key enzymes catalyzing the esterification of tRNAs with appropriate amino acids40. In general, as outlined in Figure 3, cellular growth is performed in a defined medium until the mid-logarithmic growth phase is reached. In the next step, the amino acid to be replaced is intracellularly depleted from the expression system during fermentation and subsequently exchanged by the desired analog or ncAA. Target protein expression is then induced for residue-specific non-canonical amino acid incorporation. The substitution of the cognate amino acid with its analog occurs in a proteome-wide manner. Although this side effect may have a negative impact on the growth of the host strain, the quality of target protein production is mostly not affected, since, in recombinant expression, the cellular resources are mainly directed to the production of the target protein41,42. Therefore, a tightly regulated, inducible expression system and strong promoters are crucial for high incorporation efficiency43. Our approach is based on multiple residue-specific incorporation of ncAAs in response to sense codons (sense codon reassignment), whereby within the target gene, the number of positions for Pro analog insertion can be manipulated via site-directed mutagenesis44. A similar approach was applied in our previous report on the preparation of recombinant peptides with antimicrobial properties45. In this work, we have applied the SPI method, which allows all proline residues to be replaced by related analogs, to generate proteins expected to possess distinct physicochemical properties not present in proteins synthesized with the canonical amino acid repertoire. By characterizing the folding and fluorescence profile of resulting variants, we aim to showcase the effects of atomic substitutions in variants of GFP.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>VWR</td>
			<td>HiPerSolv CHROMANORM ULTRA for LC-MS, 83642</td>
			<td>LC-MS grade required</td>
		</tr>
		<tr>
			<td>Acrylamide and bisacrylamide aqueous stock solution at a ratio of 37.5:1 (ROTIPHORESE Gel 30)</td>
			<td>Carl Roth</td>
			<td>3029.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar-agar</td>
			<td>Carl Roth</td>
			<td>5210</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium molybdate ((NH4)2MoO4)</td>
			<td>Sigma-Aldrich</td>
			<td>277908</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium peroxydisulphate (APS)</td>
			<td>Carl Roth</td>
			<td>9592.2</td>
			<td>&#8805;98 %, p.a., ACS grade required</td>
		</tr>
		<tr>
			<td>Ammonium sulfate ((NH4)2SO4)</td>
			<td>Sigma-Aldrich</td>
			<td>A4418</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Carl Roth</td>
			<td>K029</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4501</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brillant Blue R 250</td>
			<td>Carl Roth</td>
			<td>3862</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper sulfate (CuSO4)</td>
			<td>Carl Roth</td>
			<td>CP86.1</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Carl Roth</td>
			<td>6780</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-Bis-(dimethylamino)-ethane,&#160;N,N,N&#39;,N&#39;-Tetramethylethylenediamine (TEMED)</td>
			<td>Carl Roth</td>
			<td>2367.3</td>
			<td>&#8805;99 %, p.a., for electrophoresis</td>
		</tr>
		<tr>
			<td>1,4-dithiothreitol (DTT)</td>
			<td>Carl Roth</td>
			<td>6908</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td></td>
		</tr>
		<tr>
			<td>di-potassium hydrogen phosphate (K2HPO4)</td>
			<td>Carl Roth</td>
			<td>P749.1</td>
			<td></td>
		</tr>
		<tr>
			<td>di-sodium hydrogen phosphate (Na2HPO4)</td>
			<td>Carl Roth</td>
			<td>X987</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Dowex 50WX8-100 (hydrogen form)</td>
			<td>Acros Organics / Thermo Fisher Scientific (Waltham, U.S.A.)</td>
			<td>10731181</td>
			<td>cation exchange resin</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carl Roth</td>
			<td>9065.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>VWR</td>
			<td>HiPerSolv CHROMANORM for LC-MS, 84865</td>
			<td>LC-MS grade required</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Carl Roth</td>
			<td>3738.5</td>
			<td>100 %, p. a.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Carl Roth</td>
			<td>3783</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Carl Roth</td>
			<td>X998</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen chlroide (HCl)</td>
			<td>Merck</td>
