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><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 ((NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;)</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>&amp;ge;98 %, p.a., ACS grade required</td></tr><tr><td>Ammonium sulfate ((NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</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 (CaCl&lt;sub&gt;2&lt;/sub&gt;)</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 (CuSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth</td><td>CP86.1</td><td></td></tr><tr><td>&lt;em&gt;D&lt;/em&gt;-glucose</td><td>Carl Roth</td><td>6780</td><td></td></tr><tr><td>1,2-Bis-(dimethylamino)-ethane,&amp;nbsp;N,N,N&#039;,N&#039;-Tetramethylethylenediamine (TEMED)</td><td>Carl Roth</td><td>2367.3</td><td>&amp;ge;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 (K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth</td><td>P749.1</td><td></td></tr><tr><td>di-sodium hydrogen phosphate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</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 (FeCl&lt;sub&gt;2&lt;/sub&gt;)</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 &amp;beta;-&lt;em&gt;D&lt;/em&gt;-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 (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Carl Roth</td><td>KK36.1</td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth</td><td>8283.2</td><td></td></tr><tr><td>Manganese chloride (MnCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>63535</td><td></td></tr><tr><td>&amp;beta;-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 (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</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 (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth</td><td>T879</td><td></td></tr><tr><td>Sodium dodecyl sulphate (NaC&lt;sub&gt;12&lt;/sub&gt;H&lt;sub&gt;25&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</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 (ZnCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>229997</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-alanine</td><td>Sigma-Aldrich</td><td>A7627</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-arginine</td><td>Sigma-Aldrich</td><td>A5006</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-asparagine</td><td>Sigma-Aldrich</td><td>A8381</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-aspartic acid</td><td>Sigma-Aldrich</td><td>A0884</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-cysteine</td><td>Sigma-Aldrich</td><td>C7352</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-glutamic acid</td><td>Sigma-Aldrich</td><td>G2128</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-glutamine</td><td>Sigma-Aldrich</td><td>G3126</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-glycine</td><td>Sigma-Aldrich</td><td>G7126</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-histidine</td><td>Sigma-Aldrich</td><td>H8000</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-isoleucine</td><td>Sigma-Aldrich</td><td>I2752</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-leucine</td><td>Sigma-Aldrich</td><td>L8000</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-lysine</td><td>Sigma-Aldrich</td><td>L5501</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-methionine</td><td>Sigma-Aldrich</td><td>M9625</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-phenylalanine</td><td>Sigma-Aldrich</td><td>P2126</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-proline</td><td>Sigma-Aldrich</td><td>P0380</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-serine</td><td>Sigma-Aldrich</td><td>S4500</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-threonine</td><td>Sigma-Aldrich</td><td>T8625</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-tryptophan</td><td>Sigma-Aldrich</td><td>T0254</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-tyrosine</td><td>Sigma-Aldrich</td><td>T3754</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-valine</td><td>Sigma-Aldrich</td><td>V0500</td><td></td></tr><tr><td>(4&lt;em&gt;S&lt;/em&gt;)-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>(4&lt;em&gt;R&lt;/em&gt;)-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 &lt;em&gt;E. coli &lt;/em&gt;strain JM83</td><td>Addgene</td><td>50348</td><td>https://www.addgene.org/50348/</td></tr><tr><td>Pro-auxotrophic &lt;em&gt;E. coli&lt;/em&gt; 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 &amp;micro;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 &amp;micro;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>&amp;Auml;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 &amp;ldquo;Z&amp;rdquo; 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 ...

residue-specific-exchange-proline-proline-analogs-fluorescent

Research

JoVE Journal

Bioengineering

2.9K Views.  Technische Universität Berlin. In de meeste gevallen is conventionele plaatsgerichte mutagenese niet geschikt om de functionele rol van prolines in eiwitten te bestuderen. Hier presenteren we een methode om proline-analogen als niet-canonieke aminozuren op te nemen in ribosomaal gesynthetiseerde eiwitten om de rol van prolineresiduen op eiwitvouwing en -functie te onderzoeken. De vervanging van prolines door standaard aminozuren is een riskante aanpak.Integratie van proline analogen maakt een soort moleculaire chiru...

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

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Biochemistry

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

Chemistry

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. However, there is still little information about how insoluble protein aggregates exert their toxic effects in vivo. Simple prokaryotic and eukaryotic model organisms, such as bacteria and yeast, have contributed significantly to our present understanding of the mechanisms behind the intracellular amyloid formation, aggregates propagation, and toxicity. In this protocol, the use of yeast is described as a model to dissect the relationship between the formation of protein aggregates and their impact on cellular oxidative stress. The method combines the detection of the intracellular soluble/aggregated state of an amyloidogenic protein with the quantification of the cellular oxidative damage resulting from its expression using flow cytometry (FC). This approach is simple, fast, and quantitative. The study illustrates the technique by correlating the cellular oxidative stress caused by a large set of amyloid-&#946; peptide variants with their respective intrinsic aggregation propensities.

Protein aggregation elicits cellular oxidative stress. This protocol describes a method for monitoring the intracellular states of amyloidogenic proteins and the oxidative stress associated with them, using flow cytometry. The approach is used to study the behavior of soluble and aggregation-prone variants of the amyloid-&#946; peptide.

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Biology

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

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

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