			<td>295426</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron(II) chloride (FeCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>380024</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Carl Roth</td>
			<td>AE73.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Sigma-Aldrich</td>
			<td>I6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Carl Roth</td>
			<td>KK36.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Carl Roth</td>
			<td>8283.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese chloride (MnCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>63535</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Carl Roth</td>
			<td>4227.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Unstained Protein Ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>26614</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Carl Roth</td>
			<td>6781.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Carl Roth</td>
			<td>7156</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Carl Roth</td>
			<td>P029</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate (NaH2PO4)</td>
			<td>Carl Roth</td>
			<td>T879</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulphate (NaC12H25SO4)</td>
			<td>Carl Roth</td>
			<td>0183</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine</td>
			<td>Sigma-Aldrich</td>
			<td>T4625</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Sigma-Aldrich</td>
			<td>T6508</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris hydrochloride (Tris-HCl)</td>
			<td>Sigma-Aldrich</td>
			<td>857645</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)-aminomethane (Tris)</td>
			<td>Carl Roth</td>
			<td>5429</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Carl Roth</td>
			<td>8952</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Carl Roth</td>
			<td>2363</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc chloride (ZnCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>229997</td>
			<td></td>
		</tr>
		<tr>
			<td>L-alanine</td>
			<td>Sigma-Aldrich</td>
			<td>A7627</td>
			<td></td>
		</tr>
		<tr>
			<td>L-arginine</td>
			<td>Sigma-Aldrich</td>
			<td>A5006</td>
			<td></td>
		</tr>
		<tr>
			<td>L-asparagine</td>
			<td>Sigma-Aldrich</td>
			<td>A8381</td>
			<td></td>
		</tr>
		<tr>
			<td>L-aspartic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A0884</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamic acid</td>
			<td>Sigma-Aldrich</td>
			<td>G2128</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G3126</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>L-histidine</td>
			<td>Sigma-Aldrich</td>
			<td>H8000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-isoleucine</td>
			<td>Sigma-Aldrich</td>
			<td>I2752</td>
			<td></td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>Sigma-Aldrich</td>
			<td>L8000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L5501</td>
			<td></td>
		</tr>
		<tr>
			<td>L-methionine</td>
			<td>Sigma-Aldrich</td>
			<td>M9625</td>
			<td></td>
		</tr>
		<tr>
			<td>L-phenylalanine</td>
			<td>Sigma-Aldrich</td>
			<td>P2126</td>
			<td></td>
		</tr>
		<tr>
			<td>L-proline</td>
			<td>Sigma-Aldrich</td>
			<td>P0380</td>
			<td></td>
		</tr>
		<tr>
			<td>L-serine</td>
			<td>Sigma-Aldrich</td>
			<td>S4500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-threonine</td>
			<td>Sigma-Aldrich</td>
			<td>T8625</td>
			<td></td>
		</tr>
		<tr>
			<td>L-tryptophan</td>
			<td>Sigma-Aldrich</td>
			<td>T0254</td>
			<td></td>
		</tr>
		<tr>
			<td>L-tyrosine</td>
			<td>Sigma-Aldrich</td>
			<td>T3754</td>
			<td></td>
		</tr>
		<tr>
			<td>L-valine</td>
			<td>Sigma-Aldrich</td>
			<td>V0500</td>
			<td></td>
		</tr>
		<tr>
			<td>(4S)-fluoroproline</td>
			<td>Bachem</td>
			<td>4033274</td>
			<td>Make sure that all proline analogs are proline free, check content. Otherwise include a step to consume proline contaminations during expression.</td>
		</tr>
		<tr>
			<td>(4R)-fluoroproline</td>
			<td>Bachem</td>
			<td>4033275</td>
			<td>Make sure that all proline analogs are proline free, check content. Otherwise include a step to consume proline contaminations during expression</td>
		</tr>
		<tr>
			<td>3,4-dehydroproline</td>
			<td>Bachem</td>
			<td>4003545</td>
			<td>Make sure that all proline analogs are proline free, check content. Otherwise include a step to consume proline contaminations during expression</td>
		</tr>
		<tr>
			<td>4,4-difluoroproline</td>
			<td>Enamine</td>
			<td>EN400-17448</td>
			<td>Make sure that all proline analogs are proline free, check content. Otherwise include a step to consume proline contaminations during expression</td>
		</tr>
		<tr>
			<td>Conical polystyrene (Falcon) tubes, 15 mL</td>
			<td>Fisher Scientific</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical polystyrene (Falcon) tubes, 50 mL</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis membrane, Molecular Weight Cut-Off (MWCO) 5,000</td>
			<td>Spectrum Medical Industries</td>
			<td>Spectra/Por MWCO 5000 dialysis membrane, 133198</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilized Metal ion Affinity Chromatography (IMAC) column 1 mL, Ni-NTA</td>
			<td>GE Healthcare</td>
			<td>HisTrap HP, 1 mL, 17-5247-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-Lock syringe, 5 mL</td>
			<td>Carl Roth</td>
			<td>EP96.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-Lock syringe, 20 mL</td>
			<td>Carl Roth</td>
			<td>T550.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-Lock syringe, 50 mL</td>
			<td>Carl Roth</td>
			<td>T552.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (polystyrene, sterile)</td>
			<td>Carl Roth</td>
			<td>TA19</td>
			<td></td>
		</tr>
		<tr>
			<td>pQE-80L plasmid vector</td>
			<td>Qiagen</td>
			<td>no longer available</td>
			<td>replaced by N-terminus pQE Vector set Cat No./ID: 32915</td>
		</tr>
		<tr>
			<td>Pro-auxotrophic E. coli strain JM83</td>
			<td>Addgene</td>
			<td>50348</td>
			<td>https://www.addgene.org/50348/</td>
		</tr>
		<tr>
			<td>Pro-auxotrophic E. coli strain JM83</td>
			<td>ATCC</td>
			<td>35607</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-bottom polystyrene tubes, 14 mL</td>
			<td>Fisher Scientific</td>
			<td>Corning Falcon, 14-959-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter 0.45 &#181;m with polyvinylidene difluoride (PVDF) membrane</td>
			<td>Carl Roth</td>
			<td>CCY1.1</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Performance Liquid Chromatography (HPLC) column for LC-ESI-TOF-MS</td>
			<td>Sigma-Aldrich</td>
			<td>Supelco Discovery BIO Wide Pore C5 HPLC column, 3 &#181;m particle size, 10 cm x 2.1 mm</td>
			<td>with conical 0.1 mL glass inserts, screw caps and septa</td>
		</tr>
		<tr>
			<td>HPLC autosampler vials 1.5 mL</td>
			<td>Sigma-Aldrich</td>
			<td>Supelco 854165</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass spectrometer for LC-ESI-TOF-MS</td>
			<td>Agilent</td>
			<td>Agilent 6530 Accurate-Mass QTOF</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass spectrometry data analysis software</td>
			<td>Agilent</td>
			<td>MassHunter Qualitative Analysis software v. B.06.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop centrifuge for 1.5 mL Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>5427 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Cooling centrifuge for 50 mL Falcon tubes</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Protein Liquid Chromatography (FPLC) system</td>
			<td>GE Healthcare</td>
			<td>&#196;KTA pure 25 L</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence spectrometer</td>
			<td>Perkin Elmer</td>
			<td>LS 55</td>
			<td></td>
		</tr>
		<tr>
			<td>High pressure microfluidizer for bacterial cell disruption</td>
			<td>Microfluidics</td>
			<td>LM series with &#8220;Z&#8221; type chamber</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker for bacterial cultivation</td>
			<td>Infors HT</td>
			<td>Minitron</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump for liquid chromatography (LC)</td>
			<td>GE Healthcare</td>
			<td>P-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic homogenizer for bacterial cell disruption</td>
			<td>Omnilab</td>
			<td>Bandelin SONOPULS HD 3200, 5650182</td>
			<td>with MS72 sonifier tip</td>
		</tr>
		<tr>
			<td>UV-Vis spectrophotometer</td>
			<td>Biochrom</td>
			<td>ULTROSPEC 2100</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis/NIR spectrophotometer</td>
			<td>Perkin Elmer</td>
			<td>LAMBDA 950 UV/Vis/NIR</td>
			<td></td>
		</tr>
	</tbody>
</table>

residue-specific exchange of proline by proline analogs in fluorescent proteins: how "molecular surgery" of the backbone affects folding and stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

Watch this Scientific Journal Video about Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability at JoVE

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...

Research

JoVE Journal

Bioengineering

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